Microfluidic engineered high cell density three-dimensional neural cultures

NASA Astrophysics Data System (ADS)

Cullen, D. Kacy; Vukasinovic, Jelena; Glezer, Ari; La Placa, Michelle C.

2007-06-01

Three-dimensional (3D) neural cultures with cells distributed throughout a thick, bioactive protein scaffold may better represent neurobiological phenomena than planar correlates lacking matrix support. Neural cells in vivo interact within a complex, multicellular environment with tightly coupled 3D cell-cell/cell-matrix interactions; however, thick 3D neural cultures at cell densities approaching that of brain rapidly decay, presumably due to diffusion limited interstitial mass transport. To address this issue, we have developed a novel perfusion platform that utilizes forced intercellular convection to enhance mass transport. First, we demonstrated that in thick (>500 µm) 3D neural cultures supported by passive diffusion, cell densities <=5.0 × 103 cells mm-3 were required for survival. In 3D neuronal and neuronal-astrocytic co-cultures with increased cell density (>=104 cells mm-3), continuous medium perfusion at 2.0-11.0 µL min-1 improved viability compared to non-perfused cultures (p < 0.01), which exhibited widespread cell death and matrix degradation. In perfused cultures, survival was dependent on proximity to the perfusion source at 2.00-6.25 µL min-1 (p < 0.05); however, at perfusion rates of 10.0-11.0 µL min-1 survival did not depend on the distance from the perfusion source, and resulted in a preservation of cell density with >90% viability in both neuronal cultures and neuronal-astrocytic co-cultures. This work demonstrates the utility of forced interstitial convection in improving the survival of high cell density 3D engineered neural constructs and may aid in the development of novel tissue-engineered systems reconstituting 3D cell-cell/cell-matrix interactions.

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

PubMed

Johnson, Tylor J; Hildreth, Michael B; Gu, Liping; Zhou, Ruanbao; Gibbons, William R

2015-06-01

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

L929 cell cytotoxicity associated with experimental and commercial dental flosses

NASA Astrophysics Data System (ADS)

Tua-ngam, P.; Supanitayanon, L.; Dechkunakorn, S.; Anuwongnukroh, N.; Srikhirin, T.; Roongrujimek, P.

2017-11-01

This aim of the study was to investigate the cytotoxicity of two commercial and two experimental dental flosses. Two commercial, Oral B® Essential Floss (nylon-waxed) and Thai Silk Floss (silk-waxed), and two experimental, Floss X (nylon-waxed) and Floss Xu (nylon-unwaxed) dental flosses were used. The cytotoxic assay was performed by using cell cultures (L929) which were subjected to cell viability test with methyl-tetrazolium. Each floss specimen (0.4 g) was placed in 1 ml of Minimum Essential Medium at 37°C with 5% CO2 at 100% humidity in an incubator for 24 hours. After incubation, the cell mitochondrial activity was evaluated for detecting viable cells using optical density as per the guidelines of ISO 10993-5:2009(E). Cytotoxic effects were evaluated by measuring percentage of cell viability at 3 points of time- 5 mins, 30 mins, and 1 hr. The results showed that two commercial dental flosses and Floss X had cell viability about 90% at the three time points; however, the experimental Floss Xu presented 80% cell viability at 5 min and <70% cell viability at 30 min and 1 hr. The results concluded that the commercial dental flosses and the experimental dental floss with wax tested in this study were acceptable for clinical use.

Engineering of cell-laden gelatin-based microgels for cell delivery and immobilization in regenerative therapies.

PubMed

Blocki, Anna; Löper, Farina; Chirico, Nino; Neffe, Axel T; Jung, Friedrich; Stamm, Christof; Lendlein, Andreas

2017-01-01

Cell-based therapies often face the challenge of low cell retention and viability upon transplantation. Hence, biomaterials, which can immobilize transplanted cells, while at the same time support cell viability, are essential for successful clinical application. Noteworthy, biomaterials in the micrometer range such as microcapsules or microspheres have the advantage of a minimally invasive introduction into tissue.Hence, we established an approach to generate gelatin-based cell carriers in the form of microspherical hydrogels. Fibroblasts were microencapsulated in glycidylmethacrylate (GMA)-functionalized gelatin by photopolymerization. While the degree of GMA-functionalization was kept constant, the hydrogel cross-linking density was adjusted by varying the time of irradiation or the average gelatin-chain length.Stable microspheres were synthesized from 10 wt% GMA-gelatin solutions for all irradiation periods tested (0.5 -2 min). Evaluation of cell viability revealed that microgels with the same weight content of biopolymer but with decreased cross-linking densities and thus decreased storage and E modulus, resulted in best cell support. Noteworthy, encapsulated cells partially migrated out of the microspheres and attached to the spherical surface.10 wt% GMA-gelatin-based hydrogels with E moduli comparable to the native cellular niche proved to be a promising biomaterial suitable for the production of cell-laden microspheres and shall be evaluated further for biomedical application.

Density gradient electrophoresis of cultured human embryonic kidney cells

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Giranda, V.; Todd, P. W.

1985-01-01

Ground based confirmation of the electrophoretic heterogeneity of human embryonic kidney cell cultures, the general characterization of their electrophoretic migration, and observations on the general properties of cultures derived from electrophoretic subpopulations were studied. Cell migration in a density gradient electrophoresis column and cell electrophoretic mobility was determined. The mobility and heterogeneity of cultured human embryonic kidney cells with those of fixed rat erythrocytes as model test particle was compared. Electrophoretically separated cell subpopulations with respect to size, viability, and culture characteristics were examined.

The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability.

PubMed

Billiet, Thomas; Gevaert, Elien; De Schryver, Thomas; Cornelissen, Maria; Dubruel, Peter

2014-01-01

In the present study, we report on the combined efforts of material chemistry, engineering and biology as a systemic approach for the fabrication of high viability 3D printed macroporous gelatin methacrylamide constructs. First, we propose the use and optimization of VA-086 as a photo-initiator with enhanced biocompatibility compared to the conventional Irgacure 2959. Second, a parametric study on the printing of gelatins was performed in order to characterize and compare construct architectures. Hereby, the influence of the hydrogel building block concentration, the printing temperature, the printing pressure, the printing speed, and the cell density were analyzed in depth. As a result, scaffolds could be designed having a 100% interconnected pore network in the gelatin concentration range of 10-20 w/v%. In the last part, the fabrication of cell-laden scaffolds was studied, whereby the application for tissue engineering was tested by encapsulation of the hepatocarcinoma cell line (HepG2). Printing pressure and needle shape was revealed to impact the overall cell viability. Mechanically stable cell-laden gelatin methacrylamide scaffolds with high cell viability (>97%) could be printed. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enhancement of viability of muscle precursor cells on 3D scaffold in a perfusion bioreactor.

PubMed

Cimetta, E; Flaibani, M; Mella, M; Serena, E; Boldrin, L; De Coppi, P; Elvassore, N

2007-05-01

The aim of this study was to develop a methodology for the in vitro expansion of skeletal-muscle precursor cells (SMPC) in a three-dimensional (3D) environment in order to fabricate a cellularized artificial graft characterized by high density of viable cells and uniform cell distribution over the entire 3D domain. Cell seeding and culture within 3D porous scaffolds by conventional static techniques can lead to a uniform cell distribution only on the scaffold surface, whereas dynamic culture systems have the potential of allowing a uniform growth of SMPCs within the entire scaffold structure. In this work, we designed and developed a perfusion bioreactor able to ensure long-term culture conditions and uniform flow of medium through 3D collagen sponges. A mathematical model to assist the design of the experimental setup and of the operative conditions was developed. The effects of dynamic vs static culture in terms of cell viability and spatial distribution within 3D collagen scaffolds were evaluated at 1, 4 and 7 days and for different flow rates of 1, 2, 3.5 and 4.5 ml/min using C2C12 muscle cell line and SMPCs derived from satellite cells. C2C12 cells, after 7 days of culture in our bioreactor, perfused applying a 3.5 ml/min flow rate, showed a higher viability resulting in a three-fold increase when compared with the same parameter evaluated for cultures kept under static conditions. In addition, dynamic culture resulted in a more uniform 3D cell distribution. The 3.5 ml/min flow rate in the bioreactor was also applied to satellite cell-derived SMPCs cultured on 3D collagen scaffolds. The dynamic culture conditions improved cell viability leading to higher cell density and uniform distribution throughout the entire 3D collagen sponge for both C2C12 and satellite cells.

Viability of HEK 293 cells on poly-β-hydroxybutyrate (PHB) biosynthesized from a mutant Azotobacter vinelandii strain. Cast film and electrospun scaffolds.

PubMed

Romo-Uribe, Angel; Meneses-Acosta, Angelica; Domínguez-Díaz, Maraolina

2017-12-01

Sterilization, cytotoxicity and cell viability are essential properties defining a material for medical applications and these characteristics were investigated for poly(β-hydroxybutyrate) (PHB) of 230kDa obtained by bacterial synthesis from a mutant strain of Azotobacter vinelandii. Cell viability was investigated for two types of PHB scaffolds, solution cast films and non-woven electrospun fibrous membranes, and the efficiency was compared against a culture dish. The biosynthesized PHB was sterilized by ultraviolet radiation and autoclave, it was found that the thermal properties and intrinsic viscosity remained unchanged indicating that the sterilization methods did not degrade the polymer. Sterilized scaffolds were then seeded with human embryonic kidney 293 (HEK 293) cells to evaluate the cytotoxic response. The cell viability of these cells was evaluated for up to six days, and the results showed that the cell morphology was normal, with no cytotoxic effects. The films and electrospun membranes exhibited over 95% cell viability whereas the viability in culture dishes reached only ca. 90%. The electrospun membrane, however, exhibited significantly higher cell density than the cast film suggesting that the fibrous morphology enables better nutrients transfer. The results indicate that the biosynthesized PHB stands UV and autoclave sterilization methods, it is biocompatible and non-toxic for cell growth of human cell lines. Furthermore, cell culture for up to 18 days showed that 62% and 90% of mass was lost for the film and fibrous electrospun scaffold, respectively. This is a favorable outcome for use in tissue engineering where material degradation, as tissue regenerates, is desirable. Copyright © 2017 Elsevier B.V. All rights reserved.

Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks

PubMed Central

Aebersold, Mathias J.; Thompson-Steckel, Greta; Joutang, Adriane; Schneider, Moritz; Burchert, Conrad; Forró, Csaba; Weydert, Serge; Han, Hana; Vörös, János

2018-01-01

Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such in vitro bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of in vitro assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days in vitro at densities ranging from 50,000 cells/cm2 down to isolated cells at 1,000 cells/cm2. Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development. PMID:29535595

Effects of chlorogenic acid on intracellular calcium regulation in lysophosphatidylcholine-treated endothelial cells.

PubMed

Jung, Hye-Jin; Im, Seung-Soon; Song, Dae-Kyu; Bae, Jae-Hoon

2017-06-01

Lysophosphatidylcholine (LPC) is a major phospholipid component of oxidized low-density lipoprotein (ox-LDL) and is implicated in its atherogenic activity. This study investigated the effects of LPC on cell viability, intracellular calcium homeostasis, and the protective mechanisms of chlorogenic acid (CGA) in human umbilical vein endothelial cells (HUVECs). LPC increased intracellular calcium ([Ca 2＋ ] i ) by releasing Ca 2＋ from intracellular stores and via Ca 2＋ influx through store-operated channels (SOCs). LPC also increased the generation of reactive oxygen species (ROS) and decreased cell viability. The mRNA expression of Transient receptor potential canonical (TRPC) channel 1 was increased significantly by LPC treatment and suppressed by CGA. CGA inhibited LPC-induced Ca 2＋ influx and ROS generation, and restored cell viability. These results suggested that CGA inhibits SOC-mediated Ca 2＋ influx and ROS generation by attenuating TRPC1 expression in LPC-treated HUVECs. Therefore, CGA might protect endothelial cells against LPC injury, thereby inhibiting atherosclerosis. [BMB Reports 2017; 50(6): 323-328].

Effects of chlorogenic acid on intracellular calcium regulation in lysophosphatidylcholine-treated endothelial cells

PubMed Central

Jung, Hye-Jin; Im, Seung-Soon; Song, Dae-Kyu; Bae, Jae-Hoon

2017-01-01

Lysophosphatidylcholine (LPC) is a major phospholipid component of oxidized low-density lipoprotein (ox-LDL) and is implicated in its atherogenic activity. This study investigated the effects of LPC on cell viability, intracellular calcium homeostasis, and the protective mechanisms of chlorogenic acid (CGA) in human umbilical vein endothelial cells (HUVECs). LPC increased intracellular calcium ([Ca2+]i) by releasing Ca2+ from intracellular stores and via Ca2+ influx through store-operated channels (SOCs). LPC also increased the generation of reactive oxygen species (ROS) and decreased cell viability. The mRNA expression of Transient receptor potential canonical (TRPC) channel 1 was increased significantly by LPC treatment and suppressed by CGA. CGA inhibited LPC-induced Ca2+ influx and ROS generation, and restored cell viability. These results suggested that CGA inhibits SOC-mediated Ca2+ influx and ROS generation by attenuating TRPC1 expression in LPC-treated HUVECs. Therefore, CGA might protect endothelial cells against LPC injury, thereby inhibiting atherosclerosis. PMID:28088946

Low-level laser irradiation induces in vitro proliferation of stem cells from human exfoliated deciduous teeth.

PubMed

Ginani, Fernanda; Soares, Diego Moura; de Oliveira Rocha, Hugo Alexandre; de Souza, Lélia Batista; Barboza, Carlos Augusto Galvão

2018-01-01

The aim of this study was to evaluate the effect of low-level laser irradiation (LLLI) on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHED). Cells were irradiated or not (control) with an InGaAlP laser diode (660 nm, 30 mW, continuous action mode) using two different energy densities (0.5 J/cm 2 -16 s; 1.0 J/cm 2 -33 s). Irradiation was performed at 0 and 48 h, with the laser probe fixed at a distance of 0.5 cm from the cells. Cell proliferation was analyzed at 0, 24, 48, and 72 h by the Trypan blue exclusion method and MTT assay. Cell cycle and Ki67 expression were analyzed by flow cytometry. Apoptosis-related events were evaluated by expression of annexin V/PI and nuclear morphological changes by staining with DAPI. Differences between groups at each time were analyzed by the Kruskal-Wallis and Mann-Whitney tests, adopting a level of significance of 5% (p < 0.05). The results showed that an energy density of 1.0 J/cm 2 promoted an increase in cell proliferation at 48 and 72 h compared to the control and 0.5 J/cm 2 groups. Cell cycle analysis revealed a predominance of cells in the S and G2/M phases in the irradiated groups. This finding was confirmed by the increased expression of Ki67. Low positive staining for annexin V and PI was observed in all groups, and no nuclear changes were detected, indicating that cell viability was not affected by the energy densities tested. It can be concluded that the LLLI parameters used (660 nm, 30 mW, 1.0 J/cm 2 ) promote the proliferation of SHEDs and the maintenance of cell viability.

Rapid, portable and cost-effective yeast cell viability and concentration analysis using lensfree on-chip microscopy and machine learning.

PubMed

Feizi, Alborz; Zhang, Yibo; Greenbaum, Alon; Guziak, Alex; Luong, Michelle; Chan, Raymond Yan Lok; Berg, Brandon; Ozkan, Haydar; Luo, Wei; Wu, Michael; Wu, Yichen; Ozcan, Aydogan

2016-11-01

Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm 2 . This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP's performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 10 5 to 1.4 × 10 6 cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.

Effect of various commercial buffers on sperm viability and capacitation.

PubMed

Andrisani, Alessandra; Donà, Gabriella; Ambrosini, Guido; Bonanni, Guglielmo; Bragadin, Marcantonio; Cosmi, Erich; Clari, Giulio; Armanini, Decio; Bordin, Luciana

2014-08-01

A wide variety of sperm preparation protocols are currently available for assisted conception. They include density gradient separation and washing methods. Both aim at isolating and capacitating as much motile sperm as possible for subsequent oocyte fertilization. The aim of this study was to examine the effects of four commercial sperm washing buffers on sperm viability and capacitation. Semen samples from 48 healthy donors (normal values of sperm count, motility, morphology, and volume) were analyzed. After separation (density gradient 40/80%), sperm were incubated in various buffers then analysed for reactive oxygen species (ROS) production, viability, tyrosine phosphorylation (Tyr-P), cholera toxin B subunit (CTB) labeling, and the acrosome reaction (AR). The buffers affected ROS generation in various ways resulting either in rapid cell degeneration (when the amount of ROS was too high for cell survival) or the inability of the cells to maintain correct functioning (when ROS were too few). Only when the correct ROS generation curve was maintained, suitable membrane reorganization, evidenced by CTB labeling was achieved, leading to the highest percentages of both Tyr-P- and acrosome-reacted-cells. Distinguishing each particular pathological state of the sperm sample would be helpful to select the preferred buffer treatment since both ROS production and membrane reorganization can be significantly altered by commercial buffers.

Impact of Pancreatic Rat Islet Density on Cell Survival during Hypoxia

PubMed Central

Rodriguez-Brotons, A.; Bietiger, W.; Peronet, C.; Magisson, J.; Sookhareea, C.; Langlois, A.; Mura, C.; Jeandidier, N.; Pinget, M.; Sigrist, S.; Maillard, E.

2016-01-01

In bioartificial pancreases (BP), the number of islets needed to restore normoglycaemia in the diabetic patient is critical. However, the confinement of a high quantity of islets in a limited space may impact islet survival, particularly in regard to the low oxygen partial pressure (PO2) in such environments. The aim of the present study was to evaluate the impact of islet number in a confined space under hypoxia on cell survival. Rat islets were seeded at three different concentrations (150, 300, and 600 Islet Equivalents (IEQ)/cm2) and cultured in normal atmospheric pressure (160 mmHg) as well as hypoxic conditions (15 mmHg) for 24 hours. Cell viability, function, hypoxia-induced changes in gene expression, and cytokine secretion were then assessed. Notably, hypoxia appeared to induce a decrease in viability and increasing islet density exacerbated the observed increase in cellular apoptosis as well as the loss of function. These changes were also associated with an increase in inflammatory gene transcription. Taken together, these data indicate that when a high number of islets are confined to a small space under hypoxia, cell viability and function are significantly impacted. Thus, in order to improve islet survival in this environment during transplantation, oxygenation is of critical importance. PMID:26824040

Indocyanine green-mediated photobiomodulation on human osteoblast cells.

PubMed

Ateş, Gamze Bölükbaşı; Ak, Ayşe; Garipcan, Bora; Gülsoy, Murat

2018-05-09

Photobiomodulation (PBM) and photodynamic therapy (PDT) share similar mechanisms but have opposite aims. Increased levels of reactive oxygen species (ROS) in the target tissue in response to light combined photosensitizer (PS) application may lead to cell proliferation or oxidative damage depending on the ROS amount. The purpose of the present study is to investigate the effect of indocyanine green (ICG)-mediated PBM on osteoblast cells by measuring cell viability, proliferation, alkaline phosphatase (ALP) activity, mineralization, and gene expressions of three phenotypic osteoblast markers. A diode laser irradiating at 809 nm (10 W output power, 50 mW/cm 2 power density) was used at 0.5, 1, and 2 J/cm 2 energy densities (10, 20, and 40 s respectively) was applied following ICG incubation. No inhibitory effect was observed in cell viability and proliferation according to the (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Alamar Blue assays. ICG-mediated PBM did not alter cell viability but increased ALP activity and enhanced mineralization of existing osteoblasts. These results were also confirmed by real-time polymerase chain reaction (RT-PCR) analysis of osteoblastic markers. PS can be combined to PBM not only to damage the malignant cells as aimed in PDT studies, but also to promote cellular activity. The findings of this in vitro study may contribute to in vivo studies and ICG-mediated PBM can have promising outcomes in bone healing and regeneration therapies in future.

A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry.

PubMed

Martyniak, Brian; Bolton, Jason; Kuksin, Dmitry; Shahin, Suzanne M; Chan, Leo Li-Ying

2017-01-01

Brettanomyces spp. can present unique cell morphologies comprised of excessive pseudohyphae and budding, leading to difficulties in enumerating cells. The current cell counting methods include manual counting of methylene blue-stained yeasts or measuring optical densities using a spectrophotometer. However, manual counting can be time-consuming and has high operator-dependent variations due to subjectivity. Optical density measurement can also introduce uncertainties where instead of individual cells counted, an average of a cell population is measured. In contrast, by utilizing the fluorescence capability of an image cytometer to detect acridine orange and propidium iodide viability dyes, individual cell nuclei can be counted directly in the pseudohyphae chains, which can improve the accuracy and efficiency of cell counting, as well as eliminating the subjectivity from manual counting. In this work, two experiments were performed to demonstrate the capability of Cellometer image cytometer to monitor Brettanomyces concentrations, viabilities, and budding/pseudohyphae percentages. First, a yeast propagation experiment was conducted to optimize software counting parameters for monitoring the growth of Brettanomyces clausenii, Brettanomyces bruxellensis, and Brettanomyces lambicus, which showed increasing cell concentrations, and varying pseudohyphae percentages. The pseudohyphae formed during propagation were counted either as multiple nuclei or a single multi-nuclei organism, where the results of counting the yeast as a single multi-nuclei organism were directly compared to manual counting. Second, a yeast fermentation experiment was conducted to demonstrate that the proposed image cytometric analysis method can monitor the growth pattern of B. lambicus and B. clausenii during beer fermentation. The results from both experiments displayed different growth patterns, viability, and budding/pseudohyphae percentages for each Brettanomyces species. The proposed Cellometer image cytometry method can improve efficiency and eliminate operator-dependent variations of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.

Validation of in vitro assays in three-dimensional human dermal constructs.

PubMed

Idrees, Ayesha; Chiono, Valeria; Ciardelli, Gianluca; Shah, Siegfried; Viebahn, Richard; Zhang, Xiang; Salber, Jochen

2018-05-01

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially "murine in vitro dermal construct" based on L929 cells was generated, leading to the development of "human in vitro dermal construct" consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue ® , RealTime-Glo ™ MT, and CellTiter-Glo ® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the "shaking time" to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

Low-level laser therapy with helium-neon laser improved viability of osteoporotic bone marrow-derived mesenchymal stem cells from ovariectomy-induced osteoporotic rats

NASA Astrophysics Data System (ADS)

Fallahnezhad, Somaye; Piryaei, Abbas; Tabeie, Faraj; Nazarian, Hamid; Darbandi, Hasan; Amini, Abdoldllah; Mostafavinia, Ataroalsadat; Ghorishi, Seyed Kamran; Jalalifirouzkouhi, Ali; Bayat, Mohammad

2016-09-01

The purpose of this study was to evaluate the influences of helium-neon (He-Ne) and infrared (IR) lasers on the viability and proliferation rate of healthy and ovariectomy-induced osteoporotic (OVX) bone marrow mesenchymal stem cells (BMMSCs) in vitro. MSCs harvested from the BM of healthy and OVX rats were culture expanded. He-Ne and IR lasers were applied three times at energy densities of 0.6, 1.2, and 2.4 J/cm2 for BMMSCs. BMMSCs viability and proliferation rate were evaluated by MTT assay on days 2, 4, 6, 14, and 21. The results showed that healthy BMMSCs responded optimally to 0.6 J/cm2 using an IR laser after three times of laser radiation. Moreover, it was found that OVX-BMMSCs responded optimally to 0.6 J/cm2 with He-Ne laser and one-time laser radiation. It is concluded that the low-level laser therapy (LLLT) effect depends on the physiological state of the BMMSCs, type of the laser, wavelength, and number of laser sessions. The biostimulation efficiency of LLLT also depends on the delivered energy density. LLLT can enhance the viability and proliferation rate of healthy and especially osteoporotic autologous BMMSCs, which could be very useful in regenerative medicine.

Influence of electric current on bacterial viability in wastewater treatment.

PubMed

Wei, V; Elektorowicz, M; Oleszkiewicz, J A

2011-10-15

Minimizing the influence of electric current on bacterial viability in the electro-technologies such as electrophoresis and electrocoagulation is crucial in designing and operating the electric hybrid wastewater treatment system. In this study the biomass from a membrane bioreactor (MBR) was subjected to constant direct current and the bacterial viability was monitored against electrical intensity, duration as well as the spatial vicinity related to the electrodes. It was found that the bacterial viability was not significantly affected (less than 10% of death percentage) when the applied electric current density (CD) was less than 6.2 A/m2 after 4 h. The percentage of live cell dropped by 15% and 29% at CD of 12.3 A/m2 and 24.7 A/m2, respectively. The pH of electrolytic biomass fluid has shifted to alkaline (from nearly neutral to around pH 10) at CD above 12.3 A/m2, which could have been the contributing factor for the bacterial inactivation. The temperature change in the electrolytic media at all current densities during 4 h of experiment was less than 2 °C, thus temperature effects were negligible. Bacteria experienced different micro-environments in the electrochemical reactor. Bacterial cells on the cathode surface exhibited highest death rate, whereas bacteria outside the space between electrodes were the least affected. It was concluded that in an electro-technology integrated wastewater treatment process, sufficient mixing should be used to avoid localized inactivation of bacterial cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Investigating the importance of flow when utilizing hyaluronan scaffolds for tissue engineering.

PubMed

Donegan, Gail C; Hunt, John A; Rhodes, Nicholas

2010-02-01

Esterified hyaluronan scaffolds offer significant advantages for tissue engineering. They are recognized by cellular receptors, interact with many other extracellular matrix proteins and their metabolism is mediated by intrinsic cellular pathways. In this study differences in the viability and structural integrity of vascular tissue models cultured on hyaluronan scaffolds under laminar flow conditions highlighted potential differences in the biodegradation kinetics, processes and end-products, depending on the culture environment. Critical factors are likely to include seeding densities and the duration and magnitude of applied biomechanical stress. Proteomic evaluation of the timing and amount of remodelling protein expression, the resulting biomechanical changes arising from this response and metabolic cell viability assay, together with examination of tissue morphology, were conducted in vascular tissue models cultured on esterified hyaluronan felt and PTFE mesh scaffolds. The vascular tissue models were derived using complete cell sheets derived from harvested and expanded umbilical cord vein cells. This seeding method utilizes high-density cell populations from the outset, while the cells are already supported by their own abundant extracellular matrix. Type I and type IV collagen expression in parallel with MMP-1 and MMP-2 expression were monitored in the tissue models over a 10 day culture period under laminar flow regimes using protein immobilization technologies. Uniaxial tensile testing and scanning electron microscopy were used to compare the resulting effects of hydrodynamic stimulation upon structural integrity, while viability assays were conducted to evaluate the effects of shear on metabolic function. The proteomic results showed that the hyaluronan felt-supported tissues expressed higher levels of all remodelling proteins than those cultured on PTFE mesh. Overall, a 21% greater expression of type I collagen, 24% higher levels of type IV collagen, 24% higher levels of MMP-1 and 34% more MMP-2 were observed during hydrodynamic stress. This was coupled with a loss of structural integrity in these models after the introduction of laminar flow, as compared to the increases in all mechanical properties observed in the PTFE mesh-supported tissues. However, under flow conditions, the hyaluronan-supported tissues showed some recovery of the viability originally lost during static culture conditions, in contrast to PTFE mesh-based models, where initial gains were followed by a decline in metabolic viability after applied shear stress. Proteomic, cell viability and mechanical testing data emphasized the need for extended in vitro evaluations to enable better understanding of multi-stage remodelling and reparative processes in tissues cultured on biodegradable scaffolds. This study also highlighted the possibility that in high-density tissue culture with a biodegradable component, dynamic conditions may be more conducive to optimal tissue development than the static environment because they facilitate the efficient removal of high concentrations of degradation end-products accumulating in the pericellular space.

Evaluating 3D bone tissue engineered constructs with different seeding densities using the alamarBlue assay and the effect on in vivo bone formation.

PubMed

Wilson, C E; Dhert, W J A; Van Blitterswijk, C A; Verbout, A J; De Bruijn, J D

2002-12-01

Bone tissue engineering using patient derived cells seeded onto porous scaffolds has gained much attention in recent years. Evaluating the viability of these 3D constructs is an essential step in optimizing the process. The alamarBlue (aB) assay was evaluated for its potential to follow in vitro cell proliferation on architecturally standardized hydroxyapatite scaffolds. The impact of the aB assayed and seeding density on subsequent in vivo bone formation was investigated. Twelve scaffolds were seeded with various densities from 250 to 2.5x10(6) cells/scaffold and assay by aB at 5 time points during the 7-day culture period. Twelve additional scaffolds were seeded with 2.5x10(5) cells/scaffold. Two control and 2 aB treated scaffolds were subcutaneously implanted into each of 6 nude mice for 6 weeks. Four observers ranked bone formation using a pair wise comparison of histological sections form each mouse. The aB assay successfully followed cell proliferation, however, the diffusion kinetics of the 3D constructs must be considered. The influence of in vitro aB treatment on subsequent in vivo bone formation cannot be ruled out but was not shown to be significant in the current study. The aB assay appears to be quite promising for evaluating a maximum or end-point viability of 3D tissue engineered constructs. Finally, higher seeding densities resulted in more observed bone formation.

Effect of low-level laser irradiation and epidermal growth factor on adult human adipose-derived stem cells.

PubMed

Mvula, B; Moore, T J; Abrahamse, H

2010-01-01

The study investigated the effects of low-level laser radiation and epidermal growth factor (EGF) on adult adipose-derived stem cells (ADSCs) isolated from human adipose tissue. Isolated cells were cultured to semi-confluence, and the monolayers of ADSCs were exposed to low-level laser at 5 J/cm(2) using 636 nm diode laser. Cell viability and proliferation were monitored using adenosine triphosphate (ATP) luminescence and optical density at 0 h, 24 h and 48 h after irradiation. Application of low-level laser irradiation at 5 J/cm(2) on human ADSCs cultured with EGF increased the viability and proliferation of these cells. The results indicate that low-level laser irradiation in combination with EGF enhances the proliferation and maintenance of ADSCs in vitro.

Premixed calcium phosphate cements: synthesis, physical properties, and cell cytotoxicity.

PubMed

Xu, Hockin H K; Carey, Lisa E; Simon, Carl G; Takagi, Shozo; Chow, Laurence C

2007-04-01

Calcium phosphate cement (CPC) is a promising material for dental, periodontal, and craniofacial repairs. However, its use requires on-site powder-liquid mixing that increases the surgical placement time and raises concerns of insufficient and inhomogeneous mixing. The objective of this study was to determine a formulation of premixed CPC (PCPC) with rapid setting, high strength, and good in vitro cell viability. PCPCs were formulated from CPC powder+non-aqueous liquid+gelling agent+hardening accelerator. Five PCPCs were thus developed: PCPC-Tartaric, PCPC-Malonic, PCPC-Citric, PCPC-Glycolic, and PCPC-Malic. Formulations and controls were compared for setting time, diametral tensile strength, and osteoblast cell compatibility. Setting time (mean+/-S.D.; n=4) for PCPC-Tartaric was 8.2+/-0.8 min, significantly less than the 61.7+/-1.5 min for the Premixed Control developed previously (p<0.001). On 7th day immersion, the diametral tensile strength of PCPC-Tartaric reached 6.5+/-0.8 MPa, higher than 4.5+/-0.8 MPa of Premixed Control (p=0.036). Osteoblast cells displayed a polygonal morphology and attached to the nano-hydroxyapatite crystals in the PCPCs. All cements had similar live cell density values (p=0.126), indicating that the new PCPCs were as cell compatible as a non-premixed CPC control known to be biocompatible. Each of the new PCPCs had a cell viability that was not significantly different (p>0.1) from that of the non-premixed CPC control. PCPCs will eliminate the powder-liquid mixing during surgery and may also improve the cement performance. The new PCPCs supported cell attachment and yielded a high cell density and viability. Their mechanical strengths approached the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. These nano-crystalline hydroxyapatite cements may be useful in dental, periodontal, and craniofacial repairs.

Lidocaine cytotoxicity to the bovine articular chondrocytes in vitro: changes in cell viability and proteoglycan metabolism.

PubMed

Miyazaki, Tsuyoshi; Kobayashi, Shigeru; Takeno, Kenichi; Yayama, Takafumi; Meir, Adam; Baba, Hisatoshi

2011-07-01

A lot of studies on the effect of intra-articular injections are clinical, but many questions on the effect of lidocaine to articular chondrocytes remain unanswered. This study was performed to determine the effects of varying concentrations and exposure times of lidocaine on the viability and proteoglycan metabolism of chondrocytes in vitro. Cartilage was obtained from metatarsal joints of adult bovines. Chondrocytes in alginate beads were cultured in medium containing 6% fetal calf serum at 370 mOsmol at cell densities of 4 million cells/ml. They were then cultured for 24 h under 21% oxygen with 0.125, 0.25, 0.5, and 1% lidocaine and without lidocaine as control. The cell viability profile across intact beads was determined by manual counting using fluorescent probes and transmission electron microscopy. Lactate production was measured enzymatically as a marker of energy metabolism. Glycosaminoglycan (GAG) accumulation was measured using a modified dimethylmethylene blue assay. Cell viability decreased in a time- and dose-dependent manner in the concentration range of 0.125-1.0% lidocaine under the confocal microscope. Under the electron microscope, apoptosis increased as the concentration of lidocaine increased. GAG accumulation/tissue volume decreases as the concentration of lidocaine increased. However, GAG produced per million cells and the rate of lactate production per live cell were significantly higher for cells cultured at 0.5 and 1% lidocaine than the control group. Bovine chondrocytes cultured in alginate beads under high oxygen pressure are negatively influenced by increasing concentrations of lidocaine. Cell viability and proteoglycan production (GAG accumulation/tissue volume) decreased as the concentration of lidocaine increased. These data suggest caution in prolonged exposure of cartilage to high concentration lidocaine. Repeated joint injection of lidocaine potentially worsens osteoarthrosis by accelerating cartilage degradation.

Generation of murine induced pluripotent stem cells by using high-density distributed electrodes network.

PubMed

Lu, Ming-Yu; Li, Zhihong; Hwang, Shiaw-Min; Linju Yen, B; Lee, Gwo-Bin

2015-09-01

This study reports a robust method of gene transfection in a murine primary cell model by using a high-density electrodes network (HDEN). By demonstrating high cell viability after gene transfection and successful expression of transgenes including fluorescent proteins, the HDEN device shows great promise as a solution in which reprogramming efficiency using non-viral induction for generation of murine induced pluripotent stem cells (iPSCs) is optimized. High and steady transgene expression levels in host cells of iPSCs can be demonstrated using this method. Moreover, the HDEN device achieved successful gene transfection with a low voltage of less than 180 V while requiring relatively low cell numbers (less than 1.5 × 10(4) cells). The results are comparable to current conventional methods, demonstrating a reasonable fluorescent-plasmid transfection rate (42.4% in single transfection and 24.5% in triple transfection) and high cell viability of over 95%. The gene expression levels of each iPSC factor was measured to be over 10-fold higher than that reported in previous studies using a single mouse embryonic fibroblast cell. Our results demonstrate that the generation of iPSCs using HDEN transfection of plasmid DNA may be a feasible and safe alternative to using viral transfection methods in the near future.

Escherichia coli viability determination using dynamic light scattering: a comparison with standard methods.

PubMed

Loske, Achim M; Tello, Elba M; Vargas, Susana; Rodriguez, Rogelio

2014-08-01

To determine the concentration of bacteria in a sample is important in the food industry, medicine and biotechnology. A disadvantage of the plate-counting method is that a microorganism colony could arise from one cell or from many cells. The other standard methodology, known as optical density determination, is based on the turbidity of a suspension and registers all bacteria, dead and alive. In this article, dynamic light scattering is proposed as a fast and reliable method to determine bacterial viability and, consequently, time evolution. Escherichia coli was selected because this microorganism is well known and easy to handle. A correlation between the data from these three techniques was obtained. We were able to calculate the growth rate, usually determined by plate counting or optical density measurement, using dynamic light scattering and to predict bacterial behavior. An analytical relationship between the colony forming units and the light scattered intensity was also deduced.

ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication

PubMed Central

Hida, Shigeaki; Fujii, Chifumi; Taniguchi, Shun’ichiro; Ito, Kensuke; Matsumura, Tomio; Okada, Nagisa; Sakaizawa, Takashi; Kobayashi, Akira; Takeoka, Michiko; Miyagawa, Shin-ichi

2017-01-01

ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy. PMID:28056049

Collagen Matrix Density Drives the Metabolic Shift in Breast Cancer Cells.

PubMed

Morris, Brett A; Burkel, Brian; Ponik, Suzanne M; Fan, Jing; Condeelis, John S; Aguirre-Ghiso, Julio A; Castracane, James; Denu, John M; Keely, Patricia J

2016-11-01

Increased breast density attributed to collagen I deposition is associated with a 4-6 fold increased risk of developing breast cancer. Here, we assessed cellular metabolic reprogramming of mammary carcinoma cells in response to increased collagen matrix density using an in vitro 3D model. Our initial observations demonstrated changes in functional metabolism in both normal mammary epithelial cells and mammary carcinoma cells in response to changes in matrix density. Further, mammary carcinoma cells grown in high density collagen matrices displayed decreased oxygen consumption and glucose metabolism via the tricarboxylic acid (TCA) cycle compared to cells cultured in low density matrices. Despite decreased glucose entry into the TCA cycle, levels of glucose uptake, cell viability, and ROS were not different between high and low density matrices. Interestingly, under high density conditions the contribution of glutamine as a fuel source to drive the TCA cycle was significantly enhanced. These alterations in functional metabolism mirrored significant changes in the expression of metabolic genes involved in glycolysis, oxidative phosphorylation, and the serine synthesis pathway. This study highlights the broad importance of the collagen microenvironment to cellular expression profiles, and shows that changes in density of the collagen microenvironment can modulate metabolic shifts of cancer cells. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

NASA Astrophysics Data System (ADS)

Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

2014-03-01

Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

PubMed

Single, Andrew; Beetham, Henry; Telford, Bryony J; Guilford, Parry; Chen, Augustine

2015-12-01

Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines. © 2015 Society for Laboratory Automation and Screening.

Research on fluorescence detection method of Microcystis aeruginosa

NASA Astrophysics Data System (ADS)

Wang, Xiao-xiong

2017-07-01

The paper studied the viability determination of Microcystis aeruginosa by FDA and PI staining. The staining results were measured by fluorescence microscopy. The results indicated that viable and dead cells were stained as bright green and red fluorescent respectively by FDA and PI. Through PI-FDA dual color fluorescence staining, the color of green and red distinct obviously by fluorescence microscope. The staining rate has relation with the cell density. If the cell density of M. aeruginosa was 1.0×107-1.0×109 cell·mL-1, the staining rate would be 100.0% or 98.0% by PI and of FDA respectively.

Quantifying the correlation between spatially defined oxygen gradients and cell fate in an engineered three-dimensional culture model.

PubMed

Ardakani, Amir G; Cheema, Umber; Brown, Robert A; Shipley, Rebecca J

2014-09-06

A challenge in three-dimensional tissue culture remains the lack of quantitative information linking nutrient delivery and cellular distribution. Both in vivo and in vitro, oxygen is delivered by diffusion from its source (blood vessel or the construct margins). The oxygen level at a defined distance from its source depends critically on the balance of diffusion and cellular metabolism. Cells may respond to this oxygen environment through proliferation, death and chemotaxis, resulting in spatially resolved gradients in cellular density. This study extracts novel spatially resolved and simultaneous data on tissue oxygenation, cellular proliferation, viability and chemotaxis in three-dimensional spiralled, cellular collagen constructs. Oxygen concentration gradients drove preferential cellular proliferation rates and viability in the higher oxygen zones and induced chemotaxis along the spiral of the collagen construct; an oxygen gradient of 1.03 mmHg mm(-1) in the spiral direction induced a mean migratory speed of 1015 μm day(-1). Although this movement was modest, it was effective in balancing the system to a stable cell density distribution, and provided insights into the natural cell mechanism for adapting cell number and activity to a prevailing oxygen regime.

Titanium is not "the most biocompatible metal" under cathodic potential: The relationship between voltage and MC3T3 preosteoblast behavior on electrically polarized cpTi surfaces.

PubMed

Ehrensberger, Mark T; Sivan, Shiril; Gilbert, Jeremy L

2010-06-15

An electrochemically controlled system has been developed which allows for cell culture directly on electrically polarized metal surfaces with simultaneous control and assessment of the electrochemical current, potential, and impedance of the interface. This system was utilized in this study to assess the interactions between electrochemically polarized commercially pure titanium (cpTi) and MC3T3 preosteoblast cells. Cells were cultured on CpTi for 24 h at static potentials between -1000 mV and +1000 mV vs. Ag/AgCl and cell morphology (SEM and cell area) and viability (MTT and Live-Dead assay) were assessed along with the electrochemical current densities and surface oxide impedance properties. The results indicate that cathodic polarization in the range of -600 mV to -1000 mV markedly reduces the spreading and viability of cells cultured directly on cpTi within 24 h, while anodic polarization (-300 mV to +1000 mV) out to 72 h shows no difference in cell behavior as compared to the OCP condition. Analysis of the relationship between the cell outcomes and the electrochemical current densities and impedance indicated the presence of voltage-dependent electrochemical thresholds (cathodic current density, i(c) > 1.0 microA/cm(2), R(p) < 10(5) Omega cm(2)) which may control the biocompatibility of cpTi. In addition, these outcomes have direct clinical significance for modular orthopedic implants whose potential can shift, via fretting corrosion, down into the range of potentials exhibiting poor cell behavior. (c) 2009 Wiley Periodicals, Inc.

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

PubMed

Ellis, Mark; Patel, Pareshkumar; Edon, Marjory; Ramage, Walter; Dickinson, Robert; Humphreys, David P

2017-01-01

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD 600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017. © 2016 American Institute of Chemical Engineers.

Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays.

PubMed

Raghavan, Shreya; Ward, Maria R; Rowley, Katelyn R; Wold, Rachel M; Takayama, Shuichi; Buckanovich, Ronald J; Mehta, Geeta

2015-07-01

Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant 3D in vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. Spheroids had uniform geometry, with projected areas (42.60×10(3)μm-475.22×10(3)μm(2) for A2780 spheroids and 37.24×10(3)μm(2)-281.01×10(3)μm(2) for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell-cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70-80% viability) to cisplatin chemotherapy compared to 2D cultures (30-50% viability). Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel flow-perfusion bioreactor supports 3D dynamic cell culture.

PubMed

Sailon, Alexander M; Allori, Alexander C; Davidson, Edward H; Reformat, Derek D; Allen, Robert J; Warren, Stephen M

2009-01-01

Bone engineering requires thicker three-dimensional constructs than the maximum thickness supported by standard cell-culture techniques (2 mm). A flow-perfusion bioreactor was developed to provide chemotransportation to thick (6 mm) scaffolds. Polyurethane scaffolds, seeded with murine preosteoblasts, were loaded into a novel bioreactor. Control scaffolds remained in static culture. Samples were harvested at days 2, 4, 6, and 8 and analyzed for cellular distribution, viability, metabolic activity, and density at the periphery and core. By day 8, static scaffolds had a periphery cell density of 67% +/- 5.0%, while in the core it was 0.3% +/- 0.3%. Flow-perfused scaffolds demonstrated peripheral cell density of 94% +/- 8.3% and core density of 76% +/- 3.1% at day 8. Flow perfusion provides chemotransportation to thick scaffolds. This system may permit high throughput study of 3D tissues in vitro and enable prefabrication of biological constructs large enough to solve clinical problems.

Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays

PubMed Central

Raghavan, Shreya; Ward, Maria R.; Rowley, Katelyn R.; Wold, Rachel M.; Takayama, Shuichi; Buckanovich, Ronald J.; Mehta, Geeta

2015-01-01

Background Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant3Din vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. Methods We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. Results Spheroids had uniform geometry, with projected areas (42.60 × 103 μm–475.22 × 103 μm2 for A2780 spheroids and 37.24 × 103 μm2–281.01 × 103 μm2 for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell–cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70–80% viability) to cisplatin chemotherapy compared to 2D cultures (30–50% viability). Conclusions Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays. PMID:25913133

Effects of low-level laser therapy on stem cells from human exfoliated deciduous teeth.

PubMed

Fernandes, Ana Paula; Junqueira, Marina de Azevedo; Marques, Nádia Carolina Teixeira; Machado, Maria Aparecida Andrade Moreira; Santos, Carlos Ferreira; Oliveira, Thais Marchini; Sakai, Vivien Thiemy

2016-01-01

This study aimed to evaluate the influence of different laser therapy energy densities on SHED viability and proliferation. SHED were irradiated according to the groups: I (1.2 J/cm2 - 0.5 mW - 10 s), II (2.5 J/cm2 - 10 mW - 10 s), III (3.7 J/cm2 - 15 mW - 10 s), IV (5.0 J/cm2 - 20 mW - 10 s), V (6.2 J/cm2 - 25 mW - 10 s), and VI (not irradiated - control group). Cell viability was assessed 6 and 24 h after irradiation measuring the mitochondrial activity and using the Crystal Violet assay. Cell proliferation was assessed after 24, 48, and 72 h of irradiation by SRB assay. MTT assay demonstrated differences from 6 to 24 hours after irradiation. After 24 h, groups I and IV showed higher absorbance values than those of control group. Crystal Violet assay showed statistically differences in the absorbance rate from 6 to 24 h after irradiation for groups III and VI. At 24 h after irradiation, Group III absorbance rate was greater than that of groups I, II, and IV. Group VI absorbance rate was greater than that of groups I and IV. SRB assay showed that the group I had higher rates than those of groups II, III, V, and VI, at 24 h after irradiation. After 48 h, group I exhibited the greatest cell proliferation rate followed by groups III, V, and VI. After 72 h, group III exhibited the lowest cell proliferation rate than those of groups II, IV, and V. The Low-Level Laser Therapy energy densities used in this study did not cause loss of cell viability and stimulated SHED proliferation within the parameters described in this study.

Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

PubMed

Kaneko, Ai; Sankai, Yoshiyuki

2014-01-01

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4) cells/mL (8.9×10(3) cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

Propofol inhibits gap junctions by attenuating sevoflurane-induced cytotoxicity against rat liver cells in vitro.

PubMed

Huang, Fei; Li, Shangrong; Gan, Xiaoliang; Wang, Ren; Chen, Zhonggang

2014-04-01

Liver abnormalities are seen in a small proportion of patients following anaesthesia with sevoflurane. To investigate whether the cytotoxicity of sevoflurane against rat liver cells was mediated by gap junction intercellular communications, and the effect of propofol on sevoflurane-induced cytotoxicity. Experimental study. The study was carried out in the central laboratory of The Third Affiliated Hospital, Sun Yat-sen University. BRL-3A rat liver cells. Immortal rat liver cells BRL-3A were grown at low and high density. Colony-forming assays were performed to determine clonogenic growth of these cells. To investigate the effect of oleamide and propofol on gap junction function, we measured fluorescence transmission between cells using parachute dye-coupling assays. Immunoblotting assays were performed to determine connexin32 and connexin43 expression. Our colony formation assays revealed that, in low-density culture, sevoflurane caused no apparent inhibition of clonogenic growth of BRL-3A cells. In high-density culture, 2.2 to 4.4% sevoflurane markedly inhibited clonogenic growth of BRL-3A cells with 67.6 (0.34)% and 61.2 (0.17)% of the cells being viable, respectively (P = 0.003 vs. low-density culture), suggesting cell density dependency of sevoflurane-induced cytotoxicity. Our colony formation assays revealed that propofol markedly attenuated the suppression by sevoflurane of the clonogenic growth of BRL-3A cells (viability: propofol and sevoflurane, 91.5 (0.014)% vs. sevoflurane, 56.6 (0.019)%; P <0.01). Blocking gap junctions with 10 μmol l oleamide significantly attenuated 4.4% sevoflurane-induced suppression with a viability of 83.6 ± 0.138% (oleamide and sevoflurane vs. sevoflurane, P < 0.01). Immunoblotting assays further showed that propofol (3.2 μg ml) markedly reduced CX32 levels and significantly inhibited gap junctional intercellular communications as revealed by parachute dye-coupling assays. Values are mean (SD). This study provides the first direct evidence that sevoflurane-induced cytotoxicity, which is mediated through gap junctions, is attenuated by propofol, possibly by its action on Cx32 homomeric or heteromeric complexes.

Isolation and purification of rabbit mesenchymal stem cells using an optimized protocol.

PubMed

Lin, Chunbo; Shen, Maorong; Chen, Weiping; Li, Xiaofeng; Luo, Daoming; Cai, Jinhong; Yang, Yuan

2015-11-01

Mesenchymal stem cells were first isolated and grown in vitro by Friedenstein over 40 yr ago; however, their isolation remains challenging as they lack unique markers for identification and are present in very small quantities in mesenchymal tissues and bone marrow. Using whole marrow samples, common methods for mesenchymal stem cell isolation are the adhesion method and density gradient fractionation. The whole marrow sample adhesion method still results in the nonspecific isolation of mononuclear cells, and activation and/or potential loss of target cells. Density gradient fractionation methods are complicated, and may result in contamination with toxic substances that affect cell viability. In the present study, we developed an optimized protocol for the isolation and purification of mesenchymal stem cells based on the principles of hypotonic lysis and natural sedimentation.

Premixed calcium phosphate cements: Synthesis, physical properties, and cell cytotoxicity

PubMed Central

Xu, Hockin H.K.; Carey, Lisa E.; Simon, Carl G.; Takagi, Shozo; Chow, Laurence C.

2009-01-01

Objectives Calcium phosphate cement (CPC) is a promising material for dental, periodontal, and craniofacial repairs. However, its use requires on-site powder–liquid mixing that increases the surgical placement time and raises concerns of insufficient and inhomogeneous mixing. The objective of this study was to determine a formulation of premixed CPC (PCPC) with rapid setting, high strength, and good in vitro cell viability. Methods PCPCs were formulated from CPC powder + non-aqueous liquid + gelling agent + hardening accelerator. Five PCPCs were thus developed: PCPC-Tartaric, PCPC-Malonic, PCPC-Citric, PCPC-Glycolic, and PCPC-Malic. Formulations and controls were compared for setting time, diametral tensile strength, and osteoblast cell compatibility. Results Setting time (mean ± S.D.; n = 4) for PCPC-Tartaric was 8.2 ± 0.8 min, significantly less than the 61.7 ± 1.5 min for the Premixed Control developed previously (p < 0.001). On 7th day immersion, the diametral tensile strength of PCPC-Tartaric reached 6.5 ± 0.8 MPa, higher than 4.5 ± 0.8 MPa of Premixed Control (p = 0.036). Osteoblast cells displayed a polygonal morphology and attached to the nano-hydroxyapatite crystals in the PCPCs. All cements had similar live cell density values (p = 0.126), indicating that the new PCPCs were as cell compatible as a non-premixed CPC control known to be biocompatible. Each of the new PCPCs had a cell viability that was not significantly different (p > 0.1) from that of the non-premixed CPC control. Significance PCPCs will eliminate the powder–liquid mixing during surgery and may also improve the cement performance. The new PCPCs supported cell attachment and yielded a high cell density and viability. Their mechanical strengths approached the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. These nano-crystalline hydroxyapatite cements may be useful in dental, periodontal, and craniofacial repairs. PMID:16678895

Photobiomodulation of wound healing via visible and infrared laser irradiation.

PubMed

Solmaz, Hakan; Ulgen, Yekta; Gulsoy, Murat

2017-05-01

Fibroblast cells are known to be one of the key elements in wound healing process, which has been under the scope of research for decades. However, the exact mechanism of photobiomodulation on wound healing is not fully understood yet. Photobiomodulation of 635 and 809 nm laser irradiation at two different energy densities were investigated with two independent experiments; first, in vitro cell proliferation and then in vivo wound healing. L929 mouse fibroblast cell suspensions were exposed with 635 and 809 nm laser irradiations of 1 and 3 J/cm 2 energy densities at 50 mW output power separately for the investigation of photobiomodulation in vitro. Viabilities of cells were examined by means of MTT assays performed at the 24th, 48th, and 72nd hours following the laser irradiations. Following the in vitro experiments, 1 cm long cutaneous incisional skin wounds on Wistar albino rats (n = 24) were exposed with the same laser sources and doses in vivo. Wound samples were examined on 3rd, 5th, and 7th days of healing by means of mechanical tensile strength tests and histological examinations. MTT assay results showed that 635 nm laser irradiation of both energy densities after 24 h were found to be proliferative. One joule per square centimeter laser irradiation results also had positive effect on cell proliferation after 72 h. However, 809 nm laser irradiation at both energy densities had neither positive nor negative affects on cell viability. In vivo experiment results showed that, 635 nm laser irradiation of both energy densities stimulated wound healing in terms of tensile strength, whereas 809 nm laser stimulation did not cause any stimulative effect. The results of mechanical tests were compatible with the histological evaluations. In this study, it is observed that 635 nm laser irradiations of low energy densities had stimulative effects in terms of cell proliferation in vitro and mechanical strength of incisions in vivo. However, 809 nm laser irradiations at the same doses did not have any positive effect.

Optimized delivery of skin keratinocytes by aerosolization and suspension in fibrin tissue adhesive.

PubMed

Harkin, Damien G; Dawson, Rebecca A; Upton, Zee

2006-01-01

Aerosolized suspensions of keratinocytes provide a potential therapy for wounds, but the effects of aerosolization on cell viability remain unclear. Likewise, little is known of the resulting cell distribution pattern and how this compares to the density required for epithelialization. The potential benefits of cospraying cells in the presence of fibrin adhesive are equally uncertain. Thus, in the present study we have optimized conditions for the aerosolization of cultured keratinocytes using a device (Tissomat) that supports the option for coapplication with fibrin (Tisseel). Cell viability was unaffected when sprayed at 10 psi, but a significant reduction in metabolic activity, as determined by the methylthiazoyldiphenol-tetrazolium assay, was observed at higher pressure. Bursts of 0.2 mL cell suspension (1.5x10(6)/mL) delivered from a height of 10 cm was sufficient to epithelialize an area of 10-15 cm2 within 7 days in vitro. Confluent areas corresponded to those with a density of 5,000-10,000 cells/cm2 at 24 hours. Optimal cell growth in Tisseel was achieved through dilution of fibrinogen (1-3 mg/mL) and thrombin (2-5 IU/mL). This optimized formulation eliminated fluid run-off postspraying and stimulated a twofold increase in cellular response. Therefore, our in vitro data supports the theory that aerosolized suspensions of keratinocytes in fibrin will benefit healing.

Fluoro-luminometric real-time measurement of bacterial viability and killing.

PubMed

Lehtinen, Janne; Virta, Marko; Lilius, Esa Matti

2003-10-01

The viability and killing of Escherichia coli was measured on a real-time basis using a fluoro-luminometric device, which allows successive measurements of fluorescence and bioluminescence without user intervention. Bacteria were made fluorescent and bioluminescent by expression of gfp and insect luciferase (lucFF) genes. The green fluorescent protein (GFP) is a highly fluorescent, extremely stable protein, which accumulates in cells during growth, and therefore the measured fluorescence signal was proportional to the total number of cells. The luciferase reaction is dependent of ATP produced by living cells, so that the bioluminescence level was a direct measure of the viable cells. In contrast to the bacterial luciferase, the insect luciferase uses a water-soluble and nonvolatile substrate, which makes automated multi-well microplate assay possible. For the validation of the assay, the proportion of living and dead cell populations was experimentally modified by incubating E. coli cells in the presence of various ethanol concentrations. Bacterial viability and killing measured by a fluoro-luminometric assay correlated fairly well with the reference methods: conventional plate counting, optical density measurement and various flow cytometric analyses. The real-time assay described here allows following the changes in bacterial cultures and assessing the bactericidal and other effects of various chemical, immunological and physical agents simultaneously in large numbers of samples.

Effects of Orange II and Sudan III azo dyes and their metabolites on Staphylococcus aureus

PubMed Central

Pan, Hongmiao; Feng, Jinhui; Cerniglia, Carl E.

2018-01-01

Azo dyes are widely used in the plastic, paper, cosmetics, food, and pharmaceutical industries. Some metabolites of these dyes are potentially genotoxic. The toxic effects of azo dyes and their potential reduction metabolites on Staphylococcus aureus ATCC BAA 1556 were studied. When the cultures were incubated with 6, 18, and 36 μg/ml of Orange II and Sudan III for 48 h, 76.3, 68.5, and 61.7% of Orange II and 97.8, 93.9, and 75.8% of Sudan III were reduced by the bacterium, respectively. In the presence of 36 μg/ml Sudan III, the cell viability of the bacterium decreased to 61.9% after 48 h of incubation, whereas the cell viability of the control culture without the dye was 71.5%. Moreover, the optical density of the bacterial cultures at 10 h decreased from 0.74 to 0.55, indicating that Sudan III is able to inhibit growth of the bacterium. However, Orange II had no significant effects on either cell growth or cell viability of the bacterium at the tested concentrations. 1-Amino-2-naphthol, a metabolite common to Orange II and Sudan III, was capable of inhibiting cell growth of the bacterium at 1 μg/ml and completely stopped bacterial cell growth at 24–48 μg/ml. On the other hand, the other metabolites of Orange II and Sudan III, namely sulfanilic acid, p-phenylenediamine, and aniline, showed no significant effects on cell growth. p-Phenylenediamine exhibited a synergistic effect with 1-amino-2-naphthol on cell growth inhibition. All of the dye metabolites had no significant effects on cell viability of the bacterium. PMID:21451978

Smad4 controls bone homeostasis through regulation of osteoblast/osteocyte viability.

PubMed

Moon, Young Jae; Yun, Chi-Young; Choi, Hwajung; Ka, Sun-O; Kim, Jung Ryul; Park, Byung-Hyun; Cho, Eui-Sic

2016-09-02

Regulation of osteoblast and osteocyte viability is essential for bone homeostasis. Smad4, a major transducer of bone morphogenetic protein and transforming growth factor-β signaling pathways, regulates apoptosis in various cell types through a mitochondrial pathway. However, it remains poorly understood whether Smad4 is necessary for the regulation of osteoblast and osteocyte viability. In this study, we analyzed Smad4Δ(Os) mice, in which Smad4 was subjected to tissue-specific disruption under the control of the 2.3-kb Col1a1 promoter, to understand the functional significance of Smad4 in regulating osteoblast/osteocyte viability during bone formation and remodeling. Smad4Δ(Os) mice showed a significant increase in osteoblast number and osteocyte density in the trabecular and cortical regions of the femur, whereas osteoclast activity was significantly decreased. The proliferation of osteoblasts/osteocytes did not alter, as shown by measuring 5'-bromo-2'deoxyuridine incorporation. By contrast, the percentage of TUNEL-positive cells decreased, together with a decrease in the Bax/Bcl-2 ratio and in the proteolytic cleavage of caspase 3, in Smad4Δ(Os) mice. Apoptosis in isolated calvaria cells from Smad4Δ(Os) mice decreased after differentiation, which was consistent with the results of the TUNEL assay and western blotting in Smad4Δ(Os) mice. Conversely, osteoblast cells overexpressing Smad4 showed increased apoptosis. In an apoptosis induction model of Smad4Δ(Os) mice, osteoblasts/osteocytes were more resistant to apoptosis than were control cells, and, consequently, bone remodeling was attenuated. These findings indicate that Smad4 has a significant role in regulating osteoblast/osteocyte viability and therefore controls bone homeostasis.

IN VITRO AND IN VIVO TOXICITY: A COMPARISON OF ACRYLAMIDE, CYCLOPHOSPHAMIDE, CHLORDECONE, AND DIETHYLSTILBESTROL

EPA Science Inventory

Four chemicals that had been tested in an in vivo toxicological screen were tested in a Chinese hamster ovary (CHO) cytotoxicity assay. Cell density, viability, ATP concentration, rate of protein synthesis, and cellular protein concentration were decreased by exposure to acrylami...

Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient.

PubMed

Ren, Xiang; Wang, Fuyou; Chen, Cheng; Gong, Xiaoyuan; Yin, Li; Yang, Liu

2016-07-20

Cartilage tissue engineering is a promising approach for repairing and regenerating cartilage tissue. To date, attempts have been made to construct zonal cartilage that mimics the cartilaginous matrix in different zones. However, little attention has been paid to the chondrocyte density gradient within the articular cartilage. We hypothesized that the chondrocyte density gradient plays an important role in forming the zonal distribution of extracellular matrix (ECM). In this study, collagen type II hydrogel/chondrocyte constructs were fabricated using a bioprinter. Three groups were created according to the total cell seeding density in collagen type II pre-gel: Group A, 2 × 10(7) cells/mL; Group B, 1 × 10(7) cells/mL; and Group C, 0.5 × 10(7) cells/mL. Each group included two types of construct: one with a biomimetic chondrocyte density gradient and the other with a single cell density. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis. We found that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes' biosynthetic ability was affected by both the total cell density and the cell distribution pattern. Our results suggested that zonal engineered cartilage could be fabricated by bioprinting collagen type II hydrogel constructs with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to achieve synergistic biological effects.

Influence of cell printing on biological characters of chondrocytes

PubMed Central

Qu, Miao; Gao, Xiaoyan; Hou, Yikang; Shen, Congcong; Xu, Yourong; Zhu, Ming; Wang, Hengjian; Xu, Haisong; Chai, Gang; Zhang, Yan

2015-01-01

Objective: To establish a two-dimensional biological printing technique of chondrocytes and compare the difference of related biological characters between printed chondrocytes and unprinted cells so as to control the cell transfer process and keep cell viability after printing. Methods: Primary chondrocytes were obtained from human mature and fetal cartilage tissues and then were regularly sub-cultured to harvest cells at passage 2 (P2), which were adjusted to the single cell suspension at a density of 1×106/mL. The experiment was divided into 2 groups: experimental group P2 chondrocytes were transferred by rapid prototype biological printer (driving voltage value 50 V, interval in x-axis 300 μm, interval in y-axis 1500 μm). Afterwards Live/Dead viability Kit and flow cytometry were respectively adopted to detect cell viability; CCK-8 Kit was adopted to detect cell proliferation viability; immunocytochemistry, immunofluorescence and RT-PCR was employed to identify related markers of chondrocytes; control group steps were the same as the printing group except that cell suspension received no printing. Results: Fluorescence microscopy and flow cytometry analyses showed that there was no significant difference between experimental group and control group in terms of cell viability. After 7-day in vitro culture, control group exhibited higher O.D values than experimental group from 2nd day to 7th day but there was no distinct difference between these two groups (P>0.05). Inverted microscope observation demonstrated that the morphology of these two groups had no significant difference either. Similarly, Immunocytochemistry, immunofluorescence and RT-PCR assays also showed that there was no significant difference in the protein and gene expression of type II collagen and aggrecan between these two groups (P>0.05). Conclusion Cell printing has no distinctly negative effect on cell vitality, proliferation and phenotype of chondrocytes. Biological printing technique may provide a novel approach for realizing the oriented, quantificational and regular distribution of chondrocytes in a two-dimensional plane and lay the foundation for the construction of three-dimensional cell printing or even organ printing system. PMID:26770337

In vitro cytotoxicity of white MTA, MTA Fillapex® and Portland cement on human periodontal ligament fibroblasts.

PubMed

Yoshino, Patrícia; Nishiyama, Celso Kenji; Modena, Karin Cristina da Silva; Santos, Carlos Ferreira; Sipert, Carla Renata

2013-01-01

The aim of this study was to compare the in vitro cytotoxicity of white mineral trioxide aggregate (MTA), MTA Fillapex® and Portland cement (PC) on human cultured periodontal ligament fibroblasts. Periodontal ligament fibroblast culture was established and the cells were used for cytotoxic tests after the fourth passage. Cell density was set at 1.25 X10 4 cells/well in 96-well plates. Endodontic material extracts were prepared by placing sealer/cement specimens (5x3mm) in 1mL of culture medium for 72 h. The extracts were then serially two-fold diluted and inserted into the cell-seeded wells for 24, 48 and 72 h. MTT assay was employed for analysis of cell viability. Cell supernatants were tested for nitric oxide using the Griess reagent system. MTA presented cytotoxic effect in undiluted extracts at 24 and 72 h. MTA Fillapex® presented the highest cytotoxic levels with important cell viability reduction for pure extracts and at ½ and ¼ dilutions. In this study, PC did not induce alterations in fibroblast viability. Nitric oxide was detected in extract-treated cell supernatants and also in the extracts only, suggesting presence of nitrite in the soluble content of the tested materials. In the present study, MTA Fillapex displayed the highest cytotoxic effect on periodontal ligament fibroblasts followed by white MTA and PC.

Effect of 99 GHz continuous millimeter wave electro-magnetic radiation on E. coli viability and metabolic activity.

PubMed

Cohen, Irena; Cahan, Rivka; Shani, Gad; Cohen, Eyal; Abramovich, Amir

2010-05-01

To investigate time exposure dependence of continuous millimeter wave (CW) 99 GHz radiation on Escherichia coli bacterial cell viability and metabolic activity. Suspensions of E. coli bacterial cells with an optical density of OD(660 nm) = 0.1 were used for viability tests and OD(660 nm) = 1.0 for metabolic activity tests. These suspensions were exposed to 99 GHz CW electromagnetic radiation, generated by a Backward Wave Oscillator (BWO) tube base instrument with a horn antenna at the BWO exit, to obtain an almost ideal Gaussian beam. Calculations of the Gaussian beam show that a power of 0.2 mW/cm(2) was obtained at the bacterial plane. The experimental results show that 1 hour of exposure to 99 GHz CW electromagnetic radiation had no effect on E. coli viability and colony characterisation. In 19 h of radiation, the number of colonies forming units was half order of magnitude higher than the sham-exposed and the control. However, 19 h of exposure did not affect the E. coli metabolic activity. Exposure of E. coli to millimeter wave (MW) CW 99 GHz radiation for a short period did not affect the viability of E. coli bacterial cells. However, exposure for 19 h caused a slight proliferation but did not influence the metabolic activities of about 90 biochemical reactions that were examined. Hence, we assume that the slight proliferation (half order of magnitude) after 19 h of exposure dose not have a biological meaning.

Improving ovarian tissue cryopreservation for oncologic patients: slow freezing versus vitrification, effect of different procedures and devices.

PubMed

Herraiz, Sonia; Novella-Maestre, Edurne; Rodríguez, Beatriz; Díaz, César; Sánchez-Serrano, María; Mirabet, Vicente; Pellicer, Antonio

2014-03-01

To compare slow freezing (SF) with four vitrification techniques (VT) for cryopreservation of ovarian tissue (OT) and to evaluate the best protocol for human OT in a xenograft model. Experimental study. University hospital. Patients undergoing fertility preservation. Ovariectomized nude mice. Cryopreservation of bovine OT after SF and four VTs (VT1, VT2, VT3, and VT4) by combining two cryoprotectant vitrification solutions (VS1 and VS2) and two devices (metallic grid and ethyl vinyl acetate bag), after which the cryopreservation of human OT by SF and VT1 and xenograft into nude mice. Follicular densities, proliferation, vascularization, fibrosis, apoptosis, tissue viability. The in vitro study in bovine OT showed a lower percentage of quiescent follicles in the SF group but not in the vitrification groups (VT1-VT4). Apoptosis increased and cell proliferation decreased in all the experimental groups except VT1 (20% ethylene glycol, 20% dimethyl sulfoxide, 0.5 M sucrose, and 20% synthetic serum substitute in HEPES-buffered M199 culture media with Cryotissue metallic grids). Tissue viability was diminished in VT3, and the SF-xenografted human samples showed reduced primordial and secondary densities and unbalanced follicular populations when compared with fresh and VT1 tissue. VT1 offers similar conditions to fresh tissue for follicular density, proliferation, viability, and cell death and preserves a larger population of quiescent follicles than SF after transplantation, thus ensuring the maintenance of graft potential fertility. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Cold storage of rat hepatocyte suspensions for one week in a customized cold storage solution--preservation of cell attachment and metabolism.

PubMed

Pless-Petig, Gesine; Singer, Bernhard B; Rauen, Ursula

2012-01-01

Primary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells. Rat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production) of adherent cells were assessed. Cold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage. In the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

PubMed Central

Newton, Joseph M.; Schofield, Desmond; Vlahopoulou, Joanna

2016-01-01

Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016 PMID:27111912

Lactobacillus acidophilus contributes to a healthy environment for vaginal epithelial cells.

PubMed

Pi, Woojin; Ryu, Jae-Sook; Roh, Jaesook

2011-09-01

Lactobacillus species in the female genital tract are thought to act as a barrier to infection. Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. acidophilus and the acidity of the culture medium were measured. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state. L. acidophilus showed exponential growth for the first 6 hr until 9 hr, and the pH was maintained close to 4.0-5.0 at 24 hr after culture, consistent with previous studies. The growth curve of L. acidophilus or the pH values were relatively unaffected by co-culture with MS74 cells, confirming that L. acidophilus maintains a low pH in the presence of MS74 cells. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. On the other hand, MS74 cells co-cultured with L. acidophilus more firmly attached to the culture plate, and a higher number of cells were present compared to cells cultured in the absence of L. acidophilus. These results indicate that L. acidophilus increases MS74 cell proliferation and viability, suggesting that lactobacilli may contribute to the healthy environment for vaginal epithelial cells.

[Detection of viable metabolically active yeast cells using a colorimetric assay].

PubMed

Růzicka, F; Holá, V

2008-02-01

The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

Poly(ethylene glycol) hydrogel microstructures encapsulating living cells

NASA Technical Reports Server (NTRS)

Koh, Won-Gun; Revzin, Alexander; Pishko, Michael V.

2002-01-01

We present an easy and effective method for the encapsulation of cells inside PEG-based hydrogel microstructures fabricated using photolithography. High-density arrays of three-dimensional microstructures were created on substrates using this method. Mammalian cells were encapsulated in cylindrical hydrogel microstructures of 600 and 50 micrometers in diameter or in cubic hydrogel structures in microfluidic channels. Reducing lateral dimension of the individual hydrogel microstructure to 50 micrometers allowed us to isolate 1-3 cells per microstructure. Viability assays demonstrated that cells remained viable inside these hydrogels after encapsulation for up to 7 days.

Impact of Cyanidin-3-Glucoside on Glycated LDL-Induced NADPH Oxidase Activation, Mitochondrial Dysfunction and Cell Viability in Cultured Vascular Endothelial Cells

PubMed Central

Xie, Xueping; Zhao, Ruozhi; Shen, Garry X.

2012-01-01

Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC. PMID:23443099

Impact of cyanidin-3-glucoside on glycated LDL-induced NADPH oxidase activation, mitochondrial dysfunction and cell viability in cultured vascular endothelial cells.

PubMed

Xie, Xueping; Zhao, Ruozhi; Shen, Garry X

2012-11-27

Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC.

Suppression effects of negative pressure on the proliferation and metastasis in human pancreatic cancer cells.

PubMed

Yang, Xiujiang; Sun, Bo; Zhu, Haihang; Jiang, Ziting

2015-01-01

The aim was to explore the effect of negative pressure on the proliferation and metastasis of human pancreatic cancer SW1990 cells. Three groups were conducted in the work: normal control group (NC group, 0 mm Hg), low negative pressure group (LN group, -300 mm Hg), and high negative pressure group (HN group, -600 mm Hg). Cell morphological assay was conducted using an inverted Nikon TE2000-S microscope. Cell viability was assayed using cell counting kit-8 solution. Cell apoptosis was evaluated with flow cytometry. Cell migration was investigated using transwell assay. Compared to LN and HN groups, SW1990 cells in NC group grew quite well, showing a higher density. The NC group represented the highest cell viability. The HN group represented the lowest cell viability, which was lower than that of the LN group (P < 0.01). The apoptosis rate in NC group, LN group and HN group was 1.91% ± 0.13%, 2.31% ± 0.06% and 15.22% ± 0.81%, respectively (P < 0.05). The average number of migration cells in NC group was 53.60 ± 4.14 (× 200), which was decreased to 18.93 ± 3.67 and 11.07 ± 3.01 in LN group and HN group, respectively (P < 0.01). The negative pressure shows suppression effects on the proliferation and metastasis of human pancreatic cancer SW1990 cells. It is indicated that negative pressure may be involved in the development of human pancreatic cancer by influencing cell biological characteristics.

Apoptotic death in cerebral hemisphere cells is density dependent and modulated by transient oxygen and glucose deprivation.

PubMed

Yavin, E; Billia, D M

1997-03-01

Flow cytometry, light and fluorescence microscopy, and designated biochemical techniques were used to examine the type of death which occurs in cerebral cortex cells when grown under crowded vs. sparse conditions or after brief anoxia/hypoglycemia. A 4 hr episode of anoxia combined with glucose deprivation enhanced apoptotic cell death as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and reduced neutral red eye uptake. An additional form of cell death involving exclusion of the nucleus was recorded by time lapse cinematography and DAPI stain. The presence of the endonuclease inhibitor aurintricarboxylic acid (0.1 mM) reduced cell death by 56.6%, while the protein and RNA synthesis inhibitors actinomycin D and cycloheximide (each at 5 micrograms/ml) effectively decreased cell death by 83.3% and 90.6%, respectively. In contrast, 5 mM glutamate had no effect on cell death in accord with the immature state of the cells. Growth of cells under crowded conditions improved cell survival; after 2 h or 4 days in culture, cells seeded at high density (34 microgram cellular DNA/cm2) showed a nearly 3-fold decline in the amount of cell death in comparison to cells seeded at low density (5 micrograms cellular DNA/cm2). At high cell density, anoxic episodes enhanced cell death most likely by preventing a cell density-mediated rescue. Neutral red dye uptake, an index for cell viability, was enhanced with increasing cell density and in vitro maturation, but was reduced in dense cultures exposed to anoxic/hypoglycemic conditions. The data suggest that cell density may play a critical role in brain organogenesis and that anoxic stress is more deleterious in dense than sparse cell assemblies.

Investigation of mitomycin-C-treated fibroblasts in 3-D collagen gel and conditioned medium for keratinocyte proliferation.

PubMed

Huang, Yi-Chau; Wang, Tzu-Wei; Sun, Jui-Sheng; Lin, Feng-Huei

2006-03-01

Fibroblasts produce a spectrum of necessary growth factors essential for growth and proliferation of a variety of cell types. In this study, the paracrine effect of mitomycin-C-treated fibroblasts with various densities in collagen gel for keratinocyte proliferation was investigated from which an optimum cell density and optimum conditioned medium would be determined to expand keratinocyte without further differentiation for skin equivalent tissue engineering. The optimum cell density in collagen feeder gel for optimum collected medium preparation will be determined by checking the level of keratinocyte growth factor and granulocyte macrophage colony-stimulating factor in conventional medium. The results showed that the cell density of 1 x 10(5) cells/gel in the feeder gel is better to produce optimum collected medium. The conditioned medium is prepared by mixing together the optimum collected medium and molecular cellular and developmental biology (MCDB) 153 medium in different ratios for keratinocyte growth. The keratinocyte viability will be measured by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the optimum conditioned medium. From the study, 67% conditioned medium was supposed as the better medium for keratinocyte proliferation. In this experiment, the optimum cell density in feeder gel to coculture with keratinocytes is also determined as 1 x 10(5) cells/gel. Keratin 10 (K10) and Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling stain will be used to check the cell differentiation and apoptosis, respectively. The results suggest that keratinocytes should not be cultured in postconfluent conditions due to undesired apoptosis and differentiation. The result of cell viability from passages to passages shows that the optimum feeder gel plays a more important role to the keratinocyte proliferation than that of optimum conditioned medium. Keratinocytes cultured with optimum feeder gel in 67% conditioned medium could effectively promote proliferation, inhibit apoptosis, and prevent differentiation. The combination of conditioned media and feeder gel to culture keratinocytes without external supplements can provide an inexpensive way for keratinocyte proliferation and construct an environment for real-time communication between the two cells. The results conclude that keratinocyte cultivation in feeder gel with modified medium should be feasible in the production of high quality keratinocytes for skin equivalents preparation.

Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells.

PubMed

Kujawska, T; Secomski, W; Bilmin, K; Nowicki, A; Grieb, P

2014-07-01

In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43 °C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity ISATA (spatial averaged, temporal averaged) varied from 0.33 W/cm(2) to 8 W/cm(2) (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43 °C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm(2). Copyright © 2014 Elsevier B.V. All rights reserved.

Fabrication of cell-benign inverse opal hydrogels for three-dimensional cell culture.

PubMed

Im, Pilseon; Ji, Dong Hwan; Kim, Min Kyung; Kim, Jaeyun

2017-05-15

Inverse opal hydrogels (IOHs) for cell culture were fabricated and optimized using calcium-crosslinked alginate microbeads as sacrificial template and gelatin as a matrix. In contrast to traditional three-dimensional (3D) scaffolds, the gelatin IOHs allowed the utilization of both the macropore surface and inner matrix for cell co-culture. In order to remove templates efficiently for the construction of 3D interconnected macropores and to maintain high cell viability during the template removal process using EDTA solution, various factors in fabrication, including alginate viscosity, alginate concentration, alginate microbeads size, crosslinking calcium concentration, and gelatin network density were investigated. Low viscosity alginate, lower crosslinking calcium ion concentration, and lower concentration of alginate and gelatin were found to obtain high viability of cells encapsulated in the gelatin matrix after removal of the alginate template by EDTA treatment by allowing rapid dissociation and diffusion of alginate polymers. Based on the optimized fabrication conditions, gelatin IOHs showed good potential as a cell co-culture system, applicable to tissue engineering and cancer research. Copyright © 2017 Elsevier Inc. All rights reserved.

Rapid determination of cell mass and density using digitally controlled electric field in a microfluidic chip.

PubMed

Zhao, Yuliang; Lai, Hok Sum Sam; Zhang, Guanglie; Lee, Gwo-Bin; Li, Wen Jung

2014-11-21

The density of a single cell is a fundamental property of cells. Cells in the same cycle phase have similar volume, but the differences in their mass and density could elucidate each cell's physiological state. Here we report a novel technique to rapidly measure the density and mass of a single cell using an optically induced electrokinetics (OEK) microfluidic platform. Presently, single cellular mass and density measurement devices require a complicated fabrication process and their output is not scalable, i.e., it is extremely difficult to measure the mass and density of a large quantity of cells rapidly. The technique reported here operates on a principle combining sedimentation theory, computer vision, and microparticle manipulation techniques in an OEK microfluidic platform. We will show in this paper that this technique enables the measurement of single-cell volume, density, and mass rapidly and accurately in a repeatable manner. The technique is also scalable - it allows simultaneous measurement of volume, density, and mass of multiple cells. Essentially, a simple time-controlled projected light pattern is used to illuminate the selected area on the OEK microfluidic chip that contains cells to lift the cells to a particular height above the chip's surface. Then, the cells are allowed to "free fall" to the chip's surface, with competing buoyancy, gravitational, and fluidic drag forces acting on the cells. By using a computer vision algorithm to accurately track the motion of the cells and then relate the cells' motion trajectory to sedimentation theory, the volume, mass, and density of each cell can be rapidly determined. A theoretical model of micro-sized spheres settling towards an infinite plane in a microfluidic environment is first derived and validated experimentally using standard micropolystyrene beads to demonstrate the viability and accuracy of this new technique. Next, we show that the yeast cell volume, mass, and density could be rapidly determined using this technology, with results comparable to those using the existing method suspended microchannel resonator.

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss.

PubMed

Newton, Joseph M; Schofield, Desmond; Vlahopoulou, Joanna; Zhou, Yuhong

2016-07-08

Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction-point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069-1076, 2016. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

Fuel Cells: A Real Option for Unmanned Aerial Vehicles Propulsion

PubMed Central

2014-01-01

The possibility of implementing fuel cell technology in Unmanned Aerial Vehicle (UAV) propulsion systems is considered. Potential advantages of the Proton Exchange Membrane or Polymer Electrolyte Membrane (PEMFC) and Direct Methanol Fuel Cells (DMFC), their fuels (hydrogen and methanol), and their storage systems are revised from technical and environmental standpoints. Some operating commercial applications are described. Main constraints for these kinds of fuel cells are analyzed in order to elucidate the viability of future developments. Since the low power density is the main problem of fuel cells, hybridization with electric batteries, necessary in most cases, is also explored. PMID:24600326

Evaluation of results of cell electrophoresis experiments on space shuttle STS-3 including pre-flight and post-flight laboratory experiments

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

The objectives of the red blood cell experiments were to provide a visual check on the electrophoretic process and especially electroosmotic flow in space as well as to provide test separations of non-degradable standard particles for comparison with the separations of the three viable cell types studied on the Apollo-Soyuz Test Project. Determination of the maximum concentrations of cells that can be separated in column electrophore was a significant goal. Two of the eight columns were available for red cell experiments, so two concentrations of human and rabbit RBC mixtures were used. The objectives of another experiment were to evaluate the reproducibility of microgravity electrophoretic separation of living kidney cells, to separate cells with highly viability despite two freeze-thaw cycles, and to optimize the physical conditions of cell separation. Owing to the uncertain heterogeneity of the starting material, the experimental design does not assess resolution in microgravity, but improved separability was sought in comparison to density-gradient electrophoresis or continuous-flow electrophoresis. Efforts were made to increase cell yield and cell viability and to assess reproducibility directly.

Factors leading to different viability predictions for a grizzly bear data set

USGS Publications Warehouse

Mills, L.S.; Hayes, S.G.; Wisdom, M.J.; Citta, J.; Mattson, D.J.; Murphy, K.

1996-01-01

Population viability analysis programs are being used increasingly in research and management applications, but there has not been a systematic study of the congruence of different program predictions based on a single data set. We performed such an analysis using four population viability analysis computer programs: GAPPS, INMAT, RAMAS/AGE, and VORTEX. The standardized demographic rates used in all programs were generalized from hypothetical increasing and decreasing grizzly bear (Ursus arctos horribilis) populations. Idiosyncracies of input format for each program led to minor differences in intrinsic growth rates that translated into striking differences in estimates of extinction rates and expected population size. In contrast, the addition of demographic stochasticity, environmental stochasticity, and inbreeding costs caused only a small divergence in viability predictions. But, the addition of density dependence caused large deviations between the programs despite our best attempts to use the same density-dependent functions. Population viability programs differ in how density dependence is incorporated, and the necessary functions are difficult to parameterize accurately. Thus, we recommend that unless data clearly suggest a particular density-dependent model, predictions based on population viability analysis should include at least one scenario without density dependence. Further, we describe output metrics that may differ between programs; development of future software could benefit from standardized input and output formats across different programs.

A rapid co-culture stamping device for studying intercellular communication.

PubMed

Hassanzadeh-Barforoushi, Amin; Shemesh, Jonathan; Farbehi, Nona; Asadnia, Mohsen; Yeoh, Guan Heng; Harvey, Richard P; Nordon, Robert E; Warkiani, Majid Ebrahimi

2016-10-18

Regulation of tissue development and repair depends on communication between neighbouring cells. Recent advances in cell micro-contact printing and microfluidics have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. Nonetheless, these techniques are still complicated to perform and as a result, are seldom used by biologists. We report here development of a temporarily sealed microfluidic stamping device which utilizes a novel valve design for patterning two adherent cell lines with well-defined interlacing configurations to study cell-cell interactions. We demonstrate post-stamping cell viability of >95%, the stamping of multiple adherent cell types, and the ability to control the seeded cell density. We also show viability, proliferation and migration of cultured cells, enabling analysis of co-culture boundary conditions on cell fate. We also developed an in-vitro model of endothelial and cardiac stem cell interactions, which are thought to regulate coronary repair after myocardial injury. The stamp is fabricated using microfabrication techniques, is operated with a lab pipettor and uses very low reagent volumes of 20 μl with cell injection efficiency of >70%. This easy-to-use device provides a general strategy for micro-patterning of multiple cell types and will be important for studying cell-cell interactions in a multitude of applications.

A rapid co-culture stamping device for studying intercellular communication

NASA Astrophysics Data System (ADS)

Hassanzadeh-Barforoushi, Amin; Shemesh, Jonathan; Farbehi, Nona; Asadnia, Mohsen; Yeoh, Guan Heng; Harvey, Richard P.; Nordon, Robert E.; Warkiani, Majid Ebrahimi

2016-10-01

Regulation of tissue development and repair depends on communication between neighbouring cells. Recent advances in cell micro-contact printing and microfluidics have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. Nonetheless, these techniques are still complicated to perform and as a result, are seldom used by biologists. We report here development of a temporarily sealed microfluidic stamping device which utilizes a novel valve design for patterning two adherent cell lines with well-defined interlacing configurations to study cell-cell interactions. We demonstrate post-stamping cell viability of >95%, the stamping of multiple adherent cell types, and the ability to control the seeded cell density. We also show viability, proliferation and migration of cultured cells, enabling analysis of co-culture boundary conditions on cell fate. We also developed an in-vitro model of endothelial and cardiac stem cell interactions, which are thought to regulate coronary repair after myocardial injury. The stamp is fabricated using microfabrication techniques, is operated with a lab pipettor and uses very low reagent volumes of 20 μl with cell injection efficiency of >70%. This easy-to-use device provides a general strategy for micro-patterning of multiple cell types and will be important for studying cell-cell interactions in a multitude of applications.

Retinoids, retinoid analogs, and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro.

PubMed

Wang, Y; Baumrucker, C R

2010-07-01

Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability (P<0.05) and exhibited a dose-dependent inhibition with 9-cisRA (9cRA; P<0.05). The MeBo cells exhibited some inhibition with these natural ligands (P<0.05), but they were not as sensitive. The addition of bLf had similar inhibitory effects (P<0.05) on cell viability of the 2 mammary cell types. Applications of RA receptor (RAR) agonist indicated that the stimulation of the RAR in both mammary cell types was highly effective in inhibition of cell viability (P<0.05), whereas the application of an RAR antagonist stimulated MeBo cell viability (P<0.05) and inhibited BME-UV1 cell viability (P<0.05). Finally, the use of the RAR antagonist in conjunction with bLf indicated a rescue of the bLf effect in the MeBo cells, suggesting that bLf is acting through the RAR receptor. Conversely, bLf reverted inhibition of cell viability by 9cRA in the BME-UV1 cell type (P<0.05). We conclude that RAR interaction in bovine mammary cell types regulates cell viability in vitro; we hypothesize that the natural ligands mediate regulation of bovine mammary cell viability in vivo and that bLf can either enhance or reverse the retinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.

Effects of low-level laser therapy on stem cells from human exfoliated deciduous teeth

PubMed Central

FERNANDES, Ana Paula; JUNQUEIRA, Marina de Azevedo; MARQUES, Nádia Carolina Teixeira; MACHADO, Maria Aparecida Andrade Moreira; SANTOS, Carlos Ferreira; OLIVEIRA, Thais Marchini; SAKAI, Vivien Thiemy

2016-01-01

ABSTRACT Low-Level Laser Therapy stimulates the proliferation of a variety of types of cells. However, very little is known about its effect on stem cells from human exfoliated deciduous teeth (SHED). Objective This study aimed to evaluate the influence of different laser therapy energy densities on SHED viability and proliferation. Material and Methods SHED were irradiated according to the groups: I (1.2 J/cm2 - 0.5 mW – 10 s), II (2.5 J/cm2 – 10 mW – 10 s), III (3.7 J/cm2 – 15 mW – 10 s), IV (5.0 J/cm2 – 20 mW – 10 s), V (6.2 J/cm2 – 25 mW – 10 s), and VI (not irradiated – control group). Cell viability was assessed 6 and 24 h after irradiation measuring the mitochondrial activity and using the Crystal Violet assay. Cell proliferation was assessed after 24, 48, and 72 h of irradiation by SRB assay. Results MTT assay demonstrated differences from 6 to 24 hours after irradiation. After 24 h, groups I and IV showed higher absorbance values than those of control group. Crystal Violet assay showed statistically differences in the absorbance rate from 6 to 24 h after irradiation for groups III and VI. At 24 h after irradiation, Group III absorbance rate was greater than that of groups I, II, and IV. Group VI absorbance rate was greater than that of groups I and IV. SRB assay showed that the group I had higher rates than those of groups II, III, V, and VI, at 24 h after irradiation. After 48 h, group I exhibited the greatest cell proliferation rate followed by groups III, V, and VI. After 72 h, group III exhibited the lowest cell proliferation rate than those of groups II, IV, and V. Conclusions The Low-Level Laser Therapy energy densities used in this study did not cause loss of cell viability and stimulated SHED proliferation within the parameters described in this study. PMID:27556203

Protective properties of Salvia lavandulifolia Vahl. essential oil against oxidative stress-induced neuronal injury.

PubMed

Porres-Martínez, María; González-Burgos, Elena; Carretero, M Emilia; Gómez-Serranillos, M Pilar

2015-06-01

Salvia lavandulifolia Vahl., known as "Spanish sage", has potential value in dementia for its sedative, antioxidant, anti-inflammatory and anticholinesterase properties. This work aimed to evaluate the in vitro neuroprotective activity of S. lavandulifolia essential oils, obtained from plants at different phenological stages (vegetative and flowering phases) and plants grown at different densities, against H2O2-induced oxidative stress in PC12 cells. The effect on cell viability and morphology, lipid peroxidation, GSH/GSSG ratio, intracellular ROS levels, antioxidant enzymes (CAT, SOD, GR, GPx, HO-1) and apoptotic enzymes was investigated. Comparing with H2O2-treated PC12 cells, pretreatments with essential oil samples attenuated morphological changes and loss of cell viability, decreased MDA levels and intracellular ROS production and increased GSH/GSSG ratio. Moreover, Spanish sage increased antioxidant status as evidenced in an increase of antioxidant enzyme activity and protein expression and inhibited caspase-3 activity. Furthermore, our results suggest that S. lavandulifolia essential oils are able to activate Nrf2 transcription factor. Collectively, the sample of essential oil obtained with the highest densities of planting and at flowering phase exerted the major neuroprotective activity. Our findings demonstrate that S. lavandulifolia essential oils may have therapeutic value for the prevention and treatment of neurodegenerative diseases associated with oxidative stress-induced neuronal injury. Copyright © 2015 Elsevier Ltd. All rights reserved.

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

PubMed

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

Protein-engineered block-copolymers as stem cell delivery vehicles

NASA Astrophysics Data System (ADS)

Heilshorn, Sarah

2015-03-01

Stem cell transplantation is a promising therapy for a myriad of debilitating diseases and injuries; however, current delivery protocols are inadequate. Transplantation by direct injection, which is clinically preferred for its minimal invasiveness, commonly results in less than 5% cell viability, greatly inhibiting clinical outcomes. We demonstrate that mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we show that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. Building on these fundamental studies, we have designed a reproducible, bio-resorbable, customizable hydrogel using protein-engineering technology. In our Mixing-Induced Two-Component Hydrogel (MITCH), network assembly is driven by specific and stoichiometric peptide-peptide binding interactions. By integrating protein science methodologies with simple polymer physics models, we manipulate the polypeptide chain interactions and demonstrate the direct ability to tune the network crosslinking density, sol-gel phase behavior, and gel mechanics. This is in contrast to many other physical hydrogels, where predictable tuning of bulk mechanics from the molecular level remains elusive due to the reliance on non-specific and non-stoichiometric chain interactions for network formation. Furthermore, the hydrogel network can be easily modified to deliver a variety of bioactive payloads including growth factors, peptide drugs, and hydroxyapatite nanoparticles. Through a series of in vitro and in vivo studies, we demonstrate that these materials may significantly improve transplanted stem cell retention and function.

Development and storage studies of high density macrocapsules containing Lactobacillus spp. strains as nutritional supplement in young calves.

PubMed

Astesana, Diego M; Zimmermann, Jorge A; Frizzo, Laureano S; Zbrun, María V; Blajman, Jesica E; Berisvil, Ayelén P; Romero-Scharpen, Analía; Signorini, Marcelo L; Rosmini, Marcelo R; Soto, Lorena P

2018-03-17

The aim of this study was to evaluate different production methodologies of probiotic macrocapsules with high bacterial densities destined to lactating calves. Three types of capsules containing Lactobacillus casei DSPV318T and Lactobacillus plantarum DSPV354T were prepared from an overnight culture in whey medium: (1) mixing the culture with calcium alginate and then, reincubating the capsules in whey (RC); (2) concentrating the biomass by centrifugation and mixing the pellet with calcium alginate (CC) at different concentrations with respect to the initial culture (5X and 12.5X); (3) CC with cryoprotectants: whey permeate (Per) and glycerol (Gly). Chitosan coating was evaluated. Capsules were freeze-dried and viability was assessed before freezing, after freeze-drying and every two weeks for 84 days of storage at room temperature, 4°C and -20°C. CC showed higher cell densities than RC. Storage temperature affected viability: greater viability at lower temperature. Moreover, the effect of temperature was influenced by other factors, such as capsule coating, culture neutralization and cryoprotectants. Coating improved viability at room temperature; however no effect was observed at 4°C. Culture neutralization allowed greater survival during storage. Cryoprotectants improved viability during freezing, but they also generated a positive or negative effect depending on storage temperature. The best results were: at refrigeration Gly12.5X exhibited counts above 10 9 CFU/capsule until day 70 and Per12.5X until day 56 of storage and at -20°C Gly12.5X showed counts above 10 9 CFU/capsule until the end of the study (84 days). A 10 9 CFU capsule is the daily dose per calf which would facilitate the administration of this probiotic inoculum to field animals. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Equine ovarian tissue viability after cryopreservation and in vitro culture.

PubMed

Gastal, G D A; Aguiar, F L N; Alves, B G; Alves, K A; de Tarso, S G S; Ishak, G M; Cavinder, C A; Feugang, J M; Gastal, E L

2017-07-15

Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmentation. Vitrification with EG had the highest rate of morphologically normal preantral follicles, while DMSO had the lowest (76.1 ± 6.1% and 40.9 ± 14.8%, respectively; P < 0.05). In slow-freezing, despite that DMSO had the highest percentage of morphologically normal follicles (77.7 ± 5.8%), no difference among the CPAs was observed. Fluorescence intensity of EGFR and Ki-67 was greater when vitrification with EG was used. Regardless of the cryopreservation treatment, DMSO had the highest (P < 0.05) Bax/Bcl-2 ratio; however, DNA fragmentation was similar (P > 0.05) among treatments after thawing. After in vitro culture, the percentage of normal follicles was similar (P > 0.05) between slow-freezing and vitrification methods; however, vitrification had greater (P < 0.05) stromal cell density than slow-freezing. In summary, equine ovarian tissue was successfully cryopreserved, increasing the viability of the cells in the ovarian tissue after thawing when using DMSO and EG for slow-freezing and vitrification methods, respectively. Therefore, these results are relevant for fertility preservation programs. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessment of eye bank-prepared posterior lamellar corneal tissue for endothelial keratoplasty.

PubMed

Rose, Linda; Briceño, César A; Stark, Walter J; Gloria, Dante G; Jun, Albert S

2008-02-01

To evaluate eye bank-prepared tissue for Descemet's stripping automated endothelial keratoplasty (DSAEK). Experimental study and retrospective case series. Seventeen human donor corneas and 4 recipient patients undergoing DSAEK surgery. Corneal-scleral discs were obtained. Specular microscopy and pachymetry were performed. A designated Tissue Banks International technician used a microkeratome to prepare a flap. Posterior bed thickness was measured. The sectioned tissue was stored, and at 24 and 48 hours, pachymetry was repeated. At 48 hours, specular microscopy was repeated, and endothelial cell viability was assessed with trypan blue. Descemet's stripping automated endothelial keratoplasty was performed in 4 patients using eye bank-prepared posterior lamellar tissue. Corneal tissue was assessed with the following parameters: corneal thickness measured with ultrasonic pachymetry, cell density counts measured with a keratoanalyzer, and cell viability as observed with trypan blue exclusion. Patient outcomes were measured by changes in visual acuity (VA) and the presence of a clear graft. Donor corneal pachymetry before sectioning averaged 599+/-52 microm. Immediately after sectioning with a microkeratome set at a depth of 300 microm, mean posterior bed thickness was 328+/-95 microm. Thus, the mean cutting depth achieved by the microkeratome when set at 300 micrometers averaged 271+/-83 microm. After storage for 24 hours, the posterior beds measured 352 microm, an average swelling of 24 (7%) microm (P = 0.14). After 48 hours, the posterior beds measured 382 microm, an average swelling of 54 (16%) microm (P = 0.02). Cell counts 48 hours after sectioning decreased by an average of 11% (P = 0.10). Endothelial cell staining confirmed improvement in postsectioning morphology and survival with increased technician experience. All 4 patients receiving eye bank-prepared DSAEK tissue showed uncomplicated postoperative results, with improvement in VA. The microkeratome cutting depth was moderately accurate. Pachymetry, cell density, and cell viability of sectioned tissue after 48 hours in storage were encouraging overall. Initial clinical results of eye bank-prepared DSAEK tissue showed uncomplicated postoperative courses and improved VA. Additional studies are needed to follow the long-term outcomes in the recipients of these tissues.

The impact of various scaffold components on vascularized bone constructs.

PubMed

Eweida, Ahmad; Schulte, Matthias; Frisch, Oliver; Kneser, Ulrich; Harhaus, Leila

2017-06-01

Bone tissue engineering is gaining more interest in the field of craniofacial surgery where continuous efforts are being made to improve the outcomes via modulation of the scaffold components. In an in vitro three dimensional (3D) culture, the effect of bone morphogenic protein 2 (BMP2, 60 μg/ml) and the effect of different cell seeding densities (0.25, 0.5, and 1 × 104) of rat mesenchymal stem cells seeded on nanocrystalline hydroxyapatite in silica gel matrix (Nanobone ® ) on the cell viability and differentiation were studied. Alkaline phosphatase and viability assays were performed at day 7, day 14, and day 21 to assess the differentiation and the relative fraction of viable cells in the 3D cell cultures. In a subsequent in vivo study, we examined the effect of axial vascularization, the scaffold's particle size and the nature of the matrix (collagen type I vs. diluted fibrin) on vascularization and tissue generation in vascularized bone construct in rats. Regarding vascularization, we compared constructs vascularized randomly by extrinsic vascularization from the periphery of the implanted construct with others vascularized axially via an implanted arteriovenous loop (AVL). Regarding the particle size, we compared constructs having a scaffold particle size of 0.2 mm (powder) with other constructs having a particle size of 2 × 0.6 mm (granules). Regarding the matrix we compared constructs having a collagen matrix with others having a fibrin matrix. Various groups were compared regarding the amount of tissue generation, vascularization, and cellular proliferation. The initial seeding density had a temporary and minimal effect on the overall osteogenic differentiation of the cells. On the contrary, adding BMP2 in a concentration of 60 μg/ml over one week led to an overall enhanced osteogenic differentiation despite depressed cell viability. Axial vascularization was mandatory for efficient tissue formation and vascularization of the bone construct. Collagen matrix and a smaller particle size provided more favorable results in terms of vascularization and tissue formation than diluted fibrin and larger Nanobone particles. Copyright © 2017 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

In vitro and in vivo evaluation of a new nanocomposite, containing high density polyethylene, tricalcium phosphate, hydroxyapatite, and magnesium oxide nanoparticles.

PubMed

Pourdanesh, Fereydoun; Jebali, Ali; Hekmatimoghaddam, Seyedhossein; Allaveisie, Azra

2014-07-01

In this study, a new nanocomposite, which contained high density polyethylene (HDPE), tricalcium phosphate (Ca3(PO4)2) nanoparticles (TCP NPs), hydroxyapatite nanoparticles (HA NPs), and magnesium oxide nanoparticles (MgO NPs) was prepared. As in vitro experiment, human osteoblasts (HOB) cells were exposed to pristine HDPE and its nanocomposite for a period of 1, 4, and 7 days at 37 °C, and then different assays were carried out, including osteoblast cell proliferation, Trypan blue staining, cell viability, alkaline phosphatase (ALP), and cell adhesion. Antibacterial property of pristine HDPE and its nanocomposite was evaluated, and also their mechanical properties were measured after 2 and 4 months. As in vivo experiment, pristine HDPE and its nanocomposite were separately implanted on calvarium bone of rabbits, and tissue inflammation and osteogenesis were investigated after 2, 4, and 6 months. In case of HOB cells treated with HDPE or nanocomposite, as incubation time was increased, cell proliferation, live/dead ratio, and cell viability were decreased. But, the ALP activity and cell adhesion of HOB cells which treated with nanocomposite were raised after increase of incubation time. This study demonstrated that although the mechanical properties of nanocomposite were similar to HDPE sheet, but their antibacterial property was not similar. The in vivo experiment showed that both pristine HDPE and its nanocomposite had same inflammation responses. Interestingly, osteogenesis was observed after 2 months at bone/nanocomposite interface, and was highly increased after 4 and 6 months. It must be noted that such pattern was not seen at bone/HDPE interface. Copyright © 2014 Elsevier B.V. All rights reserved.

Scaffold Architecture Controls Insulinoma Clustering, Viability, and Insulin Production

PubMed Central

Blackstone, Britani N.; Palmer, Andre F.; Rilo, Horacio R.

2014-01-01

Recently, in vitro diagnostic tools have shifted focus toward personalized medicine by incorporating patient cells into traditional test beds. These cell-based platforms commonly utilize two-dimensional substrates that lack the ability to support three-dimensional cell structures seen in vivo. As monolayer cell cultures have previously been shown to function differently than cells in vivo, the results of such in vitro tests may not accurately reflect cell response in vivo. It is therefore of interest to determine the relationships between substrate architecture, cell structure, and cell function in 3D cell-based platforms. To investigate the effect of substrate architecture on insulinoma organization and function, insulinomas were seeded onto 2D gelatin substrates and 3D fibrous gelatin scaffolds with three distinct fiber diameters and fiber densities. Cell viability and clustering was assessed at culture days 3, 5, and 7 with baseline insulin secretion and glucose-stimulated insulin production measured at day 7. Small, closely spaced gelatin fibers promoted the formation of large, rounded insulinoma clusters, whereas monolayer organization and large fibers prevented cell clustering and reduced glucose-stimulated insulin production. Taken together, these data show that scaffold properties can be used to control the organization and function of insulin-producing cells and may be useful as a 3D test bed for diabetes drug development. PMID:24410263

Curcumin induced autophagy anticancer effects on human lung adenocarcinoma cell line A549

PubMed Central

Liu, Furong; Gao, Song; Yang, Yuxuan; Zhao, Xiaodan; Fan, Yameng; Ma, Wenxia; Yang, Danrong; Yang, Aimin; Yu, Yan

2017-01-01

To investigate the anticancer effects of curcumin-induced autophagy and its effects on the human lung adenocarcinoma A549 cell line, inverted phase contrast microscopy was used to observe alterations to the cytomorphology of cells. An MTT assay was used to measure cell viability. Autophagy was detected using acridine orange (AO) staining and 3-methyladenine (3-MA) was used as an autophagy-specific inhibitor. Dose- and time-dependent A549 cell viability inhibition was observed following curcumin treatment. A dose-dependent increase in the red fluorescent structures in A549 cells was identified following curcumin treatment for 48 h through AO staining. In addition, the activation of autophagy was determined through changes in the number of autophagic vesicles (AVs; fluorescent particles) infected with monodansylcadaverine (MDC). The fluorescence intensity and density of AVs in the curcumin-treated groups were higher at 48 h compared with the control group. Finally, the MTT assay demonstrated that the survival rates of the curcumin-treated cells were increased when pretreated with 3-MA for 3 h, indicating that the inhibitory effect of curcumin on A549 cells is reduced following the inhibition of autophagy. Furthermore, AO and MDC staining confirmed that 3-MA does inhibit the induction of autophagy. Thus, it was hypothesized that the induction of autophagy is partially involved in the reduction of cell viability observed following curcumin treatment. The anticancer effects of curcumin on A549 cells can be reduced using autophagy inhibitors. This suggests a possible cancer therapeutic application of curcumin through the activation of autophagy. These findings have improved the understanding of the mechanism underlying the anticancer property of curcumin. PMID:28928819

Curcumin induced autophagy anticancer effects on human lung adenocarcinoma cell line A549.

PubMed

Liu, Furong; Gao, Song; Yang, Yuxuan; Zhao, Xiaodan; Fan, Yameng; Ma, Wenxia; Yang, Danrong; Yang, Aimin; Yu, Yan

2017-09-01

To investigate the anticancer effects of curcumin-induced autophagy and its effects on the human lung adenocarcinoma A549 cell line, inverted phase contrast microscopy was used to observe alterations to the cytomorphology of cells. An MTT assay was used to measure cell viability. Autophagy was detected using acridine orange (AO) staining and 3-methyladenine (3-MA) was used as an autophagy-specific inhibitor. Dose- and time-dependent A549 cell viability inhibition was observed following curcumin treatment. A dose-dependent increase in the red fluorescent structures in A549 cells was identified following curcumin treatment for 48 h through AO staining. In addition, the activation of autophagy was determined through changes in the number of autophagic vesicles (AVs; fluorescent particles) infected with monodansylcadaverine (MDC). The fluorescence intensity and density of AVs in the curcumin-treated groups were higher at 48 h compared with the control group. Finally, the MTT assay demonstrated that the survival rates of the curcumin-treated cells were increased when pretreated with 3-MA for 3 h, indicating that the inhibitory effect of curcumin on A549 cells is reduced following the inhibition of autophagy. Furthermore, AO and MDC staining confirmed that 3-MA does inhibit the induction of autophagy. Thus, it was hypothesized that the induction of autophagy is partially involved in the reduction of cell viability observed following curcumin treatment. The anticancer effects of curcumin on A549 cells can be reduced using autophagy inhibitors. This suggests a possible cancer therapeutic application of curcumin through the activation of autophagy. These findings have improved the understanding of the mechanism underlying the anticancer property of curcumin.

Genetic modification of mesenchymal stem cells overexpressing CCR1 increases cell viability, migration, engraftment, and capillary density in the injured myocardium.

PubMed

Huang, Jing; Zhang, Zhiping; Guo, Jian; Ni, Aiguo; Deb, Arjun; Zhang, Lunan; Mirotsou, Maria; Pratt, Richard E; Dzau, Victor J

2010-06-11

Although mesenchymal stem cell (MSC) transplantation has been shown to promote cardiac repair in acute myocardial injury in vivo, its overall restorative capacity appears to be restricted mainly because of poor cell viability and low engraftment in the ischemic myocardium. Specific chemokines are upregulated in the infarcted myocardium. However the expression levels of the corresponding chemokine receptors (eg, CCR1, CXCR2) in MSCs are very low. We hypothesized that this discordance may account for the poor MSC engraftment and survival. To determine whether overexpression of CCR1 or CXCR2 chemokine receptors in MSCs augments their cell survival, migration and engraftment after injection in the infarcted myocardium. Overexpression of CCR1, but not CXCR2, dramatically increased chemokine-induced murine MSC migration and protected MSC from apoptosis in vitro. Moreover, when MSCs were injected intramyocardially one hour after coronary artery ligation, CCR1-MSCs accumulated in the infarcted myocardium at significantly higher levels than control-MSCs or CXCR2-MSCs 3 days postmyocardial infarction (MI). CCR1-MSC-injected hearts exhibited a significant reduction in infarct size, reduced cardiomyocytes apoptosis and increased capillary density in injured myocardium 3 days after MI. Furthermore, intramyocardial injection of CCR1-MSCs prevented cardiac remodeling and restored cardiac function 4 weeks after MI. Our results demonstrate the in vitro and in vivo salutary effects of genetic modification of stem cells. Specifically, overexpression of chemokine receptor enhances the migration, survival and engraftment of MSCs, and may provide a new therapeutic strategy for the injured myocardium.

Separation of cells from the rat anterior pituitary gland

NASA Technical Reports Server (NTRS)

Hymer, Wesley C.; Hatfield, J. Michael

1983-01-01

Various techniques for separating the hormone-producing cell types from the rat anterior pituitary gland are examined. The purity, viability, and responsiveness of the separated cells depend on the physiological state of the donor, the tissue dissociation procedures, the staining technique used for identification of cell type, and the cell separation technique. The chamber-gradient setup and operation, the characteristics of the gradient materials, and the separated cell analysis of velocity sedimentation techniques (in particular Staput and Celsep) are described. Consideration is given to the various types of materials used in density gradient centrifugation and the operation of a gradient generating device. The use of electrophoresis to separate rat pituitary cells is discussed.

Influence of beam shape on in-vitro cellular transformations in human skin fibroblasts

NASA Astrophysics Data System (ADS)

Mthunzi, Patience; Forbes, Andrew; Hawkins, Denise; Abrahamse, Heidi; Karsten, Aletta E.

2005-08-01

A variety of strategies have been utilised for prevention and treatment of chronic wounds such as leg ulcers, diabetic foot ulcers and pressure sores1. Low Level Laser Therapy (LLLT) has been reported to be an invaluable tool in the enhancement of wound healing through stimulating cell proliferation, accelerating collagen synthesis and increasing ATP synthesis in mitochondria to name but a few2. This study focused on an in-vitro analysis of the cellular responses induced by treatment with three different laser beam profiles namely, the Gaussian (G), Super Gaussian (SG) and Truncated Gaussian (TG), on normal wounded irradiated (WI) and wounded non-irradiated (WNI) human skin fibroblast cells (WS1), to test their influence in wound healing at 632.8 nm using a helium neon (HeNe) laser. For each beam profile, measurements were made using average energy densities over the sample ranging from 0.2 to 1 J, with single exposures on normal wounded cells. The cells were subjected to different post irradiation incubation periods, ranging from 0 to 24 hours to evaluate the duration (time) dependent effects resulting from laser irradiation. The promoted cellular alterations were measured by increase in cell viability, cell proliferation and cytotoxicity. The results obtained showed that treatment with the G compared to the SG and TG beams resulted in a marked increase in cell viability and proliferation. The data also showed that when cells undergo laser irradiation some cellular processes are driven by the peak energy density rather than the energy of the laser beam. We show that there exist threshold values for damage, and suggest optimal operating regimes for laser based wound healing.

Voltage effects on cells cultured on metallic biomedical implants

NASA Astrophysics Data System (ADS)

Haerihosseini, Seyed Morteza

Electrochemical voltage shifts in metallic biomedical implants occur in-vivo due to a number of processes including mechanically assisted corrosion. Surface potential of biomedical implants and excursions from resting open circuit potential (OCP), which is the voltage they attain while in contact with an electrolyte, can significantly change the interfacial properties of the metallic surfaces and alter the behavior of the surrounding cells, compromising the biocompatibility of metallic implants. Voltages can also be controlled to modulate cell function and fate. To date, the details of the physico-chemical phenomena and the role of different biomaterial parameters involved in the interaction between cells and metallic surfaces under cathodic bias have not been fully elucidated. In this work, changes in the interfacial properties of a CoCrMo biomedical alloy (ASTM F-1537) in phosphate-buffered saline (PBS) (pH 7.4) at different voltages was studied. Step polarization impedance spectroscopy technique was used to apply 50 mV voltage steps to samples, and the time-based current transients were recorded. A new equation was derived based on capacitive discharge through a Tafel element and generalized to deal with non-ideal impedance behavior. The new function compared to the KWW-Randles function, better matched the time-transient response. The results also showed a voltage dependent oxide resistance and capacitance behavior. Additionally, the in-vitro effect of static voltages on the behavior of MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy (ASTM-1537) was studied to determine the range of cell viability and mode of cell death beyond the viable range. Cell viability and morphology, changes in actin cytoskeleton, adhesion complexes and nucleus, and mode of cell death (necrosis, or intrinsic or extrinsic apoptosis) were characterized at different voltages ranging from -1000 to +500 mV (Ag/AgCl). Moreover, electrochemical currents and metal ion concentrations at each voltage were measured and related to the observed responses. Results show that cathodic and anodic voltages outside the voltage viability range (-400 < V < +500) lead to primarily intrinsic apoptotic and necrotic cell death, respectively. Cell death is associated with cathodic current densities of 0.1 uAcm-2 and anodic current densities of 10 uAcm-2. Significant increase in metallic ions (Co, Cr, Ni, Mo) was seen at +500 mV, and -1000 mV (Cr only) compared to open circuit potential. The number and total projected area of adhesion complexes was also lower on the polarized alloy (p < 0.05). These results show that reduction reactions on CoCrMo alloys leads to apoptosis of cells on the surface and may be a relevant mode of cell death for metallic implants in-vivo. . On the other hand, we studied how surface oxide thickness of Ti affects its voltage viability range and cellular response and whether anodic oxidation can serve as a means to extend this range. Cellular behavior (cell viability, cytoskeletal organization, and cellular adhesion) on bare and anodized Ti samples, potentiostatically held at voltages at the cathodic edge of the viability range, were assessed. Surfaces were characterized using contact angle (CA) measurement technique and atomic force microscopy (AFM), and the observed cellular response was related to the changes in the electrochemical properties (electrochemical currents, open circuit potential, and impedance spectra) of the samples. Results show that anodization at a low voltage (9 V) in phosphate buffer saline (PBS) generates a compact surface oxide with comparable surface roughness and energy to the starting native oxide on the bare surface. The anodized surface extends the viability range at 24 hours by about a 100 mV in the cathodic region, and preserved the cytoskeletal integrity and cell adhesion. Broadening of the viability range corresponds to an increase in impedance of the anodized surface at -400 mV(Ag/AgCl) and the resulting low average currents (below 0.1 uAcm-2) at the interface, which diminish the harmful cathodic reactions. Finally, cellular dynamics (size, polarity, movement) and temporal changes in the number and total area of focal adhesions in transiently transfected MC3T3-E1 pre-osteoblasts cultured on a CoCrMo alloy polarized at the cathodic and anodic edges of its voltage viability range (-400 and +500 mV(Ag/AgCl) respectively) were studied. Nucleus dynamics (size, circularity, movement) and the release of reactive oxygen species (ROS) was also studied on the polarized metal at -1000, -400, and +500 mV(Ag/AgCl). The results show that at -400 mV(Ag/AgCl) a gradual loss of adhesion occurs over 24 hours while cells shrink in size during this time. At +500 mV, cells become non-viable after 5 hours without showing any significant changes in adhesion behavior right before cell death. Nucleus size of cells at -1000 mV decreased sharply within 15 minutes after electrochemical polarization, which rendered the cells completely non-viable. No significant amount of ROS was released by cells on the polarized CoCrMo at any of these voltages.

Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding.

PubMed

Mendonça, Ronaldo Z; Arrózio, Sara J; Antoniazzi, Marta M; Ferreira, Jorge M C; Pereira, Carlos A

2002-07-17

The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes. In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers. Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture. Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities. Galactose was shown to be a more critical nutrient when cultures reached a high density. In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth. These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose. The inverse was not true and led, at high densities, to a decrease of cell viability. In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium. VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures. These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection. Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.

Human dental pulp stem cell adhesion and detachment in polycaprolactone electrospun scaffolds under direct perfusion

PubMed Central

Paim, A.; Braghirolli, D.I.; Cardozo, N.S.M.; Pranke, P.; Tessaro, I.C.

2018-01-01

Cell adhesion in three-dimensional scaffolds plays a key role in tissue development. However, stem cell behavior in electrospun scaffolds under perfusion is not fully understood. Thus, an investigation was made on the effect of flow rate and shear stress, adhesion time, and seeding density under direct perfusion in polycaprolactone electrospun scaffolds on human dental pulp stem cell detachment. Polycaprolactone scaffolds were electrospun using a solvent mixture of chloroform and methanol. The viable cell number was determined at each tested condition. Cell morphology was analyzed by confocal microscopy after various incubation times for static cell adhesion with a high seeding density. Scanning electron microscopy images were obtained before and after perfusion for the highest flow rate tested. The wall pore shear stress was calculated for all tested flow rates (0.005–3 mL/min). An inversely proportional relationship between adhesion time with cell detachment under perfusion was observed. Lower flow rates and lower seeding densities reduced the drag of cells by shear stress. However, there was an operational limit for the lowest flow rate that can be used without compromising cell viability, indicating that a flow rate of 0.05 mL/min might be more suitable for the tested cell culture in electrospun scaffolds under direct perfusion. PMID:29590258

The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and memory.

PubMed

Loepke, Andreas W; Istaphanous, George K; McAuliffe, John J; Miles, Lili; Hughes, Elizabeth A; McCann, John C; Harlow, Kathryn E; Kurth, C Dean; Williams, Michael T; Vorhees, Charles V; Danzer, Steve C

2009-01-01

Volatile anesthetics, such as isoflurane, are widely used in infants and neonates. Neurodegeneration and neurocognitive impairment after exposure to isoflurane, midazolam, and nitrous oxide in neonatal rats have raised concerns regarding the safety of pediatric anesthesia. In neonatal mice, prolonged isoflurane exposure triggers hypoglycemia, which could be responsible for the neurocognitive impairment. We examined the effects of neonatal isoflurane exposure and blood glucose on brain cell viability, spontaneous locomotor activity, as well as spatial learning and memory in mice. Seven-day-old mice were randomly assigned to 6 h of 1.5% isoflurane with or without injections of dextrose or normal saline, or to 6 h of room air without injections (no anesthesia). Arterial blood gases and glucose were measured. After 2 h, 18 h, or 11 wk postexposure, cellular viability was assessed in brain sections stained with Fluoro-Jade B, caspase 3, or NeuN. Nine weeks postexposure, spontaneous locomotor activity was assessed, and spatial learning and memory were evaluated in the Morris water maze using hidden and reduced platform trials. Apoptotic cellular degeneration increased in several brain regions early after isoflurane exposure, compared with no anesthesia. Despite neonatal cell loss, however, adult neuronal density was unaltered in two brain regions significantly affected by the neonatal degeneration. In adulthood, spontaneous locomotor activity and spatial learning and memory performance were similar in all groups, regardless of neonatal isoflurane exposure. Neonatal isoflurane exposure led to an 18% mortality, and transiently increased Paco(2), lactate, and base deficit, and decreased blood glucose levels. However, hypoglycemia did not seem responsible for the neurodegeneration, as dextrose supplementation failed to prevent neuronal loss. Prolonged isoflurane exposure in neonatal mice led to increased immediate brain cell degeneration, however, no significant reductions in adult neuronal density or deficits in spontaneous locomotion, spatial learning, or memory function were observed.

Synthetic high-density lipoprotein nanoconjugate targets neuroblastoma stem cells, blocking migration and self-renewal.

PubMed

Subramanian, Chitra; White, Peter T; Kuai, Rui; Kalidindi, Avinaash; Castle, Valerie P; Moon, James J; Timmermann, Barbara N; Schwendeman, Anna; Cohen, Mark S

2018-05-09

Pathways critical for neuroblastoma cancer stem cell function are targeted by 4,19,27-triacetyl withalongolide A (WGA-TA). Because neuroblastoma cells and their cancer stem cells highly overexpress the scavenger receptor class B type 1 receptor that binds to synthetic high-density lipoprotein, we hypothesized that a novel mimetic synthetic high-density lipoprotein nanoparticle would be an ideal carrier for the delivery of 4,19,27-triacetyl withalongolide to neuroblastoma and neuroblastoma cancer stem cells. Expression of scavenger receptor class B type 1 in validated human neuroblastoma cells was evaluated by quantitative polymerase chain reaction (qPCR) and Western blot. In vitro cellular uptake of synthetic high-density lipoprotein nanoparticles was observed with a fluorescence microscope. In vivo biodistribution of synthetic high-density lipoprotein nanoparticles was investigated with IVIS imaging. Self-renewal and migration/invasion were assessed by sphere formation and Boyden chamber assays, respectively. Viability was analyzed by CellTiter-Glo assay. Cancer stem cell markers were evaluated by flow cytometry. qPCR and Western blot analysis revealed a higher level of scavenger receptor class B type 1 expression and drug uptake in N-myc amplified neuroblastoma cells. In vitro uptake of synthetic high-density lipoprotein was almost completely blocked by excess synthetic high-density lipoprotein. The synthetic high-density lipoprotein nanoparticles mainly accumulated in the tumor and liver, but not in other organs. Synthetic HDL-4,19,27-triacetyl withalongolide showed a 1,000-fold higher potency than the carrier (synthetic high-density lipoprotein) alone (P < .01) to kill neuroblastoma cells. Additionally, a dose-dependent decrease in sphere formation, invasion, migration, and cancer stem cell markers was observed after treatment of neuroblastoma cells with synthetic high-density lipoprotein-4,19,27-triacetyl withalongolide A. Synthetic high-density lipoprotein is a promising platform to improve the delivery of anticancer drug 4,19,27-triacetyl withalongolide A to neuroblastomas and neuroblastoma cancer stem cells through SR-B1 targeting in vitro and in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

Hormesis effect of trace metals on cultured normal and immortal human mammary cells.

PubMed

Schmidt, Craig M; Cheng, Chun N; Marino, Angelo; Konsoula, Roula; Barile, Frank A

2004-06-01

An in vitro study was conducted to determine the effects of variable concentrations of trace metals on human cultured mammary cells. Monolayers of human mortal (MCF-12A) and immortal (MDA-MB231) mammary epithelial cells were incubated in the absence or presence of increasing concentrations of arsenic (As), mercury (Hg) and copper (Cu) for 24-h, 72-h, 4-d, and 7-d. The MTT assay was used to assess viability for all time periods and cell proliferation was monitored for 4-d and 7-d studies. Monolayers were also labeled with rhodamine-110 (R-6501), Sytox green, and Celltiter blue fluorescent dyes as indicators for intracellular esterase activity, nucleic acid staining, and cell reduction/viability, respectively. Total incubation time with chemical plus dyes was 24 h. For 24-h and 72-h studies, cells were seeded in 96-well plates, after which confluent monolayers were exposed to increasing concentrations of chemicals. For 4-d and 7-d studies, cells were seeded in 12-well plates at 1/3 confluent density (day 0) and exposed to increasing concentrations of metals on day 1. All cells were counted on days 4 and 7. In addition, test medium was removed from select groups of cultures on day 4, replaced with fresh medium in the absence of chemical (recovery studies), and assays were performed on day 7 as above. The data suggest that there is a consistent protective and/or stimulating effect of metals at the lowest concentrations in MCF-12A cells that is not observed in immortal MDA-MB231 cells. In fact, cell viability of MCF-12A cells is stimulated by otherwise equivalent inhibitory concentrations of As, Cu, and Hg on MDA-MB231 cells at 24-h. Whereas As and Hg suppress proliferation and viability in both cell lines after 4-d and 7-d of exposure, Cu enhances cell proliferation and viability of MCF-12A cells. MDA-MB231, however, recover better after 4-days of toxic insult. In addition, nutritional manipulation of media between the cell lines, or pretreatment with penicillamine, did not alter the hormesis effect displayed by MCF-12A. Growth of these cells however was not maintained in the alternative medium. The study demonstrates that a hormesis effect from trace metals is detectable in cultured mammary cells; fluorescent indicators, however, are not as sensitive as cell proliferation or MTT in recognizing the subtle responses. Also, sensitivity of mammary cells to lower concentrations of Cu, a biologically important trace metal, may play an important role in controlling cellular processes and proliferation. The ability to detect this in vitro phenomenon implies that similar processes, occurring in vivo, may be responsible for the development, induction, or enhancement of human cancers.

Wastewater treatment to enhance the economic viability of microalgae culture.

PubMed

Pires, J C M; Alvim-Ferraz, M C M; Martins, F G; Simões, M

2013-08-01

Microalgae culture is still not economically viable and it presents some negative environmental impacts, concerning water, nutrient and energy requirements. In this context, this study aims to review the recent advances on microalgal cultures in wastewaters to enhance their economic viability. We focused on three different culture concepts: (1) suspended cell systems, (2) cell immobilization, and (3) microalgae consortia. Cultures with suspended cells are the most studied. The nutrient removal efficiencies are usually high for wastewaters of different sources. However, biomass harvesting is difficult and a costly process due to the small cell size and lower culture density. On the other hand, the cell immobilization systems showed to be the solution for this problem, having as main limitation the nutrient diffusion from bulk to cells, which results in a reduced nutrient removal efficiency. The consortium between microalgae and bacteria enhances the growth of both microorganisms. This culture concept showed to be a promising technology to improve wastewater treatment, regarding not only nutrient removal but also biomass harvesting by bioflocculation. The aggregation mechanism must be studied in depth to find the process parameters that would lead to an effective and cheap harvesting process.

Comparative toxicological effects of two antifouling biocides on the marine diatom Chaetoceros lorenzianus: Damage and post-exposure recovery.

PubMed

Chavan, Pooja; Kumar, Rajesh; Kirubagaran, Ramalingam; Venugopalan, Vayalam P

2017-10-01

Antifouling biocides are commonly used in coastal electric power stations to prevent biofouling in their condenser cooling systems. However, the environmental impact of the chemical biocides is less understood than the thermal stress effects caused by the condenser effluents. In this study, Chaetoceros lorenzianus, a representative marine diatom, was used to analyse the toxicity of two antifouling biocides, chlorine and chlorine dioxide. The diatom cells were subjected to a range of concentrations of the biocides (from 0.05 to 2mg/L, as total residual oxidants, TRO) for contact time of 30min. They were analysed for viability, genotoxicity, chlorophyll a and cell density endpoints. The cells were affected at all concentrations of the biocides (0.05-2mg/L), showing dose-dependent decrease in viability and increase in DNA damage. The treated cells were later incubated in filtered seawater devoid of biocide to check for recovery. The cells were able to recover in terms of overall viability and DNA damage, when they had been initially treated with low concentrations of the biocides (0.5mg/L of Cl 2 or 0.2mg/L of ClO 2 ). Chlorophyll a analysis showed irreparable damage at all concentrations, while cell density showed increasing trend of reduction, if treated above 0.5mg/L of Cl 2 and 0.2mg/L of ClO 2 . The data indicated that in C. lorenzianus, cumulative toxic effects and recovery potential of ClO 2 up to 0.2mg/L were comparable with those of Cl 2 , up to 0.5mg/L concentration in terms of the studied endpoints. The results indicate that at the biocide levels currently being used at power stations, recovery of the organism is feasible upon return to ambient environment. Similar studies should be carried out on other planktonic and benthic organisms, which will be helpful in the formulation of future guidelines for discharge of upcoming antifouling biocides such as chlorine dioxide. Copyright © 2017 Elsevier Inc. All rights reserved.

Noninvasive Real-Time Assessment of Cell Viability in a Three-Dimensional Tissue.

PubMed

Mahfouzi, Seyed Hossein; Amoabediny, Ghassem; Doryab, Ali; Safiabadi-Tali, Seyed Hamid; Ghanei, Mostafa

2018-04-01

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

Tobacco and e-cigarette products initiate Kupffer cell inflammatory responses.

PubMed

Rubenstein, David A; Hom, Sarah; Ghebrehiwet, Berhane; Yin, Wei

2015-10-01

Kupffer cells are liver resident macrophages that are responsible for screening and clearing blood of pathogens and foreign particles. It has recently been shown that Kupffer cells interact with platelets, through an adhesion based mechanism, to aid in pathogen clearance and then these platelets re-enter the general systemic circulation. Thus, a mechanism has been identified that relates liver inflammation to possible changes in the systemic circulation. However, the role that Kupffer cells play in cardiovascular disease initiation/progression has not been elucidated. Thus, our objective was to determine whether or not Kupffer cells are responsive to a classical cardiovascular risk factor and if these changes can be transmitted into the general systemic circulation. If Kupffer cells initiate inflammatory responses after exposure to classical cardiovascular risk factors, then this provides a potential alternative/synergistic pathway for cardiovascular disease initiation. We aimed to elucidate the prevalence of this potential pathway. We hypothesized that Kupffer cells would initiate a robust inflammatory response after exposure to tobacco cigarette or e-cigarette products and that the inflammatory response would have the potential to antagonize other salient cells for cardiovascular disease progression. To test this, Kupffer cells were incubated with tobacco smoke extracts, e-cigarette vapor extracts or pure nicotine. Complement deposition onto Kupffer cells, Kupffer cell complement receptor expression, oxidative stress production, cytokine release and viability and density were assessed after the exposure. We observed a robust inflammatory response, oxidative stress production and cytokine release after Kupffer cells were exposed to tobacco or e-cigarette extracts. We also observed a marginal decrease in cell viability coupled with a significant decrease in cell density. In general, this was not a function of the extract formulation (e.g. tobacco vs. e-cigarette products or the formulation of the cigarette product). These results indicate that Kupffer cells are responsive to classical cardiovascular risk factors and that an inflammatory response is initiated that may pass into the general systemic circulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Experimental Calcium Silicate-Based Cement with and without Zirconium Oxide Modulates Fibroblasts Viability.

PubMed

Slompo, Camila; Peres-Buzalaf, Camila; Gasque, Kellen Cristina da Silva; Damante, Carla Andreotti; Ordinola-Zapata, Ronald; Duarte, Marco Antonio Hungaro; de Oliveira, Rodrigo Cardoso

2015-01-01

The aim of this study was to verify whether the use of zirconium oxide as a radiopacifier of an experimental calcium silicate-based cement (WPCZO) leads to cytotoxicity. Fibroblasts were treated with different concentrations (10 mg/mL, 1 mg/mL, and 0.1 mg/mL) of the cements diluted in Dulbecco's modified Eagle's medium (DMEM) for periods of 12, 24, and 48 h. Groups tested were white Portland cement (WPC), white Portland cement with zirconium oxide (WPCZO), and white mineral trioxide aggregate Angelus (MTA). Control group cells were not treated. The cytotoxicity was evaluated through mitochondrial-activity (MTT) and cell-density (crystal violet) assays. All cements showed low cytotoxicity. In general, at the concentration of 10 mg/mL there was an increase in viability of those groups treated with WPC and WPCZO when compared to the control group (p<0.05). A similar profile for the absorbance values was noted among the groups: 10 mg/mL presented an increase in viability compared to the control group. On the other hand, smaller concentrations presented a similar or lower viability compared to the control group, in general. A new dental material composed of calcium silicate-based cement with 20% zirconium oxide as the radiopacifier showed low cytotoxicity as a promising material to be exploited for root-end filling.

[Influence of Cryopreservation on Human Peripheral Blood Mononuclear Cell Immunocompetence].

PubMed

Pan, Xue-Feng; Lu, Chun-Xia; Yang, Li-Li; Shu, Chang; Yao, Na; Zuo, Hong-Bin; Cui, Li-Feng

2016-08-01

To establish a method for isolation, cryopreservation and recovery of the highly viable human peripheral blood monomuclear cells (PBMNCs) so as to achieve the long-term preservation of PBMNCs. A total of 80-100 ml peripheral blood were collected from the healthy volumteers aged over 50 years old. The PBMNCs were isolated by the Ficoll density gradient technique and cryopreserved gradually by program control method in liquid nitrogen freezer of -196 °C. The serum-free medium and autoloqous plasma medium were test for preservation of PBMNCs. The cell viability was assessed at time point of 1, 2, 4, 8, 12 and 24 months after thawing. Finally, the proliferation ability, purity and cytotoxicity were compared between the autologous immune lymphocytes (AIL) induced from cryopreserved PBMNCs and AIL as control from fresh PBMNCs. After separating, the cell viability was 99.6%±0.4%, and the recovery rate of lymphocytes was 58.4%±6.52%. The cell recovery rate of lymphocyte was 89.7%±3.82% at 24 months. The quality assurance program was reliable within 2 years of running. The AIL cells induced with cryopreserved PBMNCs were not significantly different from those induced from fresh PBMNCs in terms of proliferative action, purity and cytotoxicity(CD3(+)CD8(+) ≥45%,CD3(+)CD56(+) NKT≥10%,CD4(+)CD25(+) NKT≤10%). Manual separation of lymphocytes in vitro can get enough high-quality PBMNCs. The long-term cryopreserved PBMNC still maintain their high viability. The reinfusion of the clinical autologous immune cells would be advantageous for early tumor immunotherapy. Human AIL induced from cryopreserved PBMNC maintain their anti-tumor ability. These findings have the important implications for the application of these cells to adoptive cellular therapy.

The effect of ultrasound-related stimuli on cell viability in microfluidic channels

PubMed Central

2013-01-01

Background In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. Results Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0–29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. Conclusion The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability. Results from this study may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells. PMID:23809777

The effect of ultrasound-related stimuli on cell viability in microfluidic channels.

PubMed

Ankrett, Dyan N; Carugo, Dario; Lei, Junjun; Glynne-Jones, Peter; Townsend, Paul A; Zhang, Xunli; Hill, Martyn

2013-06-28

In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0-29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability. Results from this study may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells.

Viability of primary osteoblasts after treatment with tenofovir alafenamide: Lack of cytotoxicity at clinically relevant drug concentrations

PubMed Central

Callebaut, Christian; Liu, Yang; Babusis, Darius; Ray, Adrian; Miller, Michael; Kitrinos, Kathryn

2017-01-01

Tenofovir alafenamide (TAF) is a phosphonoamidate prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV). TAF is approved for the treatment of HIV-1 infection as part of the single-tablet regimen containing elvitegravir, cobicistat, emtricitabine, and TAF. When dosed once-daily, TAF results in approximately 90% lower levels of plasma TFV and a 4-fold increase in intracellular TFV-diphosphate (TFV-DP) in PBMCs compared with the TFV prodrug tenofovir disoproxil fumarate (TDF). Several antiretrovirals, including TDF, have been associated with bone mineral density decreases in patients; the effect of clinically relevant TAF concentrations on primary osteoblast viability was therefore assessed in vitro. Studies in PBMCs determined that a 2-hour TAF exposure at concentrations similar to human plasma Cmax achieved intracellular TFV-DP levels comparable to those observed after the maximum recommended human dose of 25 mg TAF. Comparable intracellular TFV-DP levels were achieved in primary osteoblasts with 2-hour TAF exposure daily for 3 days at concentrations similar to those used for PBMCs (100–400 nM). No change in cell viability was observed in either primary osteoblasts or PBMCs. The mean TAF CC50 in primary osteoblasts after 3 days of daily 2-hour pulses was >500 μM, which is >1033 times higher than the TAF maximum recommended human dose plasma Cmax. In summary, primary osteoblasts were not preferentially loaded by TAF compared with PBMCs, with comparable TFV-DP levels achieved in both cell types. Furthermore, there was no impact on osteoblast cell viability at clinically relevant TAF concentrations. PMID:28182625

Viability of primary osteoblasts after treatment with tenofovir alafenamide: Lack of cytotoxicity at clinically relevant drug concentrations.

PubMed

Callebaut, Christian; Liu, Yang; Babusis, Darius; Ray, Adrian; Miller, Michael; Kitrinos, Kathryn

2017-01-01

Tenofovir alafenamide (TAF) is a phosphonoamidate prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV). TAF is approved for the treatment of HIV-1 infection as part of the single-tablet regimen containing elvitegravir, cobicistat, emtricitabine, and TAF. When dosed once-daily, TAF results in approximately 90% lower levels of plasma TFV and a 4-fold increase in intracellular TFV-diphosphate (TFV-DP) in PBMCs compared with the TFV prodrug tenofovir disoproxil fumarate (TDF). Several antiretrovirals, including TDF, have been associated with bone mineral density decreases in patients; the effect of clinically relevant TAF concentrations on primary osteoblast viability was therefore assessed in vitro. Studies in PBMCs determined that a 2-hour TAF exposure at concentrations similar to human plasma Cmax achieved intracellular TFV-DP levels comparable to those observed after the maximum recommended human dose of 25 mg TAF. Comparable intracellular TFV-DP levels were achieved in primary osteoblasts with 2-hour TAF exposure daily for 3 days at concentrations similar to those used for PBMCs (100-400 nM). No change in cell viability was observed in either primary osteoblasts or PBMCs. The mean TAF CC50 in primary osteoblasts after 3 days of daily 2-hour pulses was >500 μM, which is >1033 times higher than the TAF maximum recommended human dose plasma Cmax. In summary, primary osteoblasts were not preferentially loaded by TAF compared with PBMCs, with comparable TFV-DP levels achieved in both cell types. Furthermore, there was no impact on osteoblast cell viability at clinically relevant TAF concentrations.

Correlating measured transient temperature rises with damage rate processes in cultured cells

NASA Astrophysics Data System (ADS)

Denton, Michael L.; Tijerina, Amanda J.; Gonzalez, Cherry C.; Gamboa, B. Giovana; Noojin, Gary D.; Ahmed, Elharith M.; Rickman, John M.; Dyer, Phillip H.; Rockwell, Benjamin A.

2017-02-01

Thermal damage rate processes in biological tissues are usually characterized by a kinetics approach. This stems from experimental data that show how the transformation of a specified biological property of cells or biomolecule (plating efficiency for viability, change in birefringence, tensile strength, etc.) is dependent upon both time and temperature. Here, two disparate approaches were used to study thermal damage rate processes in cultured retinal pigment epithelial cells. Laser exposure (photothermal) parameters included 2-μm laser exposure of non-pigmented cells and 532-nm exposures of cells possessing a variety of melanosome particle densities. Photothermal experiments used a mid-IR camera to record temperature histories with spatial resolution of about 8 μm, while fluorescence microscopy of the cell monolayers identified threshold damage at the boundary between live and dead cells. Photothermal exposure durations ranged from 0.05-20 s, and the effects of varying ambient temperature were investigated. Temperature during heat transfer using a water-jacketed cuvette was recorded with a fast microthermister, while damage and viability of the suspended cells were determined as percentages. Exposure durations for the heat transfer experiments ranged from 50- 60 s. Empirically-determined kinetic parameters for the two heating methods were compared with each other, and with values found in the literature.

Release of tissue inhibitor of metalloproteinase-2 from alginate microcapsule encapsulating genetically engineered cells

PubMed Central

Kim, Yeon Seong; Jeong, Young-II; Jin, Shu-Guang; Pei, Jian; Wen, Min; Kim, In-Young; Moon, Kyung-Sub; Jung, Tae-Young; Ryu, Hyang-Hwa; Jung, Shin

2013-01-01

Background In this study, 293T cells were genetically engineered to secrete tissue inhibitor of metalloproteinase-2 (TIMP2) and encapsulated into alginate microcapsules to continuously release TIMP2 protein. Methods The anti-invasive potential of the microcapsules was studied in vitro using brain tumor cells. The TIMP2 gene was transfected to 293T cells, and genetically engineered 293TIMP2 cells were encapsulated into alginate microcapsules. Release of TIMP2 protein was detected with Western blot analysis and the anti-invasive potential against U87MG cells was tested using gelatin zymography and a Matrigel assay. Results Cell viability within the alginate microcapsules was maintained at a cell density of 5 × 106. Because polycationic polymers are helpful for maintaining the mechanical strength of microcapsules with good cell viability, the alginate microcapsules were reinforced with chitosan (0.1% w/v). Expression of TIMP2 protein in cell lysates and secretion of TIMP2 into the conditioned medium was confirmed by Western blot analysis. Alginate microcapsules encapsulating 293TIMP2 cells released TIMP2 protein into the medium efficiently, where the TIMP2 protein participated in degradation of the matrix metalloproteinase-2 enzyme and inhibited invasion of U87MG cells. Conclusion Alginate microcapsules encapsulating 293TIMP2 cells are promising candidates for anti-invasive treatment of glioma. PMID:24231999

Effects of copper supplement on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture.

PubMed

Rodríguez, L Mato; Alatossava, T

2008-10-01

To determine the effects of supplemented copper (Cu2+) on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture. Thirteen strains belonging to Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus rhamnosus, Streptococcus thermophilus or Propionibacterium freudenreichii species were exposed to various copper concentrations in the proper growth medium at relevant growth temperatures, and the effects of supplemented copper on bacterial growth and cell viability were determined by optical density and pH measurements, also by platings. Among the species considered, L. delbrueckii was the most copper resistant and S. thermophilus the most sensitive to copper. Anaerobic conditions increased this sensitivity significantly. There was also a considerable amount of variation in copper resistance at strain level. Copper resistance is both a species- and strain-dependent property and may reflect variability in copper-binding capacities by cell wall components among species and strains. In addition, the chemical state of copper may be involved. This study revealed that copper resistance is a highly variable property among starter and adjunct strains, and this variability should be considered when strains are selected for Emmental cheese manufacture.

Diesel oil removal by immobilized Pseudoxanthomonas sp. RN402.

PubMed

Nopcharoenkul, Wannarak; Netsakulnee, Parichat; Pinyakong, Onruthai

2013-06-01

Pseudoxanthomonas sp. RN402 was capable of degrading diesel, crude oil, n-tetradecane and n-hexadecane. The RN402 cells were immobilized on the surface of high-density polyethylene plastic pellets at a maximum cell density of 10(8) most probable number (MPN) g(-1) of plastic pellets. The immobilized cells not only showed a higher efficacy of diesel oil removal than free cells but could also degrade higher concentrations of diesel oil. The rate of diesel oil removal by immobilized RN402 cells in liquid culture was 1,050 mg l(-1) day(-1). Moreover, the immobilized cells could maintain high efficacy and viability throughout 70 cycles of bioremedial treatment of diesel-contaminated water. The stability of diesel oil degradation in the immobilized cells resulted from the ability of living RN402 cells to attach to material surfaces by biofilm formation, as was shown by CLSM imaging. These characteristics of the immobilized RN402 cells, including high degradative efficacy, stability and flotation, make them suitable for the purpose of continuous wastewater bioremediation.

Metabolic activity of odontoblast-like cells irradiated with blue LED (455 nm).

PubMed

de Almeida, Leopoldina Fátima Dantas; Basso, Fernanda Gonçalves; Turrioni, Ana Paula Silveira; de-Souza-Costa, Carlos Alberto; Hebling, Josimeri

2016-01-01

Blue light emitting diodes (LEDs) are frequently used in dentistry for light activation of resin-based materials; however, their photobiostimulatory effects have not yet been fully investigated. This study aimed to investigate the effect of blue LED (455 nm) on the metabolism of odontoblast-like cells MDPC-23. Energy doses of 2 and 4 J/cm(2) were used at 20 mW/cm(2) fixed power density. MDPC-23 cells were seeded at 10,000 cells/cm(2) density in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum (FBS). After 12 h, the culture medium was replaced with new DMEM supplemented with 0.5 % of FBS, and the cells were incubated for further 12 h. After that, single irradiation was performed to the culture, under selected parameters. Cell viability evaluations (Alamar Blue Assay, n = 12), number of viable cells (Trypan Blue Assay, n = 12), morphological analysis by scanning electron microscopy (SEM, n = 2), gene expression (n = 6) of alkaline phosphatase (Alp), collagen (Col-1a1), and dental matrix protein (Dmp-1) (quantitative polymerase chain reaction (qPCR)) were performed 72 h after irradiation. Data were analyzed by Kruskal-Wallis, ANOVA, and Tukey tests (p < 0.05). Direct light application at 4 J/cm(2) energy dose had no negative effects on cell viability, while irradiation with 2 J/cm(2) reduced cell metabolism. None of doses affected the number of viable cells compared with the control group. The two energy doses downregulated the expression of Alp; however, expression of Col-1a1 and Dmp-1 had no alteration. Cells presented change in the cytoskeleton only when irradiated with 2 J/cm(2). In conclusion, the blue LED (455 nm) irradiation, under the evaluated parameters, had no biostimulatory effects on MDPC-23 cells.

Northern contaminant mixtures induced morphological and functional changes in human coronary artery endothelial cells under culture conditions typifying high fat/sugar diet and ethanol exposure.

PubMed

Florian, Maria; Yan, Jin; Ulhaq, Saad; Coughlan, Melanie; Laziyan, Mahemuti; Willmore, William; Jin, Xiaolei

2013-11-16

It has been reported that Northern populations are exposed to mixtures of various environmental contaminants unique to the Arctic (Northern contaminant mixtures - NCM) at a large range of concentrations, depending on their geological location, age, lifestyle and dietary habits. To determine if these contaminants may contribute to a cardiovascular health risk, especially when combined with a high fat and sugar diet and ethanol exposure, we treated human coronary artery endothelial cells (HCAEC) with two mixtures of 4 organic (NCM1) or 22 organic and inorganic (NCM2) chemicals detected in Northerners' blood during 2004-2005 in the presence or absence of low-density lipoprotein (1.5mg/ml), very-low-density lipoprotein (1.0mg/ml) and glucose (10mmol/L) (LVG), and in the absence or presence of 0.1% ethanol. After 24h of exposure, cell morphology and markers of cytotoxicity and endothelial function were examined. NCM1 treatment did not affect cell viability, but increased cell size, disrupted cell membrane integrity, and decreased cell density, uptake of small peptides, release of endothelin-1 (ET-1) and plasminogen activator inhibitor (PAI), while causing no changes in endothelial nitric oxide synthase (eNOS) protein expression and nitric oxide (NO) release. In contrast, NCM2 decreased cell viability, total protein yield, uptake of small peptides, eNOS protein expression, and NO release and caused membrane damage, but caused no changes in the secretion of ET-1, prostacyclin and PAI. The presence of LVG and/or alcohol did or did not influence the effects of NCM1 or NCM2 depending on the endpoint and the mixture examined. These results suggested that the effects of one or one group of contaminants may be altered by the presence of other contaminants, and that with or without the interaction of high fat and sugar diet and/or ethanol exposure, NCMs at the concentrations used caused endothelial dysfunction in vitro. It remains to be investigated if these effects of NCMs also occur in vivo. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

Superhydrophilic-Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection.

PubMed

Ueda, Erica; Feng, Wenqian; Levkin, Pavel A

2016-10-01

High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal transfection reagent, ScreenFect A, the method of reverse cell transfection is optimized on the patterned substrates and several factors that affect transfection efficiency and cytotoxicity are identified. Higher levels of transfection are achieved on HOOC- versus NH 2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the transfection mixture, while minimizing the amount of transfection reagent improves cell viability. Almost no diffusion of the printed transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing reverse cell transfection conditions before performing further cell screenings. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Density, Viability Conidia And Symptoms of Metarhizium anisopliae infection on Oryctes rhinoceros larvae

NASA Astrophysics Data System (ADS)

Indriyanti, D. R.; Putri, R. I. P.; Widiyaningrum, P.; Herlina, L.

2017-04-01

M. anisopliae is parasitic fungus on insect pests; it is used as a biocontrol agent. M. anisopliae can be propagated on maize or rice substrate. M. anisopliae is currently sold in the form of kaolin powder formulations. Before it is used to check the density, viability and pathogenicity of M. anisopliae. However the problem is the kaolin powder very soft, so it difficult to distinguish between kaolin and conidia. This article gives information on how to calculate conidia density, viability and symptoms of M. anisopliae infection on Oryctes rhinoceros larvae. The study was conducted in the laboratory to determine the density and viability. The pathogenicity testing was done using pots. The Pot is containing soil substrate mixed with M. Anispoliae and ten tails O. Rhinoceros larvae per pot. The results showed that the density of M. anisopliae conidia was 1.81 x 108 conidia mL-1 and the viability was 94% within 24 hours. The larval mortality began to emerge in the 1st week, and all larvae died at the sixth week. The symptom of M. anisopliae infection on Oryctes rhinoceros larvae, there was a black spot on the larval integument. The larvae movements become slow and poor appetite; it will die within 3-7 days. The larvae die hard, and the white hyphae grow on the body surface that turns green.

Serenoa repens extracts promote hair regeneration and repair of hair loss mouse models by activating TGF-β and mitochondrial signaling pathway.

PubMed

Zhu, H-L; Gao, Y-H; Yang, J-Q; Li, J-B; Gao, J

2018-06-01

Plenty of plant extracts have been used for treating hair loss. This study aims to investigate the effects of liposterolic extracts of Serenoa repens (LSESr) on hair cell growth and regeneration of hair, and clarify the associated mechanisms. Human keratinocyte cells (HACAT) were cultured, incubated with dihydrotestosterone (DHT) and treated with LSESr. Cell viability was examined by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide (MTT) assay. Hair loss C57BL/6 mouse model was established by inducing with DHT. Hair growth, density, and thickness were evaluated. Back skin samples were collected and stained with hematoxylin and eosin (HE) assay. B-cell lymphoma-2 (Bcl-2), Bcl-2 associated protein X (Bax), cleaved caspase 3 and transforming growth factor β2 (TGF-β2) were examined using Western blot assay. LSESr treatment significantly increased HACAT cell viabilities compared to DHT-only treated cells (p<0.05). LSESr treatment post injection of DHT significantly converted skin color from pink to gray and increased hair density, weight and thickness compared to DHT-only treated mice (p<0.05). LSESr treatment significantly triggered follicle growth and decreased inflammatory response. LSESr treatment significantly decreased TGF-β2 and cleaved caspase 3 expression of hair loss mouse models compared to that of DHT treated mice (p<0.05). LSESr treatment significantly enhanced Bcl-2 expression and reduced Bax expression compared to that of DHT treated mice (p<0.05). Meanwhile, effects of LSESr were substantial even achieving to the potential of finasteride. LSESr promoted the hair regeneration and repair of hair loss mouse models by activating TGF-β signaling and mitochondrial signaling pathway.

Novel MET/TIE2/VEGFR2 inhibitor altiratinib inhibits tumor growth and invasiveness in bevacizumab-resistant glioblastoma mouse models

PubMed Central

Piao, Yuji; Park, Soon Young; Henry, Verlene; Smith, Bryan D.; Tiao, Ningyi; Flynn, Daniel L.

2016-01-01

Background Glioblastoma highly expresses the proto-oncogene MET in the setting of resistance to bevacizumab. MET engagement by hepatocyte growth factor (HGF) results in receptor dimerization and autophosphorylation mediating tumor growth, invasion, and metastasis. Evasive revascularization and the recruitment of TIE2-expressing macrophages (TEMs) are also triggered by anti-VEGF therapy. Methods We investigated the activity of altiratinib (a novel balanced inhibitor of MET/TIE2/VEGFR2) against human glioblastoma stem cell lines in vitro and in vivo using xenograft mouse models. The biological activity of altiratinib was assessed in vitro by testing the expression of HGF-stimulated MET phosphorylation as well as cell viability after altiratinib treatment. Tumor volume, stem cell and mesenchymal marker levels, microvessel density, and TIE2-expressing monocyte infiltration were evaluated in vivo following treatment with a control, bevacizumab alone, bevacizumab combined with altiratinib, or altiratinib alone. Results In vitro, HGF-stimulated MET phosphorylation was completely suppressed by altiratinib in GSC17 and GSC267, and altiratinib markedly inhibited cell viability in several glioblastoma stem cell lines. More importantly, in multiple xenograft mouse models, altiratinib combined with bevacizumab dramatically reduced tumor volume, invasiveness, mesenchymal marker expression, microvessel density, and TIE2-expressing monocyte infiltration compared with bevacizumab alone. Furthermore, in the GSC17 xenograft model, altiratinib combined with bevacizumab significantly prolonged survival compared with bevacizumab alone. Conclusions Together, these data suggest that altiratinib may suppress tumor growth, invasiveness, angiogenesis, and myeloid cell infiltration in glioblastoma. Thus, altiratinib administered alone or in combination with bevacizumab may overcome resistance to bevacizumab and prolong survival in patients with glioblastoma. PMID:26965451

Biodegradation of low-density polyethylene by marine bacteria from pelagic waters, Arabian Sea, India.

PubMed

Harshvardhan, Kumar; Jha, Bhavanath

2013-12-15

Sixty marine bacteria isolated from pelagic waters were screened for their ability to degrade low-density polyethylene; among them, three were positive and able to grow in a medium containing polythene as the sole carbon source. The positive isolates were identified as Kocuria palustris M16, Bacillus pumilus M27 and Bacillus subtilis H1584 based on the 16S rRNA gene sequence homology. The weight loss of polyethylene was 1%, 1.5% and 1.75% after 30 days of incubation with the M16, M27 and H1584 isolates, respectively. The maximum (32%) cell surface hydrophobicity was observed in M16, followed by the H1584 and M27 isolates. The viability of the isolates growing on the polyethylene surface was confirmed using a triphenyltetrazolium chloride reduction test. The viability was also correlated with a concomitant increase in the protein density of the biomass. Polyethylene biodegradation was further confirmed by an increase in the Keto Carbonyl Bond Index, the Ester Carbonyl Bond Index and the Vinyl Bond Index, which were calculated from FT-IR spectra. Copyright © 2013 Elsevier Ltd. All rights reserved.

Differential effects of lipopolysaccharide on energy metabolism in murine microglial N9 and cholinergic SN56 neuronal cells.

PubMed

Klimaszewska-Łata, Joanna; Gul-Hinc, Sylwia; Bielarczyk, Hanna; Ronowska, Anna; Zyśk, Marlena; Grużewska, Katarzyna; Pawełczyk, Tadeusz; Szutowicz, Andrzej

2015-04-01

There are significant differences between acetyl-CoA and ATP levels, enzymes of acetyl-CoA metabolism, and toll-like receptor 4 contents in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Exposition of N9 cells to lipopolysaccharide caused concentration-dependent several-fold increases of nitrogen oxide synthesis, accompanied by inhibition of pyruvate dehydrogenase complex, aconitase, and α-ketoglutarate dehydrogenase complex activities, and by nearly proportional depletion of acetyl-CoA, but by relatively smaller losses in ATP content and cell viability (about 5%). On the contrary, SN56 cells appeared to be insensitive to direct exposition to high concentration of lipopolysaccharide. However, exogenous nitric oxide resulted in marked inhibition pyruvate dehydrogenase and aconitase activities, depletion of acetyl-CoA, along with respective loss of SN56 cells viability. These data indicate that these two common neurodegenerative signals may differentially affect energy-acetyl-CoA metabolism in microglial and cholinergic neuronal cell compartments in the brain. Moreover, microglial cells appeared to be more resistant than neuronal cells to acetyl-CoA and ATP depletion evoked by these neurodegenerative conditions. Together, these data indicate that differential susceptibility of microglia and cholinergic neuronal cells to neurotoxic signals may result from differences in densities of toll-like receptors and degree of disequilibrium between acetyl-CoA provision in mitochondria and its utilization for energy production and acetylation reactions in each particular group of cells. There are significant differences between acetyl-CoA and ATP levels and enzymes of acetyl-CoA metabolism in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Pathological stimulation of microglial toll-like receptors (TLRs) triggered excessive synthesis of microglia-derived nitric oxide (NO)/NOO radicals that endogenously inhibited pyruvate dehydrogenase complex (PDHC), aconitase, and α-ketoglutarate dehydrogenase complex. However, it caused none or small suppressions of acetyl-CoA and microglial viability, respectively. Microglia-derived NO inhibited same enzymes in cholinergic neuronal cells causing marked viability loss because of acetyl-CoA deficits evoked by its competitive consumption by energy producing and acetylcholine/N-acetyl-l-aspartate (NAA) synthesizing pathways. © 2014 International Society for Neurochemistry.

Investigation of photobiomodulation potentiality by 635 and 809 nm lasers on human osteoblasts.

PubMed

Bölükbaşı Ateş, Gamze; Ak Can, Ayşe; Gülsoy, Murat

2017-04-01

Photobiomodulation (PBM) describes light-induced photochemical reactions achieved by the application of red or near infrared lasers/LED light with low energy densities. This noninvasive and painless method has been used in some clinical areas but controversial outcomes demand a skeptical look for its promising and potential effects. In this detailed in vitro study, the osteoblast cells were irradiated with 635 and 809 nm diode lasers at energy densities of 0.5, 1, and 2 J/cm 2 . Cell viability, proliferation, bone formation, and osteoblast differentiation were evaluated by methylthiazole tetrazolium (MTT) assay, Alamar Blue assay, acridine orange/propidium iodide staining, alkaline phosphatase (ALP) activity, Alizarin red staining, and reverse-transcription polymerase chain reaction (RT-PCR) to test the expression of collagen type I, ALPL, and osteocalcin. The results indicate that studied energy doses have a transient effect (48 h after laser irradiation) on the osteoblast viability and proliferation. Similarly, laser irradiation did not appear to have any effect on ALP activity. These results were confirmed by RT-PCR analysis of osteoblast markers. This study suggests that several irradiation parameters and variations in the methods should be clearly established in the laboratory before laser treatment becomes a postulated application for bone tissue regeneration in clinical level.

Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

NASA Astrophysics Data System (ADS)

Zhao, Guoping; Chen, Shaopeng; Zhao, Ye; Zhu, Lingyan; Huang, Pei; Bao, Lingzhi; Wang, Jun; Wang, Lei; Wu, Lijun; Wu, Yuejin; Xu, An

2010-02-01

Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

Comparison of Human Denuded Amniotic Membrane and Porcine Small Intestine Submucosa as Scaffolds for Limbal Mesenchymal Stem Cells.

PubMed

Sous Naasani, Liliana I; Rodrigues, Cristiano; Azevedo, Jéssica Gonçalves; Damo Souza, Aline F; Buchner, Silvio; Wink, Márcia R

2018-04-29

Blinding corneal scarring is usually treated with allogeneic graft tissue. Nevertheless, the global shortage of donors leaves millions of patients in need of therapy. Traditional tissue engineering strategies involves the combination of cells, growth factors, and scaffolds that can supply cellular biological components allowing to restore the tissue function. The mesenchymal stem cells found in the limbal stroma (L-MSCs) have a self-renewal potential for multilineage differentiation. Thus, in this work we compared the potential of human amniotic membrane (hAM) and porcine small intestine submucosa (SIS) as scaffolds for L-MSCs, aiming at potential applications in corneal regeneration. For that, L-MSCs were seeded on hAM and SIS and we analyzed their viability, actin cytoskeleton, nuclei morphology, cell density, adhesion and surface markers. Our results showed that cells adhered and integrated into both membranes with a high cell density, an important characteristic for cell therapy. However, due to its transparency, the hAM allowed a better observation of L-MSCs. In addition, the analysis of surface markers expression on L-MSCs after two weeks showed a slight increase in the percentages of negative markers for MSCs grown on SIS membrane. Thus, considering a long-term culture, the hAM was considered better in maintaining the MSCs phenotype. Regarding the function as scaffolds, SIS was as efficient as the amniotic membrane, considering that these two types of biological matrices maintained the cell viability, actin cytoskeleton, nuclei morphology and mesenchymal phenotype, without causing cell death. Therefore, our data in vitro provides evidence for future pre-clinical studies were these membranes can be used as a support to transport mesenchymal stem cells to the injured area, creating a kind of temporary curative, allowing the release of bioactive molecules, such as cytokines and growth factors and then promoting the tissue regeneration, both in human and veterinary medicine.

N-Acetylcysteine Increases Corneal Endothelial Cell Survival in a Mouse Model of Fuchs Endothelial Corneal Dystrophy

PubMed Central

Kim, Eun Chul; Meng, Huan; Jun, Albert S.

2014-01-01

The present study evaluated survival effects of N-acetylcysteine (NAC) on cultured corneal endothelial cells exposed to oxidative and endoplasmic reticulum (ER) stress and in a mouse model of early-onset Fuchs endothelial corneal dystrophy (FECD). Cultured bovine corneal endothelial cell viability against oxidative and ER stress was determined by CellTiter-Glo® luminescent reagent. Two-month-old homozygous knock-in Col8a2L450W/L450W mutant (L450W) and C57/Bl6 wild-type (WT) animals were divided into two groups of 15 mice. Group I received 7 mg/mL NAC in drinking water and Group II received control water for 7 months. Endothelial cell density and morphology were evaluated with confocal microscopy. Antioxidant gene (iNos) and ER stress/unfolded protein response gene (Grp78 and Chop) mRNA levels and protein expression were measured in corneal endothelium by real time PCR and Western blotting. Cell viability of H2O2 and thapsigargin exposed cells pre-treated with NAC was significantly increased compared to untreated controls (pitalic>0.01). Corneal endothelial cell density (CD) was higher (p=0.001) and percent polymegathism was lower (p=0.04) in NAC treated L450W mice than in untreated L450W mice. NAC treated L450W endothelium showed significant upregulation of iNos, whereas Grp78 and Chop were downregulated compared to untreated L450W endothelium by real time PCR and Western blotting. NAC increases survival in cultured corneal endothelial cells exposed against ER and oxidative stress. Systemic NAC ingestion increases corneal endothelial cell survival which is associated with increased antioxidant and decreased ER stress markers in a mouse model of early-onset FECD. Our study presents in vivo evidence of a novel potential medical treatment for FECD. PMID:24952277

N-Acetylcysteine increases corneal endothelial cell survival in a mouse model of Fuchs endothelial corneal dystrophy.

PubMed

Kim, Eun Chul; Meng, Huan; Jun, Albert S

2014-10-01

The present study evaluated survival effects of N-acetylcysteine (NAC) on cultured corneal endothelial cells exposed to oxidative and endoplasmic reticulum (ER) stress and in a mouse model of early-onset Fuchs endothelial corneal dystrophy (FECD). Cultured bovine corneal endothelial cell viability against oxidative and ER stress was determined by CellTiter-Glo(®) luminescent reagent. Two-month-old homozygous knock-in Col8a2(L450W/L450W) mutant (L450W) and C57/Bl6 wild-type (WT) animals were divided into two groups of 15 mice. Group I received 7 mg/mL NAC in drinking water and Group II received control water for 7 months. Endothelial cell density and morphology were evaluated with confocal microscopy. Antioxidant gene (iNos) and ER stress/unfolded protein response gene (Grp78 and Chop) mRNA levels and protein expression were measured in corneal endothelium by real time PCR and Western blotting. Cell viability of H2O2 and thapsigargin exposed cells pre-treated with NAC was significantly increased compared to untreated controls (p < 0.01). Corneal endothelial cell density (CD) was higher (p = 0.001) and percent polymegathism was lower (p = 0.04) in NAC treated L450W mice than in untreated L450W mice. NAC treated L450W endothelium showed significant upregulation of iNos, whereas Grp78 and Chop were downregulated compared to untreated L450W endothelium by real time PCR and Western blotting. NAC increases survival in cultured corneal endothelial cells exposed against ER and oxidative stress. Systemic NAC ingestion increases corneal endothelial cell survival which is associated with increased antioxidant and decreased ER stress markers in a mouse model of early-onset FECD. Our study presents in vivo evidence of a novel potential medical treatment for FECD. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enhanced viability of corneal epithelial cells for efficient transport/storage using a structurally modified calcium alginate hydrogel.

PubMed

Wright, Bernice; Cave, Richard A; Cook, Joseph P; Khutoryanskiy, Vitaliy V; Mi, Shengli; Chen, Bo; Leyland, Martin; Connon, Che J

2012-05-01

Therapeutic limbal epithelial stem cells could be managed more efficiently if clinically validated batches were transported for 'on-demand' use. In this study, corneal epithelial cell viability in calcium alginate hydrogels was examined under cell culture, ambient and chilled conditions for up to 7 days. Cell viability improved as gel internal pore size increased, and was further enhanced with modification of the gel from a mass to a thin disc. Ambient storage conditions were optimal for supporting cell viability in gel discs. Cell viability in gel discs was significantly enhanced with increases in pore size mediated by hydroxyethyl cellulose. Our novel methodology of controlling alginate gel shape and pore size together provides a more practical and economical alternative to established corneal tissue/cell storage methods.

Efficient Intracellular Delivery of Molecules with High Cell Viability Using Nanosecond-Pulsed Laser-Activated Carbon Nanoparticles

PubMed Central

2015-01-01

Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5–9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability. PMID:24547946

Combination of hyperthermia and photodynamic therapy on mesenchymal stem cell line treated with chloroaluminum phthalocyanine magnetic-nanoemulsion

NASA Astrophysics Data System (ADS)

de Paula, Leonardo B.; Primo, Fernando L.; Pinto, Marcelo R.; Morais, Paulo C.; Tedesco, Antonio C.

2015-04-01

The present study reports on the preparation and the cell viability assay of two nanoemulsions loaded with magnetic nanoparticle and chloroaluminum phthalocyanine. The preparations contain equal amount of chloroaluminum phthalocyanine (0.05 mg/mL) but different contents of magnetic nanoparticle (0.15×1013 or 1.50×1013 particle/mL). The human bone marrow mesenchymal stem cell line was used as the model to assess the cell viability and this type of cell can be used as a model to mimic cancer stem cells. The cell viability assays were performed in isolated as well as under combined magnetic hyperthermia and photodynamic therapy treatments. We found from the cell viability assay that under the hyperthermia treatment (1 MHz and 40 Oe magnetic field amplitude) the cell viability reduction was about 10%, regardless the magnetic nanoparticle content within the magnetic nanoparticle/chloroaluminum phthalocyanine formulation. However, cell viability reduction of about 50% and 60% were found while applying the photodynamic therapy treatment using the magnetic nanoparticle/chloroaluminum phthalocyanine formulation containing 0.15×1013 or 1.50×1013 magnetic particle/mL, respectively. Finally, an average reduction in cell viability of about 66% was found while combining the hyperthermia and photodynamic therapy treatments.

Towards a quantitative understanding of oxygen tension and cell density evolution in fibrin hydrogels.

PubMed

Demol, Jan; Lambrechts, Dennis; Geris, Liesbet; Schrooten, Jan; Van Oosterwyck, Hans

2011-01-01

The in vitro culture of hydrogel-based constructs above a critical size is accompanied by problems of unequal cell distribution when diffusion is the primary mode of oxygen transfer. In this study, an experimentally-informed mathematical model was developed to relate cell proliferation and death inside fibrin hydrogels to the local oxygen tension in a quantitative manner. The predictive capacity of the resulting model was tested by comparing its outcomes to the density, distribution and viability of human periosteum derived cells (hPDCs) that were cultured inside fibrin hydrogels in vitro. The model was able to reproduce important experimental findings, such as the formation of a multilayered cell sheet at the hydrogel periphery and the occurrence of a cell density gradient throughout the hydrogel. In addition, the model demonstrated that cell culture in fibrin hydrogels can lead to complete anoxia in the centre of the hydrogel for realistic values of oxygen diffusion and consumption. A sensitivity analysis also identified these two parameters, together with the proliferation parameters of the encapsulated cells, as the governing parameters for the occurrence of anoxia. In conclusion, this study indicates that mathematical models can help to better understand oxygen transport limitations and its influence on cell behaviour during the in vitro culture of cell-seeded hydrogels. Copyright © 2010 Elsevier Ltd. All rights reserved.

[Islet isolation outcome is influenced by pancreas preparation method].

PubMed

Pokrywczyńska, Marta; Drewa, Tomasz; Cieślak, Zaneta

2008-09-01

Pancreatic islet transplantation is a treatment method for type I diabetes. Its outcome is influenced by numerous factors, islet quantity and function being important ones of them. was to estimate the influence of pancreas preparation method on the outcome of islet isolation in rat. 6 pancreata harvested from Lewis rats were used in this research. Pancreatic duct was cannulated and pancreas was injected with 1 mg/ml collagenase P solution (Sigma) and then excised. After cutting into smaller fragments, it was digested in collagenase P solution for 15-20 min. Enzyme activity was then stopped by adding dilution medium. Heterogenous cell suspension was centrifuged in density gradient (Gradisol) to isolate islets. Pancreatic islets were collected and islet equivalent was calculated. Islet purity degree was estimated as islet cells to all cells, including exocrine, ratio. Islet viability was estimated using propidium iodide and fluorescein diacetate staining. Photographic documentation was made. Proper islet morphology, highest number and viability was obtained when pancreas was excised properly (isolation 3 and 4). Pancreas preparation method is one of which influences on islet isolation outcome.

Cytotoxicity evaluation of sodium alendronate on cultured human periodontal ligament fibroblasts.

PubMed

Correia, Vera de Fátima Padrão; Caldeira, Celso L; Marques, Márcia Martins

2006-12-01

External root resorption processes are usually associated with dental trauma, mainly avulsion and intrusion. In such cases endodontic therapy aims to prevent this process by using medications that can inhibit osteoclastic activity, such as bisphosphonates. However, these drugs must be biocompatible to the periapical tissues. The aim of this study was to analyze the cytotoxicity of a bisphosphonate (sodium alendronate) on human periodontal ligament fibroblasts (PDL cells). Cells were plated in a density of 1 x 10(3) cells per dish. The experimental groups were GI (control) no sodium alendronate, and GII, GIII, and GIV with sodium alendronate at the concentrations of 10(-5), 10(-6), and 10(-7) M, respectively. The experimental times were 1, 6, 12, and 24 h (short-term) for viability and 2, 4, 6, and 8 days (long-term) for cell survival. Data in triplicate were statistically analyzed. Cultures treated with the highest alendronate concentration (GII) showed cell viability percentages significantly lower (P < 0.01) than those of the other groups (GI, GIII, and GIV), at 12 and 24 h. Cell growth on GII and GIII groups was similar. GII presented smaller growth than the other groups (P < 0.05). We concluded that sodium alendronate, on direct contact with human periodontal ligament fibroblasts, is cytotoxic in concentrations higher than of 10(-6) M.

Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability.

PubMed

Flanagan, Judith Louise; Khandekar, Neeta; Zhu, Hua; Watanabe, Keizo; Markoulli, Maria; Flanagan, John Terence; Papas, Eric

2016-02-01

We sought to determine the relative lipase production of a range of ocular bacterial isolates and to assess the efficacy of glycerol monolaurate (GML) in inhibiting this lipase production in high lipase-producing bacteria without affecting bacterial cell growth. Staphylococcus aureus,Staphylococcus epidermidis,Propionibacterium acnes, and Corynebacterium spp. were inoculated at a density of 10(6)/mL in varying concentrations of GML up to 25 μg/mL for 24 hours at 37 °C with constant shaking. Bacterial suspensions were centrifuged, bacterial cell density was determined, and production of bacterial lipase was quantified using a commercial lipase assay kit. Staphylococcus spp. produced high levels of lipase activity compared with P. acnes and Corynebacterium spp. GML inhibited lipase production by Staphylococcal spp. in a dose-dependent manner, with S. epidermidis lipase production consistently more sensitive to GML than S. aureus. Glycerol monolaurate showed significant (P < 0.05) lipase inhibition above concentrations of 15 μg/mL in S. aureus and was not cytotoxic up to 25 μg/mL. For S. epidermidis, GML showed significant (P < 0.05) lipase inhibition above 7.5 μg/mL. Lipase activity varied between species and between strains. Staphylococcal spp. produced higher lipase activity compared with P. acnes and Corynebacterium spp. Glycerol monolaurate inhibited lipase production by S. aureus and S. epidermidis at concentrations that did not adversely affect bacterial cell growth. GML can be used to inhibit ocular bacterial lipase production without proving detrimental to commensal bacteria viability.

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

PubMed Central

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-01-01

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

PubMed

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-07-07

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.

Increased illumination uniformity and reduced photodamage offered by the Lissajous scanning in fiber-optic two-photon endomicroscopy

NASA Astrophysics Data System (ADS)

Liang, Wenxuan; Murari, Kartikeya; Zhang, Yuying; Chen, Yongping; Li, Ming-Jun; Li, Xingde

2012-02-01

We compare the illumination uniformity and the associated effects of the spiral and Lissajous scanning patterns that are commonly used in an endomicroscope. Theoretical analyses and numerical simulations were first performed to quantitatively investigate the area illumination density in the spiral scanning pattern. The results revealed the potential problem of manifest photodamage due to the very high illumination density in the center of the spiral scan. Similar analyses of the Lissajous scanning pattern, which can be conveniently implemented on the same endomicroscope with no hardware modifications, showed a more uniform illumination density with about an 80-fold reduction in the peak illumination density. To underscore the benefit offered by the improved illumination uniformity, we conducted in vitro two-photon fluorescence imaging of cultured cells stained with a LIVE/DEAD viability assay using our home-built, fiber-optic, two-channel endomicroscopy system. Both the spiral and the Lissajous scans were implemented. Our experimental results showed that cells near the spiral scan center experienced obvious photodamage, whereas cells remained alive over the entire region under the Lissajous beam scanning, confirming the predicted advantage offered by the Lissajous scan over this spiral scan in an endomicroscopy setting.

Biofuel cell operating on activated THP-1 cells: A fuel and substrate study.

PubMed

Javor, Kristina; Tisserant, Jean-Nicolas; Stemmer, Andreas

2017-01-15

It is known that electrochemical energy can be harvested from mammalian cells, more specifically from white blood cells (WBC). This study focuses on an improved biofuel cell operating on phorbol myristate acetate (PMA) activated THP-1 human monocytic cells. Electrochemical investigation showed strong evidence pointing towards hydrogen peroxide being the primary current source, confirming that the current originates from NADPH oxidase activity. Moreover, an adequate substrate for differentiation and activation of THP-1 cells was examined. ITO, gold, platinum and glass were tested and the amount of superoxide anion produced by NADPH oxidase was measured by spectrophotometry through WST-1 reduction at 450nm and used as an indicator of cellular activity and viability. These substrates were subsequently used in a conventional two-compartment biofuel cell where the power density output was recorded. The material showing the highest cell activity compared to the reference cell culture plate and the highest power output was ITO. Under our experimental conditions, a power density of 4.5μW/cm 2 was reached. To the best of our knowledge, this is a threefold higher power output than other leukocyte biofuel cells. Copyright © 2016 Elsevier B.V. All rights reserved.

The potential role of polyphenols in the modulation of skin cell viability by Aspalathus linearis and Cyclopia spp. herbal tea extracts in vitro.

PubMed

Magcwebeba, Tandeka Unathi; Riedel, Sylvia; Swanevelder, Sonja; Swart, Pieter; De Beer, Dalene; Joubert, Elizabeth; Andreas Gelderblom, Wentzel Christoffel

2016-11-01

The relationship between polyphenol constituents, antioxidant properties of aqueous and methanol extracts of green tea (Camellia sinensis), the herbal teas, rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.), against skin cell viability was investigated in vitro. The effect of extracts, characterised in terms of polyphenol content and antioxidant properties, on cell viability of premalignant, normal and malignant skin cells was determined. Phenolic composition, particularly high levels of potent antioxidants, of rooibos and green tea methanol extracts was associated with a strong reduction in cell viability specifically targeting premalignant cells. In contrast, the aqueous extracts of Cyclopia spp. were more effective in reducing cell viability. This correlated with a relatively high flavanol/proanthocyanidin content and ABTS radical cation scavenging capacity. The major green tea flavanol (epigallocatechin gallate) and rooibos dihydrochalcone (aspalathin) exhibited differential effects against cell viability, while the major honeybush xanthone (mangiferin) and flavanone (hesperidin) lacked any effect presumably due to a cytoprotective effect. The underlying mechanisms against skin cell viability are likely to involve mitochondrial dysfunction resulting from polyphenol-iron interactions. The polyphenol constituents and antioxidant parameters of herbal tea extracts are useful tools to predict their activity against skin cell survival in vitro and potential chemopreventive effects in vivo. © 2016 Royal Pharmaceutical Society.

Inkjet printing Schwann cells and neuronal analogue NG108-15 cells.

PubMed

Tse, Christopher; Whiteley, Robert; Yu, Tong; Stringer, Jonathan; MacNeil, Sheila; Haycock, John W; Smith, Patrick J

2016-03-01

Porcine Schwann cells and neuronal analogue NG108-15 cells were printed using a piezoelectric-inkjet-printer with a nozzle diameter of 60 μm, within the range of 70-230 V, with analysis of viability and quality after printing. Neuronal and glial cell viabilities of >86% and >90% were detected immediately after printing and no correlation between voltage applied and cell viability could be seen. Printed neuronal cells were shown to produce neurites earlier compared to controls, and over several days, produced longer neurites which become most evident by day 7. The number of neurites becomes similar by day 7 also, and cells proliferate with a similar viability to that of non-printed cells (controls). This method of inkjet printing cells provides a technical platform for investigating neuron-glial cell interactions with no significant difference to cell viability than standard cell seeding. Such techniques can be utilized for lab-on-a-chip technologies and to create printed neural networks for neuroscience applications.

Long term cryopreservation in 5% DMSO maintains unchanged CD34(+) cells viability and allows satisfactory hematological engraftment after peripheral blood stem cell transplantation.

PubMed

Abbruzzese, L; Agostini, F; Durante, C; Toffola, R T; Rupolo, M; Rossi, F M; Lleshi, A; Zanolin, S; Michieli, M; Mazzucato, M

2013-07-01

Peripheral blood stem cell cryopreservation is associated with cell damage and decreased viability. We evaluated the impact of up to 10 years of cryopreservation (5% DMSO) on viability of CD34(+) cells utilizing graft samples of consecutive patients (2002-2012) with different malignancies who underwent stem cell collection and transplantation. Viability of CD34(+) cells from oncohaematological patients measured after 5 weeks (97·2 ± 0·6%) or after 9-10 years of cryopreservation (95·9 ± 0·5%) was unaffected. Haemoglobin, granulocyte and platelet recovery after transplantation of long-term cryopreserved grafts occurred within 8-13 days. CD34(+) stem cells can be safely stored up to 9-10 years, without affecting cell viability and clinical effectiveness. © 2013 International Society of Blood Transfusion.

Effect of culture density on biomass production and light utilization efficiency of Synechocystis sp. PCC 6803.

PubMed

Straka, Levi; Rittmann, Bruce E

2018-02-01

The viability of large-scale microalgae cultivation depends on providing optimal growth conditions, for which a key operational parameter is culture density. Using Synechocystis sp. PCC 6803, we conducted a series of fixed-density, steady-state experiments and one batch-growth experiment to investigate the role of culture density on biomass production and light utilization efficiency. In all cases, the fixed-density, steady-state experiments and batch-growth experiment showed good agreement. The highest biomass production rates (260 mg L -1  d -1 ) and efficiency for converting light energy to biomass (0.80 μg (μmol photons) -1 ) occurred together at a culture density near 760 mg L -1 , which approximately corresponded to the lowest culture density where almost all incident light was absorbed. The ratio of OD 680 /OD 735 increased with culture density up to the point of maximum productivity, where it plateaued (at a value of 2.4) for higher culture densities. This change in OD 680 /OD 735 indicates a photoacclimation effect that depended on culture density. Very high culture densities led to a sharp decline in efficiency of biomass production per photons absorbed, likely due to a combination of increased decay relative to growth, metabolic changes due to cell-cell interactions, and photodamage due to mixing between regions with high light intensity and zero light intensity. © 2017 Wiley Periodicals, Inc.

High-Density ZnO Nanowires as a Reversible Myogenic-Differentiation Switch.

PubMed

Errico, Vito; Arrabito, Giuseppe; Fornetti, Ersilia; Fuoco, Claudia; Testa, Stefano; Saggio, Giovanni; Rufini, Stefano; Cannata, Stefano; Desideri, Alessandro; Falconi, Christian; Gargioli, Cesare

2018-04-25

Mesoangioblasts are outstanding candidates for stem-cell therapy and are already being explored in clinical trials. However, a crucial challenge in regenerative medicine is the limited availability of undifferentiated myogenic progenitor cells because growth is typically accompanied by differentiation. Here reversible myogenic-differentiation switching during proliferation is achieved by functionalizing the glass substrate with high-density ZnO nanowires (NWs). Specifically, mesoangioblasts grown on ZnO NWs present a spherical viable undifferentiated cell state without lamellopodia formation during the entire observation time (8 days). Consistently, the myosin heavy chain, typically expressed in skeletal muscle tissue and differentiated myogenic progenitors, is completely absent. Remarkably, NWs do not induce any damage while they reversibly block differentiation, so that the differentiation capabilities are completely recovered upon cell removal from the NW-functionalized substrate and replating on standard culture glass. This is the first evidence of a reversible myogenic-differentiation switch that does not affect the viability. These results can be the first step toward for the in vitro growth of a large number of undifferentiated stem/progenitor cells and therefore can represent a breakthrough for cell-based therapy and tissue engineering.

An approach for cell viability online detection based on the characteristics of lensfree cell diffraction fingerprint.

PubMed

Li, Guoxiao; Zhang, Rongbiao; Yang, Ning; Yin, Changsheng; Wei, Mingji; Zhang, Yecheng; Sun, Jian

2018-06-01

To overcome the drawbacks such as low automation and high cost, an approach for cell viability online detection is proposed, based on the extracted lensfree cell diffraction fingerprint characteristics. The cell fingerprints are acquired by a constructed large field-of-view (FOV) diffraction imaging platform without any lenses. The approach realizes distinguishing live and dead cells online and calculating cell viability index based on the number of live cells. With theoretical analysis and simulation, diffraction fingerprints of cells with different morphology are simulated and two characteristics are discovered to be able to reflect cell viability status effectively. Two parameters, fringe intensity contrast (FIC) and fringe dispersion (FD), are defined to quantify these two characteristics. They are verified to be reliable to identify live cells. In a cytotoxicity assay of different methyl mercury concentration on BRL cells, the proposed approach is used to detect cell viability. MTT method is also employed and the results of correlational analysis and Bland-Altman analysis prove the validity of the proposed approach. By comparison, it can be revealed that the proposed approach has some advantages over other present techniques. Therefore it may be widely used as a cell viability measurement method in drug screening, nutritional investigation and cell toxicology studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Cell viability monitoring using Fano resonance in gold nanoslit array

NASA Astrophysics Data System (ADS)

Wu, Shu-Han; Hsieh, Shu-Yi; Lee, Kuang-Li; Weng, Ruei-Hung; Chiou, Arthur; Wei, Pei-Kuen

2013-09-01

Cell viability is a crucial issue in biological research. We present label-free monitoring of adhesion cells viability by gold nanoslits-based Fano resonance biosensors. Plastic multiple wells with gold nanoslits substrate were made using a thermal nanoimprint method. Adhesion cells in the wells were treated with doxorubicin for inducing cell death and compared with conventional colorimetric assay. The nanoslits method shows better respones of viability tests under low concentration and short interaction time due to its high surface sensitivies. The vinculin labelling indicates that the measured signals are in good agreement with the adhesion abilities of cells.

Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation.

PubMed

Gamalier, Juliana P; Silva, Thiago P; Zarantonello, Victor; Dias, Felipe F; Melo, Rossana C N

2017-01-01

Secretion of membrane vesicles is an important biological process of both eukaryotic and prokaryotic cells. This process has been characterized in pathogenic bacteria, but is less clear in non-pathogenic bacteria from aquatic ecosystems. Here, we investigated, for the first time, the process of formation of outer membranes vesicles (OMVs), nanoscale vesicles extruded from the outer membrane (OM) of gram-negative bacteria, in cultures of freshwater bacteria after exposure or not to ultraviolet radiation (UVR) as an environmental stressor. Non-axenic cultures of freshwater bacteria isolated from a Brazilian aquatic ecosystem (Funil reservoir) were exposed or not to UVR (UVA+UVB) over a 3h period, during which cell density, viability and ultrastructure were analyzed. First, we showed that UVR induce bacterial death. UVR triggered significant negative effect on cell density after 3h of UVR treatment. This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe that enables the distinction of live/dead bacteria. Transmission electron microscopy (TEM) revealed changes indicative of cell death after 3h of UVR exposure, with significant increase of damaged cells compared to the control group. Second, we demonstrated that gram-negative bacteria release OMVs during normal growth and after UVR exposure. OMVs were clearly identified as round, membrane-bound vesicles budding off from the bacterial OM as isolated or clustered vesicles or free in the extracellular medium. Remarkably, quantitative TEM analyses showed that bacteria respond to UVR with increased formation of OMVs. Moreover, while OMVs numbers per intact or damaged cell did not differ in the untreated group, UVR led to a higher vesiculation by bacteria in process of death. This means that degenerating bacteria release OMVs before lysis and that this secretion might be an adaptive/protective response to rapid changes in environmental conditions such as UV radiation. Copyright © 2016 Elsevier GmbH. All rights reserved.

The role of adrenergic activation on murine luteal cell viability and progesterone production.

PubMed

Wang, Jing; Tang, Min; Jiang, Huaide; Wu, Bing; Cai, Wei; Hu, Chuan; Bao, Riqiang; Dong, Qiming; Xiao, Li; Li, Gang; Zhang, Chunping

2016-09-15

Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO. Copyright © 2016 Elsevier Inc. All rights reserved.

Induction of oxidative stress, DNA damage, and apoptosis in a malignant human skin melanoma cell line after exposure to zinc oxide nanoparticles

PubMed Central

Alarifi, Saud; Ali, Daoud; Alkahtani, Saad; Verma, Ankit; Ahamed, Maqusood; Ahmed, Mukhtar; Alhadlaq, Hisham A

2013-01-01

The widespread use of zinc oxide (ZnO) nanoparticles worldwide exposes humans to their adverse effects, so it is important to understand their biological effects and any associated risks. This study was designed to investigate the cytotoxicity, oxidative stress, and apoptosis caused by ZnO nanoparticles in human skin melanoma (A375) cells. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] and lactate dehydrogenase-based cell viability assays showed a significant decrease in cell viability after exposure to ZnO nanoparticles, and phase contrast images revealed that cells treated with these nanoparticles had a lower density and a rounded morphology. ZnO nanoparticles were also found to induce oxidative stress, evidenced by generation of reactive oxygen species and depletion of the antioxidant, glutathione. Induction of apoptosis was confirmed by chromosomal condensation assay and caspase-3 activation. Further, more DNA damage was observed in cells exposed to the highest concentration of ZnO nanoparticles. These results demonstrate that ZnO nanoparticles have genotoxic potential in A375 cells, which may be mediated via oxidative stress. Our short-term exposure study showing induction of a genotoxic and apoptotic response to ZnO nanoparticles needs further investigation to determine whether there may be consequences of long-term exposure to ZnO nanoparticles. PMID:23493450

High-efficient and high-content cytotoxic recording via dynamic and continuous cell-based impedance biosensor technology.

PubMed

Hu, Ning; Fang, Jiaru; Zou, Ling; Wan, Hao; Pan, Yuxiang; Su, Kaiqi; Zhang, Xi; Wang, Ping

2016-10-01

Cell-based bioassays were effective method to assess the compound toxicity by cell viability, and the traditional label-based methods missed much information of cell growth due to endpoint detection, while the higher throughputs were demanded to obtain dynamic information. Cell-based biosensor methods can dynamically and continuously monitor with cell viability, however, the dynamic information was often ignored or seldom utilized in the toxin and drug assessment. Here, we reported a high-efficient and high-content cytotoxic recording method via dynamic and continuous cell-based impedance biosensor technology. The dynamic cell viability, inhibition ratio and growth rate were derived from the dynamic response curves from the cell-based impedance biosensor. The results showed that the biosensors has the dose-dependent manners to diarrhetic shellfish toxin, okadiac acid based on the analysis of the dynamic cell viability and cell growth status. Moreover, the throughputs of dynamic cytotoxicity were compared between cell-based biosensor methods and label-based endpoint methods. This cell-based impedance biosensor can provide a flexible, cost and label-efficient platform of cell viability assessment in the shellfish toxin screening fields.

Effect of sodium hypochlorite on human pulp cells: an in vitro study

PubMed Central

Essner, Mark D.; Javed, Amjad; Eleazer, Paul D.

2014-01-01

Background The purpose of this study was to determine the effect of sodium hypochlorite (NaOCl) on human pulp cells to provide an aid in determining its optimum concentration in maintaining the viability of remaining pulp cells in the revascularization of immature permanent teeth with apical periodontitis. Study design Human pulp tissue cells taken from extracted third molars were plated, incubated, and subjected to various concentrations of NaOCl (0.33%, 0.16%, 0.08%, and 0.04%) for 5-, 10-, and 15-minute time intervals to simulate possible contact times in vivo. The Cell Titer–Glo Luminescent Cell Viability Assay was used to determine the number of viable cells present in culture following treatment. Results The results showed an increase in cell viability with the lowering of NaOCl concentration. The use of 0.04% NaOCl was similar to the control, indicating nearly complete preservation of cell viability at all time intervals tested. As sodium hypochlorite concentration increased from 0.04% to 0.33%, cell viability decreased correspondingly. Conclusions The results indicate that the lowest concentration of NaOCl tested did not affect the viability of cells. This may prove beneficial in developing a new treatment protocol to help preserve existing vital pulp cells in revascularization cases. PMID:21821446

Early effects of extracorporeal shock wave treatment on osteoblast-like cells: a comparative study between electromagnetic and electrohydraulic devices.

PubMed

Martini, Lucia; Giavaresi, Gianluca; Fini, Milena; Borsari, Veronica; Torricelli, Paola; Giardino, Roberto

2006-11-01

Extracorporeal shockwave therapy (ESWT) has been increasingly applied to treat orthopedic and musculoskeletal pathologies. ESWT involves mechanical perturbations that, as with other physical therapies, can result in mechanical stimuli to a large number of cells, including bone cells. The aim of this study was to evaluate the effects of shock waves on osteoblast-like cells (MG63) when using two different generators of shock waves (electrohydraulic and electromagnetic devices), in terms of cell damage, cell viability, osteogenic phenotype expression, and cytokine production. MG63 cells were suspended in 1.5 mL screw-cap cryotubes (1 x 10 cells/mL), containing phosphate buffer solution (PBS), which were maintained at 37 degrees C during all the experimental times. Two levels of energy flux density (EFD) were evaluated for each device: 0.15 to 0.18 mJ/mm2 and 0.40 mJ/mm2. Cells were then cultivated for 72 hours starting from a concentration of 1 x 10 cells/mL, and biological activity and viability were evaluated 24 and 72 hours after treatment. The results obtained demonstrate that the factors most affecting osteoblast activity involve both the device and the level of EFD selected, and they must be considered all together. The use of the electromagnetic device and a level of EFD lower than 0.40 mJ/mm2 would appear to induce fewer immediate cytodestructive effects and better stimulate subsequent proliferation and the synthetic activity of MG63.

Antimicrobial and cell viability measurement of bovine serum albumin capped silver nanoparticles (Ag/BSA) loaded collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film.

PubMed

Bakare, Rotimi; Hawthrone, Samantha; Vails, Carmen; Gugssa, Ayele; Karim, Alamgir; Stubbs, John; Raghavan, Dharmaraj

2016-03-01

Bacterial infection of orthopedic devices has been a major concern in joint replacement procedures. Therefore, this study is aimed at formulating collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film loaded with bovine serum albumin capped silver nanoparticles (Ag/BSA NPs) to inhibit bacterial growth while retaining/promoting osteoblast cells viability. The nanoparticles loaded collagen immobilized PHBV film was characterized for its composition by X-ray Photoelectron Spectroscopy and Anodic Stripping Voltammetry. The extent of loading of Ag/BSA NPs on collagen immobilized PHBV film was found to depend on the chemistry of the functionalized PHBV film and the concentration of Ag/BSA NPs solution used for loading nanoparticles. Our results showed that more Ag/BSA NPs were loaded on higher molecular weight collagen immobilized PHEMA-g-PHBV film. Maximum loading of Ag/BSA NPs on collagen immobilized PHBV film was observed when 16ppm solution was used for adsorption studies. Colony forming unit and optical density measurements showed broad antimicrobial activity towards Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa at significantly lower concentration i.e., 0.19 and 0.31μg/disc, compared to gentamicin and sulfamethoxazole trimethoprim while MTT assay showed that released nanoparticles from Ag/BSA NPs loaded collagen immobilized PHBV film has no impact on MCTC3-E1 cells viability. Copyright © 2015 Elsevier Inc. All rights reserved.

Knockdown of long noncoding RNA XIST alleviates oxidative low-density lipoprotein-mediated endothelial cells injury through modulation of miR-320/NOD2 axis.

PubMed

Xu, Xiaohui; Ma, Congmin; Liu, Chao; Duan, Zhihui; Zhang, Li

2018-06-14

Atherosclerosis remains to be one of the most common vascular disorders resulting in morbidity and mortality in the world. Recent studies suggested that endothelial cells (ECs) injury caused by oxidative low-density lipoprotein (ox-LDL) is an early marker for atherosclerosis. Nevertheless, the mechanisms of ox-LDL-induced ECs injury are complicated and largely unknown. Here, we found lncRNA XIST (X-inactive specific transcript) was upregulated in human umbilical vein endothelial cells (HUVECs) stimulated by ox-LDL. Knockdown of XIST boosted the cell viability and suppressed cell apoptosis under ox-LDL stimuli. Further experiments identified XIST regulated the expression of Nucleotide-Binding Oligomerization Domain 2 (NOD2) by sponging miR-320. XIST silencing exerted a protective effect on ox-LDL-induced HUVECs injury via miR-320/NOD2 regulatory network. Our data provide insight into the role of the lncRNA XIST in ox-LDL mediated ECs injury, which can aid in developing new therapeutic strategies for the treatment of atherosclerosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

PubMed

Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

2015-02-15

We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers. Copyright © 2014 Elsevier B.V. All rights reserved.

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

PubMed

Sediq, A S; Klem, R; Nejadnik, M R; Meij, P; Jiskoot, Wim

2018-05-30

To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control.

PubMed

Albrecht, Simone; Kaisermayer, Christian; Reinhart, David; Ambrose, Monica; Kunert, Renate; Lindeberg, Anna; Bones, Jonathan

2018-05-01

The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MS E was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 10 6  dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control. Graphical abstract Overview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death.

The effect of simultaneous exposure of HEMn-DP and HEMn-LP melanocytes to nicotine and UV-radiation on the cell viability and melanogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delijewski, Marcin; Wrześniok, Dorota; Beberok, Ar

Nicotine is a main compound of tobacco plants and may affect more than a billion people all over the world that are permanently exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. It is known that nicotine forms complexes with melanin what may lead to accumulation of this alkaloid in tissues of living organisms containing the pigment. This may affect the viability of cells and process of melanin biosynthesis that takes place in melanocytes. Although UV radiation is known to be a particular inductor of melanin biosynthesis, its simultaneous effect with nicotine onmore » this process as well as the viability of human cells containing melanin have not been assessed so far. The aim of this study was to examine the simultaneous impact of nicotine and UV radiation on viability and melanogenesis in cultured normal human melanocytes dark (HEMn-DP) and light (HEMn-LP) pigmented. Nicotine together with UV radiation induced concentration-dependent loss in melanocytes viability. The higher cell loss was observed in dark pigmented melanocytes in comparison to light pigmented cells. Simultaneous exposure of cells to nicotine and UV radiation also caused changes in melanization process in both tested cell lines. The data suggest that simultaneous exposure of melanocytes to nicotine and UV radiation up-regulates melanogenesis and affects cell viability. Observed processes are more pronounced in dark pigmented cells. - Highlights: • Nicotine and UVA induced concentration-dependent loss in melanocytes viability. • Nicotine and UVA modulated melanization process in melanocytes. • Changes in viability and melanization were more pronounced in dark pigmented cells.« less

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

PubMed

Wigg, Alan J; Phillips, John W; Wheatland, Loretta; Berry, Michael N

2003-06-01

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Effects of surface finishing conditions on the biocompatibility of a nickel-chromium dental casting alloy.

PubMed

McGinley, Emma Louise; Coleman, David C; Moran, Gary P; Fleming, Garry J P

2011-07-01

To assess the effects of surface finishing condition (polished or alumina particle air abraded) on the biocompatibility of direct and indirect exposure to a nickel-chromium (Ni-Cr) d.Sign®10 dental casting alloy on oral keratinocytes. Biocompatibility was performed by assessing cellular viability and morphology, metabolic activity, cellular toxicity and presence of inflammatory cytokine markers. Discs of d.Sign®10 were cast, alumina particle air abraded and half were polished before surface roughness was determined by profilometry. Biocompatibility was assessed by placing the discs directly or indirectly (with immersion solutions) into contact with TR146 monolayers. Metal ion release was determined by ICP-MS. Cell viability was assessed by trypan blue dye exclusion, metabolic activity by XTT and cellular toxicity by LDH. Inflammatory cytokine analysis was performed using sandwich ELISAs. The mean polished Ra value was significantly reduced (P<0.001) compared with the alumina particle air abraded discs but metal ion release was significantly increased for the polished discs. Significant reductions in cell density of polished compared with alumina particle air abraded discs was observed following direct or indirect exposure. A significant reduction in metabolic activity, increase in cellular toxicity and an increase in the presence of inflammatory cytokine markers was highlighted for the polished relative to the alumina particle air abraded discs at 24h. Finishing condition of the Ni-Cr dental alloy investigated has important clinical implications. The approach of employing cell density and morphology, metabolic activity, cellular toxicity levels and inflammatory marker responses to TR146 epithelial cells combined with ICP-MS afforded the authors an increased insight into the complex processes dental alloys undergo in the oral environment. Copyright © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Coconut milk and probiotic milk as storage media to maintain periodontal ligament cell viability: an in vitro study.

PubMed

Saini, Divya; Gadicherla, Prahlad; Chandra, Prakash; Anandakrishna, Latha

2017-06-01

The viability of periodontal ligament (PDL) cells is a significant determinant of the long-term prognosis of replanted avulsed teeth. A storage medium is often required to maintain the viability of these cells during the extra-alveolar period. Many studies have been carried out to search for the most suitable storage medium for avulsed teeth, but an ideal solution has not yet been found. The purpose of the study was to compare and analyze the ability of coconut milk and probiotic milk to maintain PDL cell viability. In an in vitro setting, 69 caries free human premolars with normal periodontium that had been extracted for orthodontic purposes were randomly divided into two experimental groups on the basis of storage media used (i.e., coconut milk or probiotic milk) and a Hanks' balanced salt solution (HBSS) control group (23 samples per group). Immediately after extraction, the teeth were stored dry for 20 min and then immersed for 30 min in one of the storage media. The teeth were then subjected to collagenase-dispase assay and labeled with 0.5% trypan blue staining solution for determination of cell viability. The number of viable cells was counted under a light microscope and statistically analyzed using anova and post hoc Tukey test (P ≤ 0.05). Statistical analysis demonstrated there was a significant difference (P < 0.001) between coconut milk and probiotic milk as well as HBSS in maintaining cell viability. However, there was no significant difference between probiotic milk and HBSS in ability to maintain PDL cell viability (P > 0.05). Coconut milk may not be suitable as an interim transport media due to poor maintenance of cell viability. However, probiotic milk was able to maintain PDL cell viability as well as HBSS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Biofunctionalized Lysophosphatidic Acid/Silk Fibroin Film for Cornea Endothelial Cell Regeneration

PubMed Central

Jeon, Hayan; Oliveira, Joaquim Miguel; Reis, Rui Luis; Khang, Gilson

2018-01-01

Cornea endothelial cells (CEnCs) tissue engineering is a great challenge to repair diseased or damaged CEnCs and require an appropriate biomaterial to support cell proliferation and differentiation. Biomaterials for CEnCs tissue engineering require biocompatibility, tunable biodegradability, transparency, and suitable mechanical properties. Silk fibroin-based film (SF) is known to meet these factors, but construction of functionalized graft for bioengineering of cornea is still a challenge. Herein, lysophosphatidic acid (LPA) is used to maintain and increase the specific function of CEnCs. The LPA and SF composite film (LPA/SF) was fabricated in this study. Mechanical properties and in vitro studies were performed using a rabbit model to demonstrate the characters of LPA/SF. ATR-FTIR was characterized to identify chemical composition of the films. The morphological and physical properties were performed by SEM, AFM, transparency, and contact angle. Initial cell density and MTT were performed for adhesion and cell viability in the SF and LPA/SF film. Reverse transcription polymerase chain reactions (RT-PCR) and immunofluorescence were performed to examine gene and protein expression. The results showed that films were designed appropriately for CEnCs delivery. Compared to pristine SF, LPA/SF showed higher biocompatibility, cell viability, and expression of CEnCs specific genes and proteins. These indicate that LPA/SF, a new biomaterial, offers potential benefits for CEnCs tissue engineering for regeneration. PMID:29710848

Viability of human fibroblasts in coconut water as a storage medium.

PubMed

Moreira-Neto, J J S; Gondim, J O; Raddi, M S G; Pansani, C A

2009-09-01

To evaluate the effectiveness of a new storage medium for avulsed teeth, coconut water, in maintaining the viability of human fibroblasts. Cell viability after different time periods was evaluated in the following storage media: coconut water, coconut water with sodium bicarbonate, milk, saline and still mineral water. Human fibroblasts were seeded in Eagle's minimal essential medium (EMEM) supplemented with 7.5% foetal calf serum. After trypsinisation, 100 microL of culture medium containing approximately 10(4) cells mL(-1) were collected and pipetted into the wells of 96-well plates, which were incubated overnight in 5% CO(2) and 95% air mixture at 37 degrees C. EMEM was then replaced by the storage media and the plates were incubated at 37 degrees C for 1, 2 and 4 h. Cell viability was determined using the neutral red assay. The proportions of viable cells after exposure to the storage media were analysed statistically by anova and the least significant difference (LSD) test (alpha = 5%). Milk had the greatest capacity to maintain cell viability (P < 0.05), followed by coconut water with sodium bicarbonate and saline. Coconut water was significantly worse at maintaining cell viability compared to milk, coconut water with sodium bicarbonate and saline. The smallest number of viable cells was observed for mineral water (P < 0.05). Coconut water was worse than milk in maintaining human fibroblast cell viability.

Dental pulp stem cell-derived chondrogenic cells demonstrate differential cell motility in type I and type II collagen hydrogels.

PubMed

Yao, Li; Flynn, Nikol

2018-06-01

Advances in the development of biomaterials and stem cell therapy provide a promising approach to regenerating degenerated discs. The normal nucleus pulposus (NP) cells exhibit similar phenotype to chondrocytes. Because dental pulp stem cells (DPSCs) can be differentiated into chondrogenic cells, the DPSCs and DPSCs-derived chondrogenic cells encapsulated in type I and type II collagen hydrogels can potentially be transplanted into degenerated NP to repair damaged tissue. The motility of transplanted cells is critical because the cells need to migrate away from the hydrogels containing the cells of high density and disperse through the NP tissue after implantation. The purpose of this study was to determine the motility of DPSC and DPSC-derived chondrogenic cells in type I and type II collagen hydrogels. The time lapse imaging that recorded cell migration was analyzed to quantify the cell migration velocity and distance. The cell viability of DPSCs in native or poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG)-crosslinked type I and type II collagen hydrogels was determined using LIVE/DEAD cell viability assay and AlamarBlue assay. DPSCs were differentiated into chondrogenic cells. The migration of DPSCs and DPSC-derived chondrogenic cells in these hydrogels was recorded using a time lapse imaging system. This study was funded by the Regional Institute on Aging and Wichita Medical Research and Education Foundation, and the authors declare no competing interest. DPSCs showed high cell viability in non-crosslinked and crosslinked collagen hydrogels. DPSCs migrated in collagen hydrogels, and the cell migration speed was not significantly different in either type I collagen or type II collagen hydrogels. The migration speed of DPSC-derived chondrogenic cells was higher in type I collagen hydrogel than in type II collagen hydrogel. Crosslinking of type I collagen with 4S-StarPEG significantly reduced the cell migration speed of DPSC-derived chondrogenic cells. After implantation of collagen hydrogels encapsulating DPSCs or DPSC-derived chondrogenic cells, the cells can potentially migrate from the hydrogels and migrate into the NP tissue. This study also explored the differential cell motility of DPSCs and DPSC-derived chondrogenic cells in these collagen hydrogels. Copyright © 2018 Elsevier Inc. All rights reserved.

BID is a critical factor controlling cell viability regulated by IFN-α.

PubMed

Tsuno, Takaya; Mejido, Josef; Zhao, Tongmao; Phillips, Terry; Myers, Timothy G; Bekisz, Joseph; Zoon, Kathryn C

2012-01-01

Clinical applications of human interferon (IFN)-α have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-α have not been clearly identified. Our previous study indicated that IFN (α, β, and ω) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-α treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-α. In this study, IFNAR1/2- and IFN regulatory factor 9-RNAi inhibited the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not of Fas ligand, after IFN-α treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-α notably upregulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. After TRAIL signaling, caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-α and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-α and TRAIL from collapsing the mitochondrial membrane potential (ΔΨm). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-α or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-α. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-α which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-α resistant tumors.

Tunable Volumetric Density and Porous Structure of Spherical Poly-ε-caprolactone Microcarriers, as Applied in Human Mesenchymal Stem Cell Expansion.

PubMed

Li, Jian; Lam, Alan Tin-Lun; Toh, Jessica Pei Wen; Reuveny, Shaul; Oh, Steve Kah-Weng; Birch, William R

2017-03-28

Polymeric microspheres may serve as microcarrier (MC) matrices, for the expansion of anchorage-dependent stem cells. They require surface properties that promote both initial cell adhesion and the subsequent spreading of cells, which is a prerequisite for successful expansion. When implemented in a three-dimensional culture environment, under agitation, their suspension under low shear rates depends on the MCs having a modest negative buoyancy, with a density of 1.02-1.05 g/cm 3 . Bioresorbable poly-ε-caprolactone (PCL), with a density of 1.14 g/cm 3 , requires a reduction in volumetric density, for the microspheres to achieve high cell viability and yields. Uniform-sized droplets, from solutions of PCL dissolved in dichloromethane (DCM), were generated by coaxial microfluidic geometry. Subsequent exposure to ethanol rapidly extracted the DCM solvent, solidifying the droplets and yielding monodisperse microspheres with a porous structure, which was demonstrated to have tunable porosity and a hollow inner core. The variation in process parameters, including the molecular weight of PCL, its concentration in DCM, and the ethanol concentration, served to effectively alter the diffusion flux between ethanol and DCM, resulting in a broad spectrum of volumetric densities of 1.04-1.11 g/cm 3 . The solidified microspheres are generally covered by a smooth thin skin, which provides a uniform cell culture surface and masks their internal porous structure. When coated with a cationic polyelectrolyte and extracellular matrix protein, monodisperse microspheres with a diameter of approximately 150 μm and densities ranging from 1.05-1.11 g/cm 3 are capable of supporting the expansion of human mesenchymal stem cells (hMSCs). Validation of hMSC expansion was carried out with a positive control of commercial Cytodex 3 MCs and a negative control of uncoated low-density PCL MCs. Static culture conditions generated more than 70% cell attachment and similar yields of sixfold cell expansion on all coated MCs, with poor cell attachment and growth on the negative control. Under agitation, coated porous microspheres, with a low density of 1.05 g/cm 3 , achieved robust cell attachment and resulted in high cell yields of ninefold cell expansion, comparable with those generated by commercial Cytodex 3 MCs.

Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

PubMed

Ouyang, Liliang; Yao, Rui; Zhao, Yu; Sun, Wei

2016-09-16

3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and acceptable viability ranges, a combined parameters region was obtained. This study provides guidance for parameter optimization and the fine-tuning of 3D cell printing processes regarding both bioink printability and cell viability after bioplotting, especially for easily damaged cells, like ESCs.

Oxygen consumption rate of cells in 3D culture: the use of experiment and simulation to measure kinetic parameters and optimise culture conditions.

PubMed

Streeter, Ian; Cheema, Umber

2011-10-07

Understanding the basal O(2) and nutrient requirements of cells is paramount when culturing cells in 3D tissue models. Any scaffold design will need to take such parameters into consideration, especially as the addition of cells introduces gradients of consumption of such molecules from the surface to the core of scaffolds. We have cultured two cell types in 3D native collagen type I scaffolds, and measured the O(2) tension at specific locations within the scaffold. By changing the density of cells, we have established O(2) consumption gradients within these scaffolds and using mathematical modeling have derived rates of consumption for O(2). For human dermal fibroblasts the average rate constant was 1.19 × 10(-17) mol cell(-1) s(-1), and for human bone marrow derived stromal cells the average rate constant was 7.91 × 10(-18) mol cell(-1) s(-1). These values are lower than previously published rates for similar cells cultured in 2D, but the values established in this current study are more representative of rates of consumption measured in vivo. These values will dictate 3D culture parameters, including maximum cell-seeding density and maximum size of the constructs, for long-term viability of tissue models.

The newly identified K+ channel blocker talatisamine attenuates beta-amyloid oligomers induced neurotoxicity in cultured cortical neurons.

PubMed

Wang, Yanxia; Song, Mingke; Hou, Lina; Yu, Zhihua; Chen, Hongzhuan

2012-06-19

Loss of cytosolic K(+) through up-regulated delayed rectifier K(+) channels play an important role in beta-amyloid (Aβ) induced neurotoxicity. Potent K(+) channel blocker, particular specific for I(K) channels has been suggested as an attractive candidate for the treatment of Alzheimer's disease (AD). Talatisamine is a novel I(K) channel blocker discovered by virtual screening and electrophysiological characterization. In the present study, we examined the neuroprotective effect of talatisamine against Aβ oligomers induced cytotoxicity in primarily cultured cortical neurons. The neurotoxicity related to K(+) loss caused by Aβ40 oligomers included enhanced I(K) density, increased cell membrane permeability, reduced cell viability, and impaired mitochondrial transmembrane potential. Decreased Bcl-2 and increased Bax level, activation of Caspase-3 and Caspase-9 were also observed after Aβ40 oligomers incubation. Talatisamine (120 μM) and TEA (5mM) inhibited the enhanced I(K) caused by Aβ40 oligomers, attenuated cytotoxicity of Aβ oligomers by restoring cell viability and suppressing K(+) loss related apoptotic response. Our results suggested that talatisamine may become a leading compound as I(K) channel blocker for neuroprotection. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effect of the oncolytic ECHO-7 virus Rigvir® on the viability of cell lines of human origin in vitro.

PubMed

Tilgase, Andra; Patetko, Liene; Blāķe, Ilze; Ramata-Stunda, Anna; Borodušķis, Mārtiņš; Alberts, Pēteris

2018-01-01

Background: The role of oncolytic viruses in cancer treatment is increasingly studied. The first oncolytic virus (Rigvir®, ECHO-7) was registered in Latvia over a decade ago. In a recent retrospective study Rigvir® decreased mortality 4.39-6.57-fold in stage IB-IIC melanoma patients. The aims of the present study are to test the effect of Rigvir® on cell line viability in vitro and to visualize the cellular presence of Rigvir® by immunocytochemistry. Methods: The cytolytic effect of Rigvir® on the viability of FM-9, RD, AGS, A549, HDFa, HPAF‑II, MSC, MCF7, HaCaT, and Sk-Mel-28 cell lines was measured using live cell imaging. PBMC viability was measured using flow cytometry. The presence of ECHO-7 virus was visualized using immunocytochemistry. Statistical difference between treatment groups was calculated using two-way ANOVA. Results: Rigvir® (10%, volume/volume) reduced cell viability in FM-9, RD, AGS, A549, HDFa, HPAF‑II and MSC cell lines by 67-100%. HaCaT cell viability was partly affected while Rigvir® had no effect on MCF7, Sk-Mel-28 and PBMC viability. Detection of ECHO-7 by immunocytochemistry in FM-9, RD, AGS, A549, HDFa, HPAF-II and Sk-Mel-28 cell lines suggests that the presence of Rigvir® in the cells preceded or coincided with the time of reduction of cell viability. Rigvir® (10%) had no effect on live PBMC count. Conclusions: The results suggest that Rigvir® in vitro reduces the viability of cells of human melanoma, rhabdomyosarcoma, gastric adenocarcinoma, lung carcinoma, pancreas adenocarcinoma but not in PBMC. The presence of Rigvir® in the sensitive cells was confirmed using anti-ECHO-7 antibodies. The present results suggest that a mechanism of action for the clinical benefit of Rigvir® is its cytolytic properties. The present results suggest that the effect of Rigvir® could be tested in other cancers besides melanoma. Further studies of possible Rigvir® entry receptors are needed.

Chondrogenesis of Human Bone Marrow Mesenchymal Stem Cells in 3-Dimensional, Photocrosslinked Hydrogel Constructs: Effect of Cell Seeding Density and Material Stiffness

PubMed Central

Sun, Aaron X.; Lin, Hang; Fritch, Madalyn R.; Shen, He; Alexander, Pete G.; DeHart, Michael; Tuan, Rocky S.

2018-01-01

Three-dimensional hydrogel constructs incorporated with live stem cells that support chondrogenic differentiation and maintenance offer a promising regenerative route towards addressing the limited self-repair capabilities of articular cartilage. In particular, hydrogel scaffolds that augment chondrogenesis and recapitulate the native physical properties of cartilage, such as compressive strength, can potentially be applied in point-of-care procedures. We report here the synthesis of two new materials, [poly-L-lactic acid/polyethylene glycol/poly-L-lactic acid] (PLLA-PEG 1000) and [poly-D,L-lactic acid/polyethylene glycol/poly-D,L-lactic acid] (PDLLA-PEG 1000), that are biodegradable, biocompatible (>80% viability post fabrication), and possess high, physiologically relevant mechanical strength (~1,500 to 1,800 kPa). This study examined the effects of physiologically relevant cell densities (4, 8, 20, and 50 × 106/mL) and hydrogel stiffnesses (~150kPa to ~1,500 kPa Young’s moduli) on chondrogenesis of human bone marrow stem cells incorporated in hydrogel constructs fabricated with these materials and a previously characterized PDLLA-PEG 4000. Results showed that 20 × 106 cells/mL, under a static culture condition, was the most efficient cell seeding density for extracellular matrix (ECM) production on the basis of hydroxyproline and glycosaminoglycan content. Interestingly, material stiffness did not significantly affect chondrogenesis, but rather material concentration was correlated to chondrogenesis with increasing levels at lower concentrations based on ECM production, chondrogenic gene expression, and histological analysis. These findings establish optimal cell densities for chondrogenesis within three-dimensional cell-incorporated hydrogels, inform hydrogel material development for cartilage tissue engineering, and demonstrate the efficacy and potential utility of PDLLA-PEG 1000 for point-of-care treatment of cartilage defects. PMID:28611002

Kinetics of killing Listeria monocytogenes by macrophages: rapid killing accompanying phagocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davies, W.A.

1983-08-01

The kinetics of bactericidal activity of activated macrophages can be precisely described by a mathematical model in which phagocytosis, killing, digestion, and release of degraded bacterial material are considered to occur continuously. To gain a better understanding of these events, I have determined the period of time between first contact of bacteria with macrophages and the onset of killing. Activated rat peritoneal macrophages were incubated for various times up to 15 min with Listeria monocytogenes previously labeled with /sup 3/H-thymidine and the unassociated bacteria removed by two centrifugations through a density interface. Both cell-associated radioactivity and cell-associated viable bacteria, determinedmore » as colony forming units after sonication of the cell pellet, increased with time of incubation. However, the specific viability of these bacteria, expressed as the ratio of number of viable bacteria per unit radioactivity declined with time, as an approximate inverse exponential, after a lag period of 2.9 +/- 0.8 min. Evidence is given that other possible causes for this decline in specific viability, other than death of the bacteria, such as preferential ingestion of dead Listeria, clumping of bacteria, variations in autolytic activity, or release of Listericidins are unlikely. I conclude therefore that activated macrophages kill Listeria approximately 3 min after the cell and the bacterium first make contact.« less

In vitro evaluation of corrosion and cytotoxicity of orthodontic brackets.

PubMed

Costa, M T; Lenza, M A; Gosch, C S; Costa, I; Ribeiro-Dias, F

2007-05-01

The corrosion resistance of AISI 304 stainless steel (AISI 304 SS) and manganese stainless steel (low-nickel SS) brackets in artificial saliva was investigated. The cytotoxic effects of their corrosion products on L929 cell culture were compared by two assays, crystal violet, to evaluate cell viability, and MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide), for cell metabolism and proliferation. The atomic absorption spectroscopic analysis of the corrosion products demonstrated that nickel and manganese ion concentrations were higher for the AISI 304 SS-bracket immersion solution as compared with the low-nickel SS brackets. Scanning electron microscopy and energy-dispersive spectroscopy demonstrated less corrosion resistance for the AISI 304 SS brackets. Although none of the bracket extracts altered L929 cell viability or morphology, the AISI 304 SS-bracket extracts decreased cellular metabolism slightly. The results indicated that the low-nickel SS presents better in vitro biocompatibility than AISI 304 SS brackets. Abbreviations used: AISI, American Iron and Steel Institute; EDS, energy-dispersive spectroscopy; OD, optical density; ISO, International Organization for Standardization; MTT, (3-{4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NiSO(4), nickel sulfate; SEM, standard error of the mean; WHO, World Health Organization; and TNF, tumor necrosis factor.

Laser and Non-Coherent Light Effect on Peripheral Blood Normal and Acute Lymphoblastic Leukemic Cells by Using Different Types of Photosensitizers

NASA Astrophysics Data System (ADS)

El Batanouny, Mohamed H.; Khorshid, Amira M.; Arsanyos, Sonya F.; Shaheen, Hesham M.; Abdel Wahab, Nahed; Amin, Sherif N.; El Rouby, Mahmoud N.; Morsy, Mona I.

2010-04-01

Photodynamic therapy (PDT) is a novel treatment modality of cancer and non-cancerous conditions that are generally characterized by an overgrowth of unwanted or abnormal cells. Irradiation of photosensitizer loaded cells or tissues leads via the photochemical reactions of excited photosensitizer molecules to the production of singlet oxygen and free radicals, which initiate cell death. Many types of compounds have been tested as photosensitizers, such as methylene blue (MB) and photopherin seemed to be very promising. This study involved 26 cases of acute lymphoblastic leukemia and 15 normal volunteers as a control group. The cell viability was measured by Light microscope and flowcytometer. Mode of cell death was detected by flowcytometer and electron microscope in selected cases. The viability percentage of normal peripheral blood mononuclear cells (PBMC) incubated with methylene blue (MB) alone or combined with photo irradiation with diode laser (as measured by light microscope) was significantly lower than that of untreated cases either measured after 1 hour (p<0.001) or 24 hours (p<0.001) post incubation. There was a significantly lower viability percentage of normal cells incubated with MB and photoirradiated with diode laser compared to normal cells treated with MB alone for either measured after 1 hour (p<0.001) or 24 hours (p<0.001) post incubation. The decrease in viability was more enhanced with increasing the incubation time. For normal cells incubated with photopherin either for 1/2 an hour or 1 hour, there was a weak cytotoxic effect compared to the effect on untreated cells. There was a significant decrease in viability percentage of cells incubated with photopherin either for 1/2 an hour or 1 hour and photoirradiated with He:Ne laser compared to normal untreated cells. The decrease in the cell viability percentage was significantly lower with the use of PDT (photopherin and He:Ne laser ) compared to either photopherin alone or He:Ne laser alone. The decrease in viability was more enhanced with increasing the incubation time. The same effects reported on normal cells were detected on leukemic cells on comparing different methods used. However a more pronounced decrease in cell viability was detected. The most efficient ways of decreasing viability of leukemic cells with much less effect on normal cells was the use of PDT of cell incubation with MB for 1 hour then photoirradiation with diode laser and PDT of cell incubation with photopherin for 1 hour then photoirradiation with He:Ne laser. Flowcytometer (FCM) was more sensitivite than the light microscope in detecting the decrease in cell viability, it also helped in determining the mode of cell death weather apoptosis, necrosis or combined apoptosis and necrosis. Apoptotic cell percentage was higher in PDT of MB and Diode laser or photopherin and He:Ne laser, treated ALL cells compared to untreated ALL cells after 1 hour but was significantly lower after 24 hours post irradiation. A significant increase in necrotic, combined necrotic and apoptotic cell percentages either measured 1 hour or 24 hours post PDT, compared to untreated ALL cells and PDT treated normal cells. Electron microscope helped in detecting early cellular apoptotic changes occurring in response to different therapeutic modalities used in this study. In conclusion, PDT proved to be an effective clinical modality in decreasing the number of leukemic cells when irradiated in vitro with appropriate laser and photosensitizer system. Both PDT systems used in this study were efficient in inducing cell death of leukemic cells compared to untreated leukemic cells. However, photopherin PDT system was more efficient in decreasing the cell viability. A significant decrease in viability percentage was detected when studying the effect of PDT on leukemic cells compared to that on normal cells. This suggests that PDT when applied clinically will selectively differentiate between leukemic cells and normal cells, offering a successful component in ALL therapy.

Assessing the Cytotoxicity of Black Carbon As A Model for Ultrafine Anthropogenic Aerosol Across Human and Murine Cells: A Chronic Exposure Model of Nanosized Particulate Matter

NASA Astrophysics Data System (ADS)

Salinas, E.

2015-12-01

Combustion-derived nanomaterials or ultrafine (<1 μm) atmospheric aerosols are primarily products of anthropogenic activities, such as the burning of fossil fuels. Ultrafine particles (UFPs) can absorb other noxious pollutants including volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs), toxic organic compounds, and heavy metals. The combination of high population density, meteorological conditions, and industrial productivity brings high levels of air pollution to the metropolitan area of El Paso, Texas, USA/ Ciudad Juarez, Chihuahua, Mexico, comprising the Paso del Norte air basin. A study conducted by scientists from the Research Triangle Park in North Carolina, analyzed sites adjacent to heavy-traffic highways in El Paso and elucidated higher UFP concentrations in comparison to previously published work exploring pollution and adverse health effects in the basin. UFPs can penetrate deep into the alveolar sacs of the lung, reaching distant alveolar sacs and inducing a series of immune responses that are detrimental to the body: evidence suggests that UFPs can also cross the alveolar-blood barrier and potentially endanger the body's immune response. The physical properties of UFPs and the dynamics of local atmospheric and topographical conditions indicate that emissions of nanosized carbonaceous aerosols could pose significant threats to biological tissues upon inhalation by local residents of the Paso del Norte. This study utilizes Black Carbon (BC) as a model for environmental UFPs and its effects on the immunological response. An in vitro approach is used to measure the ability of BC to promote cell death upon long-term exposure. Human epithelial lung cells (A549), human peripheral-blood monocytes (THP-1), murine macrophages (RAW264.7), and murine epithelial lung cells (LA-4) were treated with BC and assessed for metabolic activity after chronic exposure utilizing three distinct and independent cell viability assays. The cell viability experiments included a chronic study at 7, 10, and 14 days of UFP exposure at six different concentrations of BC: 100μM, 300μM, 600μM, 1,250μM, 2,500μM, and 5,000μM conducting the Trypan Blue (TB) Exclusion Assay, Calcein-AM Viability Assay, and CellTiter-Glo Viability Assay.

Morphology based scoring of chromosomal instability and its correlation with cell viability.

PubMed

Yadav, Shubhlata; Bhatia, Alka

2017-09-01

The aim of this study was to devise the quantitative scoring system for Chromosomal instability (CIN) based on morphological indicators like MPM, NB, NPB, CS, La and MN in cancer cell line and to correlate it with cell viability and death. Human hepatocellular carcinoma (HepG2) cells were treated with drugs like Diethylstilbestrol 0-100μM, Griseofulvin 0-40μg/ml, Vincristine sulphate 0-25μg/ml, Mitomycin C 0-600ng/ml, Bleomycin 0-10μg/ml, Doxorubicin 0-30μg/ml for 24h. Following this, the CIN was assessed by counting the morphological indicators like Micronuclei (MN), Nuclear Buds (NB), Nucleoplasmic bridges, Laggards, Multipolar mitosis and chromatin strings/1000 cells in Giemsa stained smears by light microscopy and by determining the percentage of aneuploid cells by flow cytometry. The cell viability was assessed by MTT assay and percentage of apoptotic cells was determined by flow cytometry. The MN and NB were most frequently seen indicators and main determinants of morphological CIN. However, the morphological CIN score did not show any correlation with cell viability and apoptosis. Aneuploidy however was found to correlate positively with cell viability and NB score in our study (P-value <0.05). The study for the 1st time attempted to develop a scoring system for CIN based on morphological parameters. However, a no correlation was observed between the later and cell viability or apoptosis. More robust techniques to quantify CIN may perhaps be more helpful in exploring the true link between CIN and cell viability in future. Copyright © 2017 Elsevier GmbH. All rights reserved.

Soy milk as a storage medium to preserve human fibroblast cell viability: an in vitro study.

PubMed

Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Reis, Manuella Verdinelli de Paula; Fernandes Neto, Alfredo Júlio; Soares, Carlos José

2012-01-01

Soy milk (SM) is widely consumed worldwide as a substitute for cow milk. It is a source of vitamins, carbohydrates and sugars, but its capacity to preserve cell viability has not been evaluated. The purpose of the present study was to investigate the efficacy of SM to maintain the viability of human fibroblasts at short periods compared with different cow milks. Human mouth fibroblasts were cultured and stored in the following media at room temperature: 10% Dulbecco's Modified Eagle Medium (DMEM) (positive control group); long shelf-life ultra-high temperature whole cow milk (WM); long shelf-life ultra-high temperature skim cow milk (SKM); powdered cow milk (PM); and soy milk (SM). After 5, 15, 30 and 45 min, cell viability was analyzed using the MTT assay. Data were analyzed statistically by the Kruskal-Wallis test with post-analysis using the Dunn's method (α=0.05). SKM showed the lowest capacity to maintain cell viability in all analyzed times (p<0.05). At 30 and 45 min, the absorbance levels in control group (DMEM) and SM were significantly higher than in SKM (p<0.05). Cell viability decreased along the time (5-45 min). The results indicate that SM can be used as a more adequate storage medium for avulsed teeth. SKM was not as effective in preserving cell viability as the cell culture medium and SM.

Irradiation at 636 nm positively affects diabetic wounded and hypoxic cells in vitro.

PubMed

Sekhejane, Palesa R; Houreld, Nicolette N; Abrahamse, Heidi

2011-08-01

This study investigated the effect of low-intensity laser irradiation (LILI) on pro-inflammatory cytokines involved in wound healing processes in diabetes and hypoxia. Diabetes is associated with impaired wound healing and a prolonged inflammatory phase. Pro-inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 are elevated in diabetes. LILI has been reported to accelerate wound healing and decrease inflammatory cytokines. A human skin fibroblast cell line (WS1) was used in vitro. Cells were exposed to various insults, namely, wounding, and a diabetic or hypoxic environment. Experimental cells were exposed to an energy density of 5  J/cm(2) using a continuous wave 636-nm diode laser at an average power of 95  mW, an illuminated area of 9.05  cm(2), and an irradiance of 11 mW/cm(2) (irradiation time, 476  sec). The effect of laser irradiation on cytokine expression was examined at 1 or 24  h post-irradiation. Cellular morphology, viability, proliferation, and cytokine expression (IL-1β, IL-6, and TNF-α) were investigated. Translocation of nuclear factor-kappa B (NF-κB) was also determined. There was a higher rate of migration in irradiated wounded cultures, and irradiated hypoxic cells showed an improvement in cellular morphology. All cell models showed an increase in proliferation. Normal wounded cells showed a decrease in apoptosis, TNF-α, and IL-1β. Diabetic wounded cells showed an increase in viability and a decrease in apoptosis and IL-1β, whereas hypoxic cells showed an increase in viability and IL-6, and a decrease in apoptosis and TNF-α. NF-κB was translocated into the nucleus post-irradiation. Phototherapy resulted in hastened wound closure, increased proliferation, and normalization of cellular function. The decrease in the different pro-inflammatory cytokines and NF-κB translocation was model and time dependent. Overall, laser irradiation resulted in a reduction in inflammatory cytokines and directed cells into the cell survival pathway.

Galactoglucomannan oligosaccharides are assumed to affect tracheary element formation via interaction with auxin in Zinnia xylogenic cell culture.

PubMed

Kákošová, Anna; Digonnet, Catherine; Goffner, Deborah; Lišková, Desana

2013-04-01

Galactoglucomannan oligosaccharides seem to interact with auxin in xylogenic cell culture, thus influencing mainly metaxylem-like tracheary element differentiation depending on timing with hormones and the process kinetics. Complex mapping of Zinnia mesophyll cell transdifferentiation into tracheary elements with or without prior cell division was documented after palisade and spongy parenchyma cell immobilization during the first 4 days of culture. Here, we report a positive effect of galactoglucomannan oligosaccharides on cell viability and density and higher metaxylem-like tracheary element formation in xylogenic cell culture. The maximal positive effect was achieved by the simultaneous addition of the oligosaccharides and growth hormones (auxin, cytokinin) to the cell culture medium. Moreover, a large number of metaxylem-like tracheary elements were observed in a low-auxin medium supplemented with oligosaccharides, but not in a low-cytokinin medium, suggesting a close relationship between auxin and the oligosaccharides during tracheary element formation.

Multifunctional mussel-inspired copolymerized epigallocatechin gallate (EGCG)/arginine coating: the potential as an ad-layer for vascular materials.

PubMed

Luo, Rifang; Tang, Linlin; Xie, Lingxia; Wang, Jin; Huang, Nan; Wang, Yunbing

2016-12-01

Surface properties are considered to be important factors in addressing proper functionalities. In this paper, a multifunctional mussel-inspired coating was prepared via the direct copolymerization of epigallocatechin gallate (EGCG) and arginine. The coating formation was confirmed by X-ray photoelectron spectroscopy and Fourier transform infrared spectra. The EGCG/arginine coating contained diverse functional groups like amines, phenols and carboxyls, whose densities were also tunable. Such mussel-inspired coating could also be applied as an ad-layer for its secondary reactivity, demonstrated by quartz crystal microbalance technique. Moreover, the tunable surface density of phenols showed potential ability in modulating endothelial cell and smooth muscle cell viability. The coatings rich in phenols presented excellent free radical scavenging property. Current results strongly indicated the potential of EGCG/arginine coatings to be applied as an ad-layer for vascular materials.

Effect of berberine on the viability of adipose tissue-derived mesenchymal stem cells in nutrients deficient condition.

PubMed

Ghorbani, Ahmad; Baradaran Rahimi, Vafa; Sadeghnia, Hamid Reza; Hosseini, Azar

2018-03-01

This study was designed to examine whether berberine protects rat adipose tissue-derived stem cells (ASCs) against glucose and serum deprivation (GSD)-induced cell death. ASCs were cultured for 24 h in GSD condition in the presence of berberine and then cell viability, apoptosis and generation of reactive oxygen species (ROS) were evaluated. The GSD condition significantly decreased ASCs viability and increased ROS generation and apoptosis. Incubation with 0.75-3 μM berberine partially increased cell viability and decreased ROS generation and apoptosis in GSD condition. In conclusion, berberine partially protects ASCs in nutrients deficient condition and may help ASCs to preserve their survival during cell therapy of ischemia.

Characterization and reproducibility of HepG2 hanging drop spheroids toxicology in vitro.

PubMed

Hurrell, Tracey; Ellero, Andrea Antonio; Masso, Zelie Flavienne; Cromarty, Allan Duncan

2018-02-21

Hepatotoxicity remains a major challenge in drug development despite preclinical toxicity screening using hepatocytes of human origin. To overcome some limitations of reproducing the hepatic phenotype, more structurally and functionally authentic cultures in vitro can be introduced by growing cells in 3D spheroid cultures. Characterisation and reproducibility of HepG2 spheroid cultures using a high-throughput hanging drop technique was performed and features contributing to potential phenotypic variation highlighted. Cultured HepG2 cells were seeded into Perfecta 3D® 96-well hanging drop plates and assessed over time for morphology, viability, cell cycle distribution, protein content and protein-mass profiles. Divergent aspects which were assessed included cell stocks, seeding density, volume of culture medium and use of extracellular matrix additives. Hanging drops are advantageous due to no complex culture matrix being present, enabling background free extractions for downstream experimentation. Varying characteristics were observed across cell stocks and batches, seeding density, culture medium volume and extracellular matrix when using immortalized HepG2 cells. These factors contribute to wide-ranging cellular responses and highlights concerns with respect to generating a reproducible phenotype in HepG2 hanging drop spheroids. Copyright © 2018 Elsevier Ltd. All rights reserved.

Potential application of glycerol in the production of plant beneficial microorganisms.

PubMed

Vassilev, Nikolay; Malusa, Eligio; Requena, Antonia Reyes; Martos, Vanessa; López, Ana; Maksimovic, Ivana; Vassileva, Maria

2017-05-01

This review highlights the importance of research for development of biofertilizer and biocontrol products based on the use of glycerol for further process scale-up to industrial microbiology. Glycerol can be used successfully in all stages of production of plant beneficial microorganisms. It serves as an excellent substrate in both submerged and solid-state fermentation processes with free and immobilized microbial cells. Glycerol is also one of the most attractive formulation agents that ensures high cell density and viability including in harsh environmental conditions. Future research is discussed to make this inexpensive material a base for industrial production of plant beneficial microorganisms.

Potential of coconut water and soy milk for use as storage media to preserve the viability of periodontal ligament cells: an in vitro study.

PubMed

Moura, Camilla Cristhian Gomes; Soares, Priscilla Barbosa Ferreira; de Paula Reis, Manuella Verdinelli; Fernandes Neto, Alfredo Júlio; Zanetta Barbosa, Darceny; Soares, Carlos José

2014-02-01

There is no consensus regarding the ability of coconut water and soy milk to maintain long-term cell viability. This study investigated the ability of pH-adjusted coconut water and soy milk to maintain the viability of periodontal ligament cells over a short and a longer period and compared these abilities with those of other solutions. Dog premolar teeth were extracted, dried for 30 min, and stored in the following media for 50 min or 24 h: long shelf-life whole milk (SWM), long shelf-life skim milk (SSM), Hank's Balanced Salt Solution (HBSS), soy milk (SM), and pH-adjusted coconut water (CW). The positive and two negative control groups corresponded to 0-min, 30-min (short-term), and 24-h (long-term) dry times, respectively. Cell viability was analyzed by trypan blue exclusion. Data were statistically analyzed using the Kruskal-Wallis test with post-analysis using the Dunn method. In the short-term experiment, the SSM resulted in significantly lower cell viability than SM and CW. At 24 h, SM and CW resulted in higher viability than HBSS and SSM and in comparable performance with the positive control group. Cell viability decreased over time, except in SM and CW. Soy milk and pH-adjusted coconut water showed promising results as storage solutions for avulsed teeth, preserving the viability for up to 24 h. © 2013 John Wiley & Sons A/S.

Role of cytoskeletal mechanics and cell membrane fluidity in the intracellular delivery of molecules mediated by laser-activated carbon nanoparticles.

PubMed

Holguin, Stefany Y; Anderson, Caleb F; Thadhani, Naresh N; Prausnitz, Mark R

2017-10-01

Exposure of cells and nanoparticles to near-infrared nanosecond pulsed laser light can lead to efficient intracellular delivery of molecules while maintaining high cell viability by a photoacoustic phenomenon known as transient nanoparticle energy transduction (TNET). Here, we examined the influence of cytoskeletal mechanics and plasma membrane fluidity on intracellular uptake of molecules and loss of cell viability due to TNET. We found that destabilization of actin filaments using latrunculin A led to greater uptake of molecules and less viability loss caused by TNET. Stabilization of actin filaments using jasplakinolide had no significant effect on uptake or viability loss caused by TNET. To study the role of plasma membrane fluidity, we increased fluidity by depletion of membrane cholesterol using methyl-β-cyclodextrin and decreased fluidity by enrichment of the membrane with cholesterol using water-soluble cholesterol. Neither of these membrane fluidity changes significantly altered cellular uptake or viability loss caused by TNET. We conclude that weakening mechanical integrity of the cytoskeleton can increase intracellular uptake and decrease loss of cell viability, while plasma membrane fluidity does not appear to play a significant role in uptake or viability loss caused by TNET. The positive effects of cytoskeletal weakening may be due to an enhanced ability of the cell to recover from the effects of TNET and maintain viability. Biotechnol. Bioeng. 2017;114: 2390-2399. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Atorvastatin Promotes Cytotoxicity and Reduces Migration and Proliferation of Human A172 Glioma Cells.

PubMed

Oliveira, Karen A; Dal-Cim, Tharine; Lopes, Flávia G; Ludka, Fabiana K; Nedel, Cláudia B; Tasca, Carla I

2018-02-01

Malignant gliomas have resistance mechanisms to chemotherapy that enable tumor invasiveness and aggressiveness. Alternative therapies in cancer treatment, as statins, have been suggested to decrease proliferation, inhibit cell migration, and induce cell death. The aim of this study was to evaluate the effect of atorvastatin (ATOR) on cell viability, migration, proliferation, apoptosis, and autophagy in A172 human glioma cells. Temozolomide (TMZ), a chemotherapic used to glioma treatment, was tested as a comparison to cytotoxic effects on gliomas. Cell viability was also assessed in primary culture of cortical astrocytes. ATOR treatment (0.1 to 20 μM) did not alter astrocytic viability. However, in glioma cells, ATOR showed cytotoxic effect at 10 and 20 μM concentrations. TMZ (500 μM) reduced cell viability similarly to ATOR, and drug association did not show additive effect on cell viability. ATOR, TMZ, and their association decreased cell migration. ATOR also decreased glioma cell proliferation. ATOR increased apoptosis, and TMZ association showed a potentiation effect, enhancing it. ATOR and TMZ treatment increased acidic vesicular organelle (AVO) presence in A172 cells, an indicative of autophagy. ATOR effect of reducing A172 cell viability did not alter glutamate transport and glutamine synthetase activity, but it was partially prevented through antagonism of ionotropic and metabotropic glutamate receptors. Our data shows a cytotoxic effect of ATOR on glioma cells, whereas no toxicity was observed to astrocytes. ATOR showed similar cytotoxic effect as TMZ to glioma cells, and it may be a safer drug, regarding side effect induction, than chemotherapic agents.

MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization

PubMed Central

Hayakawa, Tohru; Yoshida, Eiji; Yoshimura, Yoshitaka; Uo, Motohiro; Yoshinari, Masao

2012-01-01

The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0 nm) and sandblasting (Ra: approximately 1.0 μm), and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells. PMID:22675359

Thermal treatment of bentonite reduces aflatoxin b1 adsorption and affects stem cell death.

PubMed

Nones, Janaína; Nones, Jader; Riella, Humberto Gracher; Poli, Anicleto; Trentin, Andrea Gonçalves; Kuhnen, Nivaldo Cabral

2015-10-01

Bentonites are clays that highly adsorb aflatoxin B1 (AFB1) and, therefore, protect human and animal cells from damage. We have recently demonstrated that bentonite protects the neural crest (NC) stem cells from the toxicity of AFB1. Its protective effects are due to the physico-chemical properties and chemical composition altered by heat treatment. The aim of this study is to prepare and characterize the natural and thermal treatments (125 to 1000 °C) of bentonite from Criciúma, Santa Catarina, Brazil and to investigate their effects in the AFB1 adsorption and in NC cell viability after challenging with AFB1. The displacement of water and mineralogical phases transformations were observed after the thermal treatments. Kaolinite disappeared at 500 °C and muscovite and montmorillonite at 1000 °C. Slight changes in morphology, chemical composition, and density of bentonite were observed. The adsorptive capacity of the bentonite particles progressively reduced with the increase in temperature. The observed alterations in the structure of bentonite suggest that the heat treatments influence its interlayer distance and also its adsorptive capacity. Therefore, bentonite, even after the thermal treatment (125 to 1000 °C), is able to increase the viability of NC stem cells previously treated with AFB1. Our results demonstrate the effectiveness of bentonite in preventing the toxic effects of AFB1. Copyright © 2015 Elsevier B.V. All rights reserved.

Lysis of Bacillus subtilis Cells by Glycerol and Sucrose Esters of Fatty Acids

PubMed Central

Tsuchido, Tetsuaki; Ahn, Yung-Hoon; Takano, Mitsuo

1987-01-01

The lytic action of glycerol and sucrose esters of fatty acids with different carbon chain lengths on the exponentially growing cells of Bacillus subtilis 168 was investigated. Of each series of esters, glycerol dodecanoate and sucrose hexadecanoate were the most active. Lysis at 1 h after the addition of 0.1 mM glycerol dodecanoate or 20 μg of sucrose hexadecanoate per ml was 81 or 79%, respectively, as evaluated by the reduction in optical density. During this treatment a great loss of viability occurred that preceded lysis. The results that were obtained suggest that autolysis is induced by these esters. The esters caused morphological changes in the cells, but a seeming adaptation of the cells to esters was seen. Images PMID:16347300

Gc-protein-derived macrophage activating factor counteracts the neuronal damage induced by oxaliplatin.

PubMed

Morucci, Gabriele; Branca, Jacopo J V; Gulisano, Massimo; Ruggiero, Marco; Paternostro, Ferdinando; Pacini, Alessandra; Di Cesare Mannelli, Lorenzo; Pacini, Stefania

2015-02-01

Oxaliplatin-based regimens are effective in metastasized advanced cancers. However, a major limitation to their widespread use is represented by neurotoxicity that leads to peripheral neuropathy. In this study we evaluated the roles of a proven immunotherapeutic agent [Gc-protein-derived macrophage activating factor (GcMAF)] in preventing or decreasing oxaliplatin-induced neuronal damage and in modulating microglia activation following oxaliplatin-induced damage. The effects of oxaliplatin and of a commercially available formula of GcMAF [oleic acid-GcMAF (OA-GcMAF)] were studied in human neurons (SH-SY5Y cells) and in human microglial cells (C13NJ). Cell density, morphology and viability, as well as production of cAMP and expression of vascular endothelial growth factor (VEGF), markers of neuron regeneration [neuromodulin or growth associated protein-43 (Gap-43)] and markers of microglia activation [ionized calcium binding adaptor molecule 1 (Iba1) and B7-2], were determined. OA-GcMAF reverted the damage inflicted by oxaliplatin on human neurons and preserved their viability. The neuroprotective effect was accompanied by increased intracellular cAMP production, as well as by increased expression of VEGF and neuromodulin. OA-GcMAF did not revert the effects of oxaliplatin on microglial cell viability. However, it increased microglial activation following oxaliplatin-induced damage, resulting in an increased expression of the markers Iba1 and B7-2 without any concomitant increase in cell number. When neurons and microglial cells were co-cultured, the presence of OA-GcMAF significantly counteracted the toxic effects of oxaliplatin. Our results demonstrate that OA-GcMAF, already used in the immunotherapy of advanced cancers, may significantly contribute to neutralizing the neurotoxicity induced by oxaliplatin, at the same time possibly concurring to an integrated anticancer effect. The association between these two powerful anticancer molecules would probably produce the dual effect of reduction of oxaliplatin-induced neurotoxicity, together with possible synergism in the overall anticancer effect.

Cytotoxic outcomes of orthodontic bands with and without silver solder in different cell lineages.

PubMed

Jacoby, Letícia Spinelli; Rodrigues Junior, Valnês da Silva; Campos, Maria Martha; Macedo de Menezes, Luciane

2017-05-01

The safety of orthodontic materials is a matter of high interest. In this study, we aimed to assess the in-vitro cytotoxicity of orthodontic band extracts, with and without silver solder, by comparing the viability outcomes of the HaCat keratinocytes, the fibroblastic cell lineages HGF and MRC-5, and the kidney epithelial Vero cells. Sterilized orthodontic bands with and without silver solder joints were added to culture media (6 cm 2 /mL) and incubated for 24 hours at 37°C under continuous agitation. Subsequently, the cell cultures were exposed to the obtained extracts for 24 hours, and an assay was performed to evaluate the cell viability. Copper strip extracts were used as positive control devices. The extracts from orthodontic bands with silver solder joints significantly reduced the viability of the HaCat, MRC-5, and Vero cell lines, whereas the viability of HGF was not altered by this material. Conversely, the extracts of orthodontic bands without silver solder did not significantly modify the viability index of all evaluated cell lines. Except for HGF fibroblasts, all tested cell lines showed decreased viability percentages after exposure to extracts of orthodontic bands containing silver solder joints. These data show the relevance of testing the toxicity of orthodontic devices in different cell lines. Copyright © 2017 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

PubMed Central

Suszynski, T.M.; Wildey, G.M.; Falde, E.J.; Cline, G.W.; Maynard, K. Stewart; Ko, N.; Sotiris, J.; Naji, A.; Hering, B.J.; Papas, K.K.

2009-01-01

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio. PMID:18374063

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

Comparative evaluation of the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin scaffold.

PubMed

Khurana, Rohit; Kudva, Praveen Bhasker; Husain, Syed Yawer

2017-01-01

The present study aims to comparatively evaluate the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin (PRF) scaffold. A total of 15 systemically healthy individuals between the age group of 15-25 years requiring third molar or orthodontic premolar extractions. Teeth were extracted atraumatically and transported to the laboratory. Stem cells were isolated from dental pulp and periodontal ligament. After attaining more than 90% confluency by the 7 th day, these cells were tested for their viability and characterization. Stem cells were also incubated with PRF and viability was assessed on the 7 th day. The mean number of cell for dental pulp stem cells (DPSCs) and periodontal ligament stem cell (PDLSC) was statistically insignificant ( P > 0.05). The mean live cell viability was compared between DPSC (98.07%) and PDLSC (98%). Both DPSC and PDLSC showed a high percentage of expression of CD73 markers, 30.40% and 29.80%, respectively. However, DPSCs and PDLSCs lacked expression of CD34 expressing only 3.47% and 3.53%, respectively. PRF membrane as a scaffold exhibited no cytotoxic effects on DPCS's or PDLSC's. The cell viability of cells cultured with PRF was statistically insignificant ( P > 0.05) when compared to the cells cultured with culture media. The study thus indicates that dental pulp and periodontal ligament are both rich sources of mesenchymal stem cells and can be successfully used for obtaining stem cells. PRF exhibits no cytotoxic effects on the cells and can be used in conjunction with dental stem cells.

The analysis of viability for mammalian cells treated at different temperatures and its application in cell shipment.

PubMed

Wang, Juan; Wei, Yun; Zhao, Shasha; Zhou, Ying; He, Wei; Zhang, Yang; Deng, Wensheng

2017-01-01

Mammalian cells are very important experimental materials and widely used in biological and medical research fields. It is often required that mammalian cells are transported from one laboratory to another to meet with various researches. Conventional methods for cell shipment are laborious and costive despite of maintaining high viability. In this study we aimed to develop a simple and low-cost method for cell shipment by investigating the viabilities of different cell lines treated at different temperatures. We show that the viability of mammalian cells incubated at 1°C or 5°C significantly reduced when compared with that at 16°C or 22°C. Colony formation assays revealed that preservation of mammalian cells at 1°C or 5°C led to a poorer recovery than that at 16°C or 22°C. The data from proliferation and apoptotic assays confirmed that M2 cells could continue to proliferate at 16°C or 22°C, but massive death was caused by apoptosis at 1°C or 5°C. The morphology of mammalian cells treated under hypothermia showed little difference from that of the untreated cells. Quantitative RT-PCR and alkaline phosphatase staining confirmed that hypothermic treatment did not change the identity of mouse embryonic stem cells. A case study showed that mammalian cells directly suspended in culture medium were able to be shipped for long distance and maintained a high level of viability and recovery. Our findings not only broaden the understanding to the effect of hypothermia on the viability of mammalian cells, but also provide an alternative approach for cell shipment.

The effects of cetrorelix and triptorelin on the viability and steroidogenesis of cultured human granulosa luteinized cells.

PubMed

Metallinou, Chryssa; Köster, Frank; Diedrich, Klaus; Nikolettos, Nikos; Asimakopoulos, Byron

2012-01-01

We investigated the effects of the gonadotropin-releasing hormone (GnRH) agonist triptorelin as well the GnRH antagonist cetrorelix those of on the viability and steroidogenesis in human granulosa luteinized (hGL) cell cultures. The hGL cells were obtained from 34 women undergoing ovarian stimulation for IVF treatment. The cells were cultured for 48 h with or without 1 nM or 3 nM of cetrorelix or triptorelin in serum-free media. The cell viability was evaluated by the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. The concentrations of estradiol and progesterone in culture supernatants were measured by ELISA. Treatment with triptorelin slightly increased cell viability, whereas treatment with 3 nM cetrorelix led to a significant decrease. Estradiol concentrations were reduced with 3 nM triptorelin. Cultures treated with high-dose of either cetrorelix or triptorelin tended to secrete less progesterone than controls. Cetrorelix significantly reduces the viability of hGL cells. Triptorelin and cetrorelix may have minor effects on steroidogenesis. These results suggest that GnRH analogues may influence ovarian functions.

Avenanthramide-C reduces the viability of MDA-MB-231 breast cancer cells through an apoptotic mechanism.

PubMed

Hastings, Jordan; Kenealey, Jason

2017-01-01

Avenanthramides (AVN) are a relatively unstudied family of phytochemicals that could be novel chemotherapeutics. These compounds, found in oats, are non-toxic to healthy cells and have been shown to reduce viability of human colon and liver cancers in vitro. However, these studies do not elucidate a molecular mechanism for individual AVN. In this study we aim to see the effects of AVN on MDA-MB-231 breast cancer cells. An MTT assay was used to determine cell viability. Staining and analysis with a flow cytometer was used to identify cell cycle progression and apoptosis. FloJo software was used to analyze the cytometric data. In all experiments, statistical significance was determined by a two-tailed t test. This study demonstrates that AVN-A, B, and C individually reduce viability in the MDA-MB-231 breast cancer cell line. AVN-C has the most potent decrease in tumor cell viability, decreasing viable cells to below 25% at 400 µM when compared to control after 96 h. We demonstrate that treatment with AVN-C causes DNA fragmentation and accumulation of over 90% of cells into a sub G 1 cell cycle population. Further, we conclude that AVN-C treated cells activate apoptosis because 97% of treated cells stain positive for annexin V while 91% have caspase-3/7 activity, a late marker of apoptosis. Breast cancer cells treated with AVN-C have a decrease in cell viability, an increase in the sub G 1 population, and stain positive for both annexin V and caspase activity, indicating that AVN-C induces apoptosis in breast cancer cells. These compounds may be able to act as chemotherapeutics as demonstrated through future in vivo studies.

Differential Effects of Bevacizumab, Ranibizumab, and Aflibercept on the Viability and Wound Healing of Corneal Epithelial Cells.

PubMed

Kang, Seungbum; Choi, Hyunsu; Rho, Chang Rae

2016-12-01

This study compared the effects of 3 antivascular endothelial growth factor (VEGF) agents (bevacizumab, ranibizumab, and aflibercept) on corneal epithelial cell viability and wound healing using human corneal epithelial cells (HCECs). To determine the cytotoxic effects of anti-VEGF agents on HCECs, HCEC viability was determined at various concentrations of these agents. An in vitro migration assay was used to investigate the migration of HCECs treated with 3 anti-VEGF agents. The protein level of extracellular signal-regulated kinase was used to evaluate the effect of anti-VEGF treatment on cell proliferation. The protein levels of p38 mitogen-activated protein kinase (MAPK) were analyzed by Western blotting to investigate cell migration. After 24 or 48 h of exposure, aflibercept treatment showed no apparent effect on cell viability; however, bevacizumab and ranibizumab treatment decreased cell viability at high concentrations (1 and 2 mg/mL). A migration assay showed that HCEC migration was different among the 3 anti-VEGF treatment groups. Bevacizumab significantly delayed HCEC migration. Western blotting showed that bevacizumab treatment decreased the expression levels of phosphorylated p38 MAPK. Bevacizumab, the most widely used and investigated anti-VEGF agent, decreased epithelial cell migration and viability. Anti-VEGF agents other than bevacizumab might therefore be better for treating corneal neovascularization complicated with epithelial defects.

Improvement in the Viability of Cryopreserved Cells by Microencapsulation

NASA Astrophysics Data System (ADS)

Matsumoto, Yoshifumi; Morinaga, Yukihiro; Ujihira, Masanobu; Oka, Kotaro; Tanishita, Kazuo

The advantages of microencapsulated cells over those of suspended cells were evaluated for improving viability in cryopreservation. Rat pheochromocytoma (PC12) cells were selected as the test biological cells and then microencapsulated in alginate-polylysine-alginate membranes. These microencapsulated PC12 cells were frozen by differential scanning calorimetry (DSC) at various cooling rates, from 0.5 to 10°C/min. Their latent heat was measured during freezing from 4 to -80°C. The post-thaw viability was evaluated by dopamine-concentration measurement and by trypan blue exclusion assay. Results showed that at cooling rates of 0.5 and 1°C/min, the latent heat of microencapsulated PC12 cells was lower than that of suspended cells. This lower latent heat is caused by the fact that the extra-microcapsule froze and the intra-capsule remained unfrozen due to the formation of ice crystals in the extra-capsule space. The post-thaw viability of microencapsulated PC12 cells was improved when the cooling rate was 0.5 or 1°C/min, compared with that of suspended cells. Therefore, in microencapsulated PC12 cells, maintaining the intra-microcapsules in an unfrozen state during freezing reduces the solution effect and thus improves the post-thaw viability.

Immunomodulatory/inflammatory effects of geopropolis produced by Melipona fasciculata Smith in combination with doxorubicin on THP-1 cells.

PubMed

Oliveira, Lucas Pires Garcia; Conte, Fernanda Lopes; Cardoso, Eliza de Oliveira; Conti, Bruno José; Santiago, Karina Basso; Golim, Marjorie de Assis; Cruz, Maria Teresa; Sforcin, José Maurício

2016-12-01

Geopropolis (GEO) in combination with doxorubicin (DOX) reduced HEp-2 cells viability compared to GEO and DOX alone. A possible effect of this combination on the innate immunity could take place, and its effects were analysed on THP-1 cell - a human leukaemia monocytic cell line used as a model to study monocyte activity and macrophage activity, assessing cell viability, expression of cell markers and cytokine production. THP-1 cells were incubated with GEO, DOX and their combination. Cell viability was assessed by MTT assay, cell markers expression by flow cytometry and cytokine production by ELISA. GEO + DOX did not affect cell viability. GEO alone or in combination increased TLR-4 and CD80 but not HLA-DR and TLR-2 expression. GEO stimulated TNF-α production while DOX alone or in combination did not affect it. GEO alone or in combination inhibited IL-6 production. GEO exerted a pro-inflammatory profile by increasing TLR-4 and CD80 expression and TNF-α production, favouring the activation of the immune/inflammatory response. GEO + DOX did not affect cell viability and presented an immunomodulatory action. Lower concentrations of DOX combined to GEO could be used in cancer patients, avoiding side effects and benefiting from the biological properties of GEO. © 2016 Royal Pharmaceutical Society.

24 CFR 971.5 - Long-term viability.

Code of Federal Regulations, 2010 CFR

2010-04-01

... amended accordingly.) (b) Density. Density reduction measures would have to result in a public housing community with a density approaching that which prevails in the community for similar types of housing (typically family), or a lower density. If the development's density already meets this description, further...

24 CFR 971.5 - Long-term viability.

Code of Federal Regulations, 2012 CFR

2012-04-01

... amended accordingly.) (b) Density. Density reduction measures would have to result in a public housing community with a density approaching that which prevails in the community for similar types of housing (typically family), or a lower density. If the development's density already meets this description, further...

24 CFR 971.5 - Long-term viability.

Code of Federal Regulations, 2014 CFR

2014-04-01

... amended accordingly.) (b) Density. Density reduction measures would have to result in a public housing community with a density approaching that which prevails in the community for similar types of housing (typically family), or a lower density. If the development's density already meets this description, further...

24 CFR 971.5 - Long-term viability.

Code of Federal Regulations, 2011 CFR

2011-04-01

... amended accordingly.) (b) Density. Density reduction measures would have to result in a public housing community with a density approaching that which prevails in the community for similar types of housing (typically family), or a lower density. If the development's density already meets this description, further...

24 CFR 971.5 - Long-term viability.

Code of Federal Regulations, 2013 CFR

2013-04-01

... amended accordingly.) (b) Density. Density reduction measures would have to result in a public housing community with a density approaching that which prevails in the community for similar types of housing (typically family), or a lower density. If the development's density already meets this description, further...

Importance of balancing membrane and electrode water in anion exchange membrane fuel cells

NASA Astrophysics Data System (ADS)

Omasta, T. J.; Wang, L.; Peng, X.; Lewis, C. A.; Varcoe, J. R.; Mustain, W. E.

2018-01-01

Anion exchange membrane fuel cells (AEMFCs) offer several potential advantages over proton exchange membrane fuel cells (PEMFCs), most notably to overcome the cost barrier that has slowed the growth and large scale implementation of fuel cells for transportation. However, limitations in performance have held back AEMFCs, specifically in the areas of stability, carbonation, and maximum achievable current and power densities. In order for AEMFCs to contend with PEMFCs for market viability, it is necessary to realize a competitive cell performance. This work demonstrates a new benchmark for a H2/O2 AEMFC with a peak power density of 1.4 W cm-2 at 60 °C. This was accomplished by taking a more precise look at balancing necessary membrane hydration while preventing electrode flooding, which somewhat surprisingly can occur both at the anode and the cathode. Specifically, radiation-grafted ETFE-based anion exchange membranes and anion exchange ionomer powder, functionalized with benchmark benzyltrimethylammonium groups, were utilized to examine the effects of the following parameters on AEMFC performance: feed gas flow rate, the use of hydrophobic vs. hydrophilic gas diffusion layers, and gas feed dew points.

Fiber optic light-scattering measurement system for evaluation of embryo viability: light-scattering characteristics from live mouse embryo

NASA Astrophysics Data System (ADS)

Itoh, Harumi; Arai, Tsunenori; Kikuchi, Makoto

1997-06-01

We measured angular distribution of the light scattering from live mouse embryo with 632.8nm in wavelength to evaluate the embryo viability. We aim to measure the mitochondrial density in human embryo which have relation to the embryo viability. We have constructed the light scattering measurement system to detect the mitochondrial density non-invasively. We have employed two optical fibers for the illumination and sensing to change the angle between these fibers. There were two dips on the scattering angular distribution from the embryo. These dips existed on 30 and 85 deg. We calculated the scattering angular pattern by Mie theory to fit the measured scattering estimated scattering size and density. The best fitting was obtained when the particle size and density were 0.9 micrometers and 1010 particles per ml, respectively. These values coincided with the approximated values of mitochondrial in the embryo. The measured light scattering may mainly originated from mitochondria in spite of the existence of the various scattering particles in the embryo. Since our simple scattering measurement may offer the mitochondrial density in the embryo, it might become the practical method of human embryo on in vitro fertilization-embryo transfer.

Cell viability in optical tweezers: high power red laser diode versus Nd:YAG laser

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert; Hendinger, Anita; Sailer, Reinhard; Gschwend, Michael H.; Strauss, Wolfgang S.; Bauer, Manfred; Schuetze, Karin

2000-01-01

Viability of cultivated Chinese hamster ovary cells in optical tweezers was measured after exposure to various light doses of red high power laser diodes ((lambda) equals 670 - 680 nm) and a Nd:yttrium-aluminum-garnet laser ((lambda) equals 1064 nm). When using a radiant exposure of 2.4 GJ/cm2, a reduction of colony formation up to a factor 2 (670 - 680 nm) or 1.6 (1064 nm) as well as a delay of cell growth were detected in comparison with nonirradiated controls. In contrast, no cell damage was found at an exposure of 340 MJ/cm2 applied at 1064 nm. Cell viabilities were correlated with fluorescence excitation spectra and with literature data of wavelength dependent cloning efficiencies. Fluorescence excitation maxima of the coenzymes NAD(P)H and flavins were detected at 365 and 450 nm, respectively. This is half of the wavelengths of the maxima of cell inactivation, suggesting that two-photon absorption by these coenzymes may contribute to cellular damage. Two-photon excitation of NAD(P)H and flavins may also affect cell viability after exposure to 670 - 680 nm, whereas one-photon excitation of water molecules seems to limit cell viability at 1064 nm.

Paraoxonase 2 modulates a proapoptotic function in LS174T cells in response to quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone

PubMed Central

Tao, Shiyu; Luo, Yanwen; Bin He; Liu, Jie; Qian, Xi; Ni, Yingdong; Zhao, Ruqian

2016-01-01

A mucus layer coats the gastrointestinal tract and serves as the first line of intestinal defense against infection. N-acyl-homoserine lactone (AHL) quorum-sensing molecules produced by gram-negative bacteria in the gut can influence the homeostasis of intestinal epithelium. In this study, we investigated the effects of two representative long- and short-chain AHLs, N-3-(oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl homoserine lactone (C4-HSL), on cell viability and mucus secretion in LS174T cells. C12-HSL but not C4-HSL significantly decreased cell viability by inducing mitochondrial dysfunction and activating cell apoptosis which led to a decrease in mucin expression. Pretreatment with lipid raft disruptor (Methyl-β-cyclodextrin, MβCD) and oxidative stress inhibitor (N-acetyl-L-cysteine, NAC) slightly rescued the viability of cells damaged by C12-HSL exposure, while the paraoxonase 2 (PON2) inhibitor (Triazolo[4,3-a]quinolone, TQ416) significantly affected recovering cells viability and mucin secretion. When LS174T cells were treated with C12-HSL and TQ416 simultaneously, TQ416 showed the maximal positive effect on cells viability. However, if cells were first treated with C12-HSL for 40 mins, and then TQ46 was added, the TQ416 had no effect on cell viability. These results suggest that the C12-HSL-acid process acts at an early step to activate apoptosis as part of C12-HSL’s effect on intestinal mucus barrier function. PMID:27364593

A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

PubMed

Garzón, Ingrid; Carriel, Victor; Marín-Fernández, Ana Belén; Oliveira, Ana Celeste; Garrido-Gómez, Juan; Campos, Antonio; Sánchez-Quevedo, María Del Carmen; Alaminos, Miguel

2012-01-01

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

Cytotoxicity Evaluation of Two Bis-Acryl Composite Resins Using Human Gingival Fibroblasts.

PubMed

Gonçalves, Fabiano Palmeira; Alves, Gutemberg; Guimarães, Vladi Oliveira; Gallito, Marco Antônio; Oliveira, Felipe; Scelza, Míriam Zaccaro

2016-01-01

Bis-acryl resins are used for temporary dental restorations and have shown advantages over other materials. The aim of this work was to evaluate the in vitro cytotoxicity of two bis-acryl composite resins (Protemp 4 and Luxatemp Star), obtained at 1, 7 and 40 days after mixing the resin components, using a standardized assay employing human primary cells closely related to oral tissues. Human gingival fibroblast cell cultures were exposed for 24 h to either bis-acryl composite resins, polystyrene beads (negative control) and latex (positive control) extracts obtained after incubation by the different periods, at 37 °C under 5% CO2. Cell viability was evaluated using a multiparametric procedure involving sequential assessment (using the same cells) of mitochondrial activity (XTT assay), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). The cells exposed to the resin extracts showed cell viability indexes exceeding 75% after 24 h. Even when cells were exposed to extracts prepared with longer conditioning times, the bis-acryl composite resins showed no significant cytotoxic effects (p>0.05), compared to the control group or in relation to the first 24 h of contact with the products. There were no differences among the results obtained for the bis-acryl composite resins evaluated 24 h, 7 days and 40 days after mixing. It may be concluded that the bis-acryl resins Protemp 4 and Luxatemp Star were cytocompatible with human gingival fibroblasts, suggesting that both materials are suitable for use in contact with human tissues.

Density and population viability of coastal marten: a rare and geographically isolated small carnivore

PubMed Central

Green, David S.; Levi, Taal

2018-01-01

Pacific martens (Martes caurina humboldtensis) in coastal forests of Oregon and northern California in the United States are rare and geographically isolated, prompting a petition for listing under the Endangered Species Act. If listed, regulations have the potential to influence land-use decisions on public and private lands, but no estimates of population size, density, or viability of remnant marten populations are available for evaluating their conservation status. We used GPS and VHF telemetry and spatial mark-resight to estimate home ranges, density, and population size of Pacific martens in the Oregon Dunes National Recreation Area, central coast Oregon, USA. We then estimated population viability at differing levels of human-caused mortality (e.g., vehicle mortality). Marten home ranges were small on average (females = 0.8 km2, males 1.5 km2) and density (1.13 martens/1 km2) was the highest reported for North American populations (M. caurina, M. americana). We estimated 71 adult martens (95% CRI [41–87]) across two subpopulations separated by a large barrier (Umpqua River). Using population viability analysis, extinction risk for a subpopulation of 30 martens, approximately the size of the subpopulation south of the Umpqua River, ranged from 32% to 99% with two or three annual human-caused mortalities within 30 years. Absent population expansion, limiting human-caused mortalities will likely have the greatest conservation impact. PMID:29637018

Human islet viability and function is maintained during high density shipment in silicone rubber membrane vessels

PubMed Central

Kitzmann, Jennifer P; Pepper, Andrew R; Lopez, Boris G; Pawlick, Rena; Kin, Tatsuya; O’Gorman, Doug; Mueller, Kathryn R; Gruessner, Angelika C; Avgoustiniatos, Efstathios S; Karatzas, Theodore; Szot, Greg L; Posselt, Andrew M; Stock, Peter G; Wilson, John R; Shapiro, AM; Papas, Klearchos K

2014-01-01

The shipment of human islets from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment). Human islets(IE) were isolated from two manufacturing centers and shipped in 10cm2 surface area SRM vessels in temperature and pressure controlled containers to a distant center following at least two days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a micro centrifuge tube negative control (NC). LD was designed to mimic the standard culture density for human islet preparations (200 IE/cm2), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1–3 vessels. Upon receipt, islets were assessed for viability, measured by oxygen consumption rate normalized to DNA content (OCR/DNA), and quantity, measured by DNA, and, when possible, potency and function with dynamic glucose-stimulated insulin secretion (GSIS) measurements and transplants in immunodeficient B6 rag mice. Post-shipment OCR/DNA was not reduced in HD versus LD, and was substantially reduced in the NC condition. HD islets exhibited normal function post-shipment. Based on the data we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function. PMID:25131090

Human islet viability and function is maintained during high-density shipment in silicone rubber membrane vessels.

PubMed

Kitzmann, J P; Pepper, A R; Gala-Lopez, B; Pawlick, R; Kin, T; O'Gorman, D; Mueller, K R; Gruessner, A C; Avgoustiniatos, E S; Karatzas, T; Szot, G L; Posselt, A M; Stock, P G; Wilson, J R; Shapiro, A M; Papas, K K

2014-01-01

The shipment of human islets (IE) from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment). IE were isolated from two manufacturing centers and shipped in 10-cm(2) surface area SRM vessels in temperature- and pressure-controlled containers to a distant center after at least 2 days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a microcentrifuge tube negative control (NC). LD was designed to mimic the standard culture density for IE preparations (200 IE/cm(2)), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1-3 vessels. Upon receipt, islets were assessed for viability (measured by oxygen consumption rate normalized to DNA content [OCR/DNA)]), quantity (measured by DNA), and, when possible, potency and function (measured by dynamic glucose-stimulated insulin secretion measurements and transplants in immunodeficient B6 Rag(+/-) mice). Postshipment OCR/DNA was not reduced in HD vs LD and was substantially reduced in the NC condition. HD islets exhibited normal function postshipment. Based on the data, we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function. Copyright © 2014 Elsevier Inc. All rights reserved.

High Modulus Biodegradable Polyurethanes for Vascular Stents: Evaluation of Accelerated in vitro Degradation and Cell Viability of Degradation Products

PubMed Central

Sgarioto, Melissa; Adhikari, Raju; Gunatillake, Pathiraja A.; Moore, Tim; Patterson, John; Nagel, Marie-Danielle; Malherbe, François

2015-01-01

We have recently reported the mechanical properties and hydrolytic degradation behavior of a series of NovoSorb™ biodegradable polyurethanes (PUs) prepared by varying the hard segment (HS) weight percentage from 60 to 100. In this study, the in vitro degradation behavior of these PUs with and without extracellular matrix (ECM) coating was investigated under accelerated hydrolytic degradation (phosphate buffer saline; PBS/70°C) conditions. The mass loss at different time intervals and the effect of aqueous degradation products on the viability and growth of human umbilical vein endothelial cells (HUVEC) were examined. The results showed that PUs with HS 80% and below completely disintegrated leaving no visual polymer residue at 18 weeks and the degradation medium turned acidic due to the accumulation of products from the soft segment (SS) degradation. As expected the PU with the lowest HS was the fastest to degrade. The accumulated degradation products, when tested undiluted, showed viability of about 40% for HUVEC cells. However, the viability was over 80% when the solution was diluted to 50% and below. The growth of HUVEC cells is similar to but not identical to that observed with tissue culture polystyrene standard (TCPS). The results from this in vitro study suggested that the PUs in the series degraded primarily due to the SS degradation and the cell viability of the accumulated acidic degradation products showed poor viability to HUVEC cells when tested undiluted, however particles released to the degradation medium showed cell viability over 80%. PMID:26000274

Pollen density on the stigma affects endogenous gibberellin metabolism, seed and fruit set, and fruit quality in Pyrus pyrifolia.

PubMed

Zhang, Caixi; Tateishi, Naoya; Tanabe, Kenji

2010-10-01

To clarify the relationship between pollen density and gametophytic competition in Pyrus pyrifolia, gametophytic performance, gibberellin metabolism, fruit set, and fruit quality were investigated by modifying P. pyrifolia pollen grain number and density with Lycopodium spores. Higher levels of pollen density improved seed viability, fruit set, and fruit quality. Treatments with the highest pollen density showed a significantly increased fruit growth rate and larger fruit at harvest. High pollen density increased germination rate and gave a faster pollen tube growth, both in vivo and in vitro. Endogenous gibberellin (GA) concentrations increased in pollen tubes soon after germination and the concentration of two growth-active GAs, GA(3), and GA(4), was positively correlated to final fruit size, cell numbers in the mesocarp, and pollen tube growth rate. These two GAs appear to be biosynthesized de novo in pollen tube and are the main pollen-derived bioactive GAs found after pollen germination. GA(1) levels in the pollen tube appear to be related to a pollen-style interaction that occurred after the pollen grains landed on the stigma.

Assessment of cryopreserved donor skin viability: the experience of the regional tissue bank of Siena.

PubMed

Pianigiani, E; Tognetti, L; Ierardi, F; Mariotti, G; Rubegni, P; Cevenini, G; Perotti, R; Fimiani, M

2016-06-01

Skin allografts from cadaver donors are an important resource for treating extensive burns, slow-healing wounds and chronic ulcers. A high level of cell viability of cryopreserved allografts is often required, especially in burn surgery, in Italy. Thus, we aimed to determine which conditions enable procurement of highly viable skin in our Regional Skin Bank of Siena. For this purpose, we assessed cell viability of cryopreserved skin allografts procured between 2011 and 2013 from 127 consecutive skin donors, before and after freezing (at day 15, 180, and 365). For each skin donor, we collected data concerning clinical history (age, sex, smoking, phototype, dyslipidemia, diabetes, cause of death), donation process (multi-tissue or multi-organ) and timing of skin procurement (assessment of intervals such as death-harvesting, harvesting-banking, death-banking). All these variables were analysed in the whole case study (127 donors) and in different groups (e.g. multi-organ donors, non refrigerated multi-tissue donors, refrigerated multi-tissue donors) for correlations with cell viability. Our results indicated that cryopreserved skin allografts with higher cell viability were obtained from female, non smoker, heartbeating donors died of cerebral haemorrhage, and were harvested within 2 h of aortic clamping and banked within 12 h of harvesting (13-14 h from clamping). Age, cause of death and dyslipidaemia or diabetes did not appear to influence cell viability. To maintain acceptable cell viability, our skin bank needs to reduce the time interval between harvesting and banking, especially for refrigerated donors.

Maintenance and assessment of cell viability in formulation of non-sporulating bacterial inoculants.

PubMed

Berninger, Teresa; González López, Óscar; Bejarano, Ana; Preininger, Claudia; Sessitsch, Angela

2018-03-01

The application of beneficial, plant-associated microorganisms is a sustainable approach to improving crop performance in agriculture. However, microbial inoculants are often susceptible to prolonged periods of storage and deleterious environmental factors, which negatively impact their viability and ultimately limit efficacy in the field. This particularly concerns non-sporulating bacteria. To overcome this challenge, the availability of protective formulations is crucial. Numerous parameters influence the viability of microbial cells, with drying procedures generally being among the most critical ones. Thus, technological advances to attenuate the desiccation stress imposed on living cells are key to successful formulation development. In this review, we discuss the core aspects important to consider when aiming at high cell viability of non-sporulating bacteria to be applied as microbial inoculants in agriculture. We elaborate the suitability of commonly applied drying methods (freeze-drying, vacuum-drying, spray-drying, fluidized bed-drying, air-drying) and potential measures to prevent cell damage from desiccation (externally applied protectants, stress pre-conditioning, triggering of exopolysaccharide secretion, 'helper' strains). Furthermore, we point out methods for assessing bacterial viability, such as colony counting, spectrophotometry, microcalorimetry, flow cytometry and viability qPCR. Choosing appropriate technologies for maintenance of cell viability and evaluation thereof will render formulation development more efficient. This in turn will aid in utilizing the vast potential of promising, plant beneficial bacteria as sustainable alternatives to standard agrochemicals. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Phenotypic and Genetic Evaluation of the Influence of Pseudomonas aeruginosa Culture Fractions on the Human Mesenchymal Stem Cells Viability, Apoptotic Pathways and Cytokine Profile.

PubMed

Holban, Alina Maria; Bleotu, Coralia; Chifiriuc, Mariana Carmen; Lazar, Veronica

2017-01-01

The objective of this study was to investigate the effects of P. aeruginosa PAO1 cellular and soluble culture fractions on human mesenchymal stem cells (MSCs) death signaling pathways and cytokine profile. The bone marrow isolated MSCs, incubated for different periods of time with one of the three P. aeruginosa PAO1 culture fractions, i.e. low density whole cultures, heat inactivated bacterial cultures sediments and sterile supernatants, were submitted to the following assays: i) fluorescence microscopy evaluation of cellular morphology and viability; ii) bax, caspase 9, relA and bcl-2 genes expression analysis by qRT-PCR; and iii) quantification of the level of IL-1β, IL-6, IL-8 and IL-10 cytokines released in the MSCs supernatants determined by ELISA. Results were statistically analyzed using the GraphPad In Stat software. The PAO1 whole cultures exhibited the most relevant influences, impacting on MSCs morphology and viability, interfering with apoptotic pathways and significantly stimulating the production of IL-1β and IL-10, while decreasing the production of IL-6 and IL-8. The culture supernatants increased the production of IL-1β and reduced the secretion of all other tested cytokines, while heat-inactivated bacterial cells significantly stimulated both IL-1β and IL-10 production. These data could suggest that in vivo, the fate of P. aeruginosa infection depends on the proportion between different bacterial culture fractions (i.e. the number of viable bacterial cells, the number of dead cells and the amount of bacterial soluble products accumulated locally) that could be influenced by the initial infective dose, by the host defense mechanisms, and also by the administered antimicrobial treatment that may thus interfere with the evolution and magnitude of the induced lesions. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Synthetic vs natural scaffolds for human limbal stem cells

PubMed Central

Tominac Trcin, Mirna; Dekaris, Iva; Mijović, Budimir; Bujić, Marina; Zdraveva, Emilija; Dolenec, Tamara; Pauk-Gulić, Maja; Primorac, Dragan; Crnjac, Josip; Špoljarić, Branimira; Mršić, Gordan; Kuna, Krunoslav; Špoljarić, Daniel; Popović, Maja

2015-01-01

Aim To investigate the impact of synthetic electrospun polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, before and after hydrolytic surface modification, on viability and differentiation of cultured human eye epithelial cells, in comparison with natural scaffolds: fibrin and human amniotic membrane. Methods Human placenta was taken at elective cesarean delivery. Fibrin scaffolds were prepared from commercial fibrin glue kits. Nanoscaffolds were fabricated by electrospinning. Limbal cells were isolated from surpluses of human cadaveric cornea and seeded on feeder 3T3 cells. The scaffolds used for viability testing and immunofluorescence analysis were amniotic membrane, fibrin, PU, and PCL nanoscaffolds, with or without prior NaOH treatment. Results Scanning electron microscope photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. There was a significant difference in viability performance between cells with highest viability cultured on tissue culture plastic and cells cultured on all other scaffolds. On the other hand, electrospun PU, PCL, and electrospun PCL treated with NaOH had more than 80% of limbal cells positive for stem cell marker p63 compared to only 27%of p63 positive cells on fibrin. Conclusion Natural scaffolds, fibrin and amniotic membrane, showed better cell viability than electrospun scaffolds. On the contrary, high percentages of p63 positive cells obtained on these scaffolds still makes them good candidates for efficient delivery systems for therapeutic purposes. PMID:26088849

Effect of laser energy, substrate film thickness and bioink viscosity on viability of endothelial cells printed by Laser-Assisted Bioprinting

NASA Astrophysics Data System (ADS)

Catros, Sylvain; Guillotin, Bertrand; Bačáková, Markéta; Fricain, Jean-Christophe; Guillemot, Fabien

2011-04-01

Biofabrication of three dimensional tissues by Laser-Assisted Bioprinting (LAB) implies to develop specific strategies for assembling the extracellular matrix (ECM) and cells. Possible strategies consist in (i) printing cells onto or in the depth of ECM layer and/or (ii) printing bioinks containing both cells and ECM-like printable biomaterial. The aim of this article was to evaluate combinatorial effects of laser pulse energy, ECM thickness and viscosity of the bioink on cell viability. A LAB workstation was used to print Ea.hy926 endothelial cells onto a quartz substrate covered with a film of ECM mimicking Matrigel™. Hence, effect of laser energy, Matrigel™ film thickness and bioink viscosity was addressed for different experimental conditions (8-24 μJ, 20-100 μm and 40-110 mPa s, respectively). Cell viability was assessed by live/dead assay performed 24 h post-printing. Results show that increasing the laser energy tends to augment the cell mortality while increasing the thickness of the Matrigel™ film and the viscosity of the bioink support cell viability. Hence, critical printing parameters influencing high cell viability have been related to the cell landing conditions and more specifically to the intensity of the cell impacts occurring at the air-ECM interface and at the ECM-glass interface.

Nano-textured fluidic biochip as biological filter for selective survival of neuronal cells.

PubMed

Han, Hsieh-Cheng; Lo, Hung-Chun; Wu, Chia-Yu; Chen, Kuei-Hsien; Chen, Li-Chyong; Ou, Keng-Liang; Hosseinkhani, Hossein

2015-06-01

This is an innovative study to engineer biological filter to evaluate the effect of template surface structure and physiochemical properties that can be used for wide variety of applications in biological, health care as well as environmental protection. Specifically, planar silicon (Si) wafer and arrayed Si nano-tips (SiNT) templates were fabricated and coated with gold for various lengths of time to study the effect of surface charge, surface roughness, and hydrophilicity on biological activity of rat pheochromocytoma cell lines PC12. The initial growth and proliferation of PC12 cells on Si and SiNT templates showed an antipathy for the ultra-sharp SiNTs templates. In contrast, the same cells demonstrated a preferable adherence to and proliferation on planar Si templates, resulting in higher cell densities by three orders of magnitude than those on SiNT templates. It is hypothesized that SiNTs array does generate nano-fluidic effect such that the effective contact region for aqueous solution on SiNTs is lower than that on planar Si templates, thus decreasing adsorbable area for cell viability and survival. Moreover, the effect of the gold coating on cell number density was analyzed in terms of the surface roughness, zeta potential and wetting properties of the templates. It was determined that surface charge, as measured by the zeta potential, strongly correlated with the trend observed in the surface cell density, whereas no such correlation was observed for surface roughness or wetting properties in the ranges of our experiment conditions. © 2014 Wiley Periodicals, Inc.

Diffusion of rhodamine B and bovine serum albumin in fibrin gels seeded with primary endothelial cells.

PubMed

Shkilnyy, Andriy; Proulx, Pierre; Sharp, Jamie; Lepage, Martin; Vermette, Patrick

2012-05-01

Scaffolds with adequate mass transport properties are needed in many tissue engineering applications. Fibrin is considered a good biological material to fabricate such scaffolds. However, very little is known about mass transport in fibrin. Therefore, a method based on the analysis of fluorescence intensity for measuring the apparent diffusion coefficient of rhodamine B and fluorescein-labelled bovine serum albumin (FITC-BSA) is described. The experiments are performed in fibrin gels with and without human umbilical vein endothelial cells (HUVEC). The apparent diffusion coefficients of rhodamine B and FITC-BSA in fibrin (fibrinogen concentration of 4 mg/mL) with different cell densities are reported. A LIVE/DEAD(®) assay is performed to confirm the viability of HUVEC seeded at high densities. Diffusion coefficients for rhodamine B remain more or less constant up to 5×10(5) cells/mL and correlate well with literature values measured by other methods in water systems. This indicates that the presence of HUVEC in the fibrin gels (up to 5×10(5) cells/mL) has almost no effect on the diffusion coefficients. Higher cell densities (>5×10(5) cells/mL) result in a decrease of the diffusion coefficients. Diffusion coefficients of rhodamine B and FITC-BSA obtained by this method agree with diffusion coefficients in water predicted by the Stokes-Einstein equation. The experimental design used in this study can be applied to measure diffusion coefficients in different types of gels seeded or not with living cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Effects of Non-Collagenous Proteins, TGF-β1, and PDGF-BB on Viability and Proliferation of Dental Pulp Stem Cells

PubMed Central

Tabatabaei, Fahimeh Sadat

2016-01-01

ABSTRACT Objectives The dentin matrix servers as a reservoir of growth factors, sequestered during dentinogenesis. The aim of this study was to assess the viability and proliferation of dental pulp stem cells in the presence of dentin matrix-derived non-collagenous proteins and two growth factors; platelet-derived growth factor BB and transforming growth factor beta 1. Material and Methods The dental pulp cells were isolated and cultured. The dentin proteins were extracted and purified. The MTT assay was performed for assessment of cell viability and proliferation in the presence of different concentrations of dentin proteins and growth factors during 24 - 72 h post-treatment. Results The cells treated with 250 ng/mL dentin proteins had the best viability and proliferation ability in comparison with other concentrations (P < 0.05). The MTT assay demonstrated that cells cultured with 5 ng/mL platelet-derived growth factor BB had the highest viability at each time point as compared to other groups (P < 0.05). However, in presence of platelet-derived growth factor BB alone and in combination with transforming growth factor beta 1 and dentin proteins (10 ng/mL), significant higher viability was seen at all time points (P < 0.05). The least viability and proliferation at each growth factor concentration was seen in cells treated with combination of transforming growth factor beta 1 and dentin proteins at 72 h (P < 0.05). Conclusions The results indicated that the triple combination of growth factors and matrix-derived non-collagenous proteins (especially at 10 ng/mL concentration) has mitogenic effect on dental pulp stem cells. PMID:27099698

Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

EPA Science Inventory

The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...

Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

EPA Science Inventory

The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

Fiber optic light-scattering measurement system for evaluation of embryo viability: model experiment

NASA Astrophysics Data System (ADS)

Itoh, Harumi; Arai, Tsunenori; Kikuchi, Makoto

1996-05-01

We evaluated the particle density detectability and particle size detectivity of our fiber-optic light-scattering measurement system. In order to prevent the multiple pregnancy on current in vitro fertilization-embryo transfer, we have aimed to develop a new quantitative and non- invasive method to select a single viable human embryo. We employed the measurement of mitochondria localization in an embryo, which may have the correlation with development ability. We applied the angular distribution measurement of the light-scattering intensity from the embryo to obtain the information originated from the mitochondria. The latex spheres with a diameter of 1.0 micrometers were used to simulate the scattering intensity of the mitochondria. The measurement probes of our system consisted of two fibers for illumination and sensing. They were arranged at a right angle to a microscope optical axis to measure the angular distribution of the light-scattering intensity. We observed that the light-scattering intensity increased monotonically in the range from 106 to 1010 particles per ml. Since the mitochondria density in a human embryo corresponded to 2.5 X 107 per ml in the measurement chamber, we may measure the mitochondria density in the human embryo. The angular dependence of light-scattering intensity changed with the sphere diameters. This result showed the possibility of the selective measurement of the mitochondria density in the embryo in spite of the presence of the other cell organelle. We think that our light-scattering measurement system might be applicable to the evaluation method for the embryo viability.

ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

PubMed

Zhang, Di; Ren, Li; Chen, Guan-Qun; Zhang, Jie; Reed, Barbara M; Shen, Xiao-Hui

2015-09-01

Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Cell morphology, viability, osteocalcin activity, and alkaline phosphatase activity in milled versus unmilled surface of the femoral head.

PubMed

Rhyu, Kee Hyung; Cho, Chang Hoon; Yoon, Kyung Sik; Chun, Young Soo

2016-12-01

To evaluate cellular activity in milled versus unmilled surface of the femoral head in 21 patients who underwent robot-assisted total hip arthroplasty(THA). The femoral head of 21 consecutive patients who underwent robot-assisted THA for osteonecrosis was used. 10 cc of trabecular bone from the entire milled surface was obtained using a curette. The same amount of trabecular bone was obtained at least 1 cm away from the milled surface and served as a matched control. Cell morphology, viability, osteocalcin activity, and alkaline phosphatase activity in milled versus unmilled surface were assessed. Cell morphology of the milled or unmilled surface was comparable; cells were smaller in the milled surface. Cell viability was a mean of 40% higher in the milled surface (107.4% vs. 67.2%, p<0.001); cell viability at 5 time points was comparable in each group. Osteocalcin activity of cells was slightly higher in the milled surface (1.43 vs. 1.24 ng/ml, p=0.69). Alkaline phosphatase activity of cells was slightly higher in the unmilled surface (150 105 vs. 141 789 U/L, p=0.078). The milled and unmilled surfaces of the femoral head were comparable in terms of cell morphology, viability, osteocalcin activity, and alkaline phosphatase activity.

Quantitative Analysis of Electrochemical and Electrode Stability with Low Self-Discharge Lithium-Sulfur Batteries

DOE PAGES

Chung, Sheng-Heng; Han, Pauline; Manthiram, Arumugam

2017-06-07

The viability of employing high-capacity sulfur cathodes in building high-energy-density lithium-sulfur batteries is limited by rapid self-discharge, short shelf life, and severe structural degradation during cell resting (static instability). Unfortunately, the static instability has largely been ignored in the literature. We present in this letter a longterm self-discharge study by quantitatively analyzing the control lithium-sulfur batteries with a conventional cathode configuration, which provides meaningful insights into the cathode failure mechanisms during resting. Lastly, utilizing the understanding obtained with the control cells, we design and present low self-discharge (LSD) lithium-sulfur batteries for investigating the long-term self-discharge effect and electrode stability.

Floating cultivation of marine cyanobacteria using coal fly ash.

PubMed

Matsumoto, M; Yoshida, E; Takeyama, H; Matsunaga, T

2000-01-01

The aim of this study was to develop improved methodologies for bulk culturing of biotechnologically useful marine cyanobacteria in the open ocean. We have investigated the viability of using coal fly ash (CFA) blocks as the support medium in a novel floating culture system for marine micro-algae. The marine cyanobacterium Synechococcus sp. NKBG 040607 was found to adhere to floating CFA blocks in liquid culture medium. Maximum density of attached cells of 2.0 x 10(8) cells/cm2 was achieved using seawater. The marine cyanobacterium Synechococcus sp. NKBG 042902 weakly adhered to floating CFA blocks in BG-11 medium. Increasing the concentration of calcium ion in the culture medium enhanced adherence to CFA blocks.

Quantitative Analysis of Electrochemical and Electrode Stability with Low Self-Discharge Lithium-Sulfur Batteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Sheng-Heng; Han, Pauline; Manthiram, Arumugam

The viability of employing high-capacity sulfur cathodes in building high-energy-density lithium-sulfur batteries is limited by rapid self-discharge, short shelf life, and severe structural degradation during cell resting (static instability). Unfortunately, the static instability has largely been ignored in the literature. We present in this letter a longterm self-discharge study by quantitatively analyzing the control lithium-sulfur batteries with a conventional cathode configuration, which provides meaningful insights into the cathode failure mechanisms during resting. Lastly, utilizing the understanding obtained with the control cells, we design and present low self-discharge (LSD) lithium-sulfur batteries for investigating the long-term self-discharge effect and electrode stability.

Taurine Protected Against the Impairments of Neural Stem Cell Differentiated Neurons Induced by Oxygen-Glucose Deprivation.

PubMed

Xiao, Bo; Liu, Huazhen; Gu, Zeyun; Liu, Sining; Ji, Cheng

2015-11-01

Cell transplantation of neural stem cells (NSCs) is a promising approach for neurological recovery both structurally and functionally. However, one big obstacle is to promote differentiation of NSCs into neurons and the followed maturation. In the present study, we aimed to investigate the protective effect of taurine on the differentiation of NSCs and subsequent maturation of their neuronal lineage, when exposed to oxygen-glucose deprivation (OGD). The results suggested that taurine (5-20 mM) promoted the viability and proliferation of NSCs, and it protected against 8 h of OGD induced impairments. Furthermore, 20 mM taurine promoted NSCs to differentiate into neurons after 7 days of culture, and it also protected against the suppressive impairments of 8 h of OGD. Consistently, taurine (20 mM) promoted the neurite sprouting and outgrowth of the NSC differentiated neurons after 14 days of differentiation, which were significantly inhibited by OGD (8 h). At D21, the mushroom spines and spine density were promoted or restored by 20 mM taurine. Taken together, the enhanced viability and proliferation of NSCs, more differentiated neurons and the promoted maturation of neurons by 20 mM taurine support its therapeutic application during stem cell therapy to enhance neurological recovery. Moreover, it protected against the impairments induced by OGD, which may highlight its role for a more direct therapeutic application especially in an ischemic stroke environment.

Multiplexing a high-throughput liability assay to leverage efficiencies.

PubMed

Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

2009-06-01

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

Predicting cell viability within tissue scaffolds under equiaxial strain: multi-scale finite element model of collagen-cardiomyocytes constructs.

PubMed

Elsaadany, Mostafa; Yan, Karen Chang; Yildirim-Ayan, Eda

2017-06-01

Successful tissue engineering and regenerative therapy necessitate having extensive knowledge about mechanical milieu in engineered tissues and the resident cells. In this study, we have merged two powerful analysis tools, namely finite element analysis and stochastic analysis, to understand the mechanical strain within the tissue scaffold and residing cells and to predict the cell viability upon applying mechanical strains. A continuum-based multi-length scale finite element model (FEM) was created to simulate the physiologically relevant equiaxial strain exposure on cell-embedded tissue scaffold and to calculate strain transferred to the tissue scaffold (macro-scale) and residing cells (micro-scale) upon various equiaxial strains. The data from FEM were used to predict cell viability under various equiaxial strain magnitudes using stochastic damage criterion analysis. The model validation was conducted through mechanically straining the cardiomyocyte-encapsulated collagen constructs using a custom-built mechanical loading platform (EQUicycler). FEM quantified the strain gradients over the radial and longitudinal direction of the scaffolds and the cells residing in different areas of interest. With the use of the experimental viability data, stochastic damage criterion, and the average cellular strains obtained from multi-length scale models, cellular viability was predicted and successfully validated. This methodology can provide a great tool to characterize the mechanical stimulation of bioreactors used in tissue engineering applications in providing quantification of mechanical strain and predicting cellular viability variations due to applied mechanical strain.

Is cell viability always directly related to corrosion resistance of stainless steels?

PubMed

Salahinejad, E; Ghaffari, M; Vashaee, D; Tayebi, L

2016-05-01

It has been frequently reported that cell viability on stainless steels is improved by increasing their corrosion resistance. The question that arises is whether human cell viability is always directly related to corrosion resistance in these biostable alloys. In this work, the microstructure and in vitro corrosion behavior of a new class of medical-grade stainless steels were correlated with adult human mesenchymal stem cell viability. The samples were produced by a powder metallurgy route, consisting of mechanical alloying and liquid-phase sintering with a sintering aid of a eutectic Mn-Si alloy at 1050 °C for 30 and 60 min, leading to nanostructures. In accordance with transmission electron microscopic studies, the additive particles for the sintering time of 30 min were not completely melted. Electrochemical impedance spectroscopic experiments suggested the higher corrosion resistance for the sample sintered for 60 min; however, a better cell viability on the surface of the less corrosion-resistant sample was unexpectedly found. This behavior is explained by considering the higher ion release rate of the Mn-Si additive material, as preferred sites to corrosion attack based on scanning electron microscopic observations, which is advantageous to the cells in vitro. In conclusion, cell viability is not always directly related to corrosion resistance in stainless steels. Typically, the introduction of biodegradable and biocompatible phases to biostable alloys, which are conventionally anticipated to be corrosion-resistant, can be advantageous to human cell responses similar to biodegradable metals. Copyright © 2016 Elsevier B.V. All rights reserved.

Hyaluronic acid increases tendon derived cell viability and proliferation in vitro: comparative study of two different hyaluronic acid preparations by molecular weight.

PubMed

Gallorini, Marialucia; Berardi, Anna C; Berardocco, Martina; Gissi, Clarissa; Maffulli, Nicola; Cataldi, Amelia; Oliva, Francesco

2017-01-01

Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate the effects of two different HA on human rotator cuff tendon derived cells in terms of cell viability, proliferation and apoptosis. An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of two different HA preparations: Sinovial HL® (High-Low molecular weight) (MW: 80-100 kDa) and KDa Sinovial Forte SF (MW: 800-1200), at various concentrations. Tendon derived cells morphology was evaluated after 0, 7 and 14 d of culture. Viability and proliferation were analyzed after 0, 24, and 48 h of culture and apoptosis occurrence was assessed after 24 h of culture. All the HAPs tested here increased viability and proliferation, in a dose-dependent manner and they reduced apoptosis at early stages (24 h) compared to control cells (without HAPs). HAPs enhanced viability and proliferation and counteracted apoptosis in tendon derived cells.

Study of wettability and cell viability of H implanted stainless steel

NASA Astrophysics Data System (ADS)

Shafique, Muhammad Ahsan; Ahmad, Riaz; Rehman, Ihtesham Ur

2018-03-01

In the present work, the effect of hydrogen ion implantation on surface wettability and biocompatibility of stainless steel is investigated. Hydrogen ions are implanted in the near-surface of stainless steel to facilitate hydrogen bonding at different doses with constant energy of 500 KeV, which consequently improve the surface wettability. Treated and untreated sample are characterized for surface wettability, incubation of hydroxyapatite and cell viability. Contact angle (CA) study reveals that surface wettability increases with increasing H-ion dose. Raman spectroscopy shows that precipitation of hydroxyapatite over the surface increase with increasing dose of H-ions. Cell viability study using MTT assay describes improved cell viability in treated samples as compared to the untreated sample. It is found that low dose of H-ions is more effective for cell proliferation and the cell count decreases with increasing ion dose. Our study demonstrates that H ion implantation improves the surface wettability and biocompatibility of stainless steel.

Bioinspired Pollen-Like Hierarchical Surface for Efficient Recognition of Target Cancer Cells.

PubMed

Wang, Wenshuo; Yang, Gao; Cui, Haijun; Meng, Jingxin; Wang, Shutao; Jiang, Lei

2017-08-01

The efficient recognition and isolation of rare cancer cells holds great promise for cancer diagnosis and prognosis. In nature, pollens exploit spiky structures to realize recognition and adhesion to stigma. Herein, a bioinspired pollen-like hierarchical surface is developed by replicating the assembly of pollen grains, and efficient and specific recognition to target cancer cells is achieved. The pollen-like surface is fabricated by combining filtering-assisted assembly and soft lithography-based replication of pollen grains of wild chrysanthemum. After modification with a capture agent specific to cancer cells, the pollen-like surface enables the capture of target cancer cells with high efficiency and specificity. In addition, the pollen-like surface not only assures high viability of captured cells but also performs well in cell mixture system and at low cell density. This study represents a good example of constructing cell recognition biointerfaces inspired by pollen-stigma adhesion. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels.

PubMed

Bertassoni, Luiz E; Cardoso, Juliana C; Manoharan, Vijayan; Cristino, Ana L; Bhise, Nupura S; Araujo, Wesleyan A; Zorlutuna, Pinar; Vrana, Nihal E; Ghaemmaghami, Amir M; Dokmeci, Mehmet R; Khademhosseini, Ali

2014-06-01

Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least eight days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

NASA Astrophysics Data System (ADS)

Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V.; Noll, Thomas; Funk, Richard H. W.; Engelmann, Katrin; Werner, Carsten

2015-08-01

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

In vitro immunomodulatory potential of Artemisia indica Willd. in chicken lymphocytes.

PubMed

Ruwali, Pushpa; Ambwani, Tanuj Kumar; Gautam, Pankaj

2018-01-01

Evaluation of the in vitro immunomodulatory potential of Artemisia indica Willd. methanolic extract in chicken lymphocyte culture system through lymphocyte (B and T cells) proliferation assay, after standardizing the maximum non-cytotoxic dose (MNCD) in chicken lymphocytes. Fresh aerial parts of A. indica Willd. (family: Asteraceae) specimens were collected (altitude 1560 m), gotten authenticated, processed, dried, and Soxhlet extracted to yield methanolic extract (AME). Chicken splenocytes were isolated from spleens collected from healthy birds; lymphocytes were separated by density gradient centrifugation, percentage cell viability determined and final cell count adjusted to 10 7 cells/ml in RPMI-1640 medium. MNCD of AME in chicken lymphocytes was determined through 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide dye reduction assay. Immunomodulatory potential of AME was evaluated through lymphocytes proliferation or B and T cells blastogenesis assay in the presence of appropriate mitogens, namely, lipopolysaccharide (LPS) and concanavalin A (Con A), respectively. Maximum concentration of AME exhibiting 100% cell viability (MNCD) was 200 μg/ml and was selected for further in vitro analysis. The in vitro exposure of chicken lymphocytes to 200 µg/ml dose of AME, resulted in significant (p<0.05) upregulation of 11.76% in B cell proliferation in the presence of B cell mitogen (LPS) and a significant (p<0.05) increase of 12.018% T cells proliferation in the presence of the mitogen (Con A), as compared to the control. The significant upregulation in the proliferation of two major cell types modulating the immune system is an indication of the immunostimulatory potential of the plant. It would be worthwhile to further evaluate A. indica on relevant immunomodulatory aspects, especially the in vivo studies in a poultry system.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)-the effect of biomolecular ligands to balance cell adhesion and stimulated detachment.

PubMed

Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V; Noll, Thomas; Funk, Richard H W; Engelmann, Katrin; Werner, Carsten

2015-08-01

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly( N -isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na + /K + -ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro . The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

PubMed Central

Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V; Noll, Thomas; Funk, Richard H W; Engelmann, Katrin; Werner, Carsten

2015-01-01

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty. PMID:27877823

Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells

PubMed Central

Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W.; Ras, Mat; Allbritton, Nancy L.; Sims, Christopher E.; Venugopalan, Vasan

2012-01-01

We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass–pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s−1 through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s−1 and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining optimal operating conditions for laser microbeam-based pallet release systems for the isolation and selection of adherent cells. PMID:22158840

Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells.

PubMed

Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W; Ras, Mat; Allbritton, Nancy L; Sims, Christopher E; Venugopalan, Vasan

2012-06-07

We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass-pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s(-1) through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s(-1) and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining optimal operating conditions for laser microbeam-based pallet release systems for the isolation and selection of adherent cells.

Nanocomposites of high-density polyethylene with amorphous calcium phosphate: in vitro biomineralization and cytocompatibility of human mesenchymal stem cells.

PubMed

Hild, Nora; Fuhrer, Roland; Mohn, Dirk; Bubenhofer, Stephanie B; Grass, Robert N; Luechinger, Norman A; Feldman, Kirill; Dora, Claudio; Stark, Wendelin J

2012-10-01

Polyethylene is widely used as a component of implants in medicine. Composites made of high-density polyethylene (HDPE) containing different amounts of amorphous calcium phosphate nanoparticles were investigated concerning their in vitro biomedical performance. The nanoparticles were produced by flame spray synthesis and extruded with HDPE, the latter complying with Food and Drug Administration regulations. Mechanical properties such as Young's modulus and contact angle as well as in vitro biomineralization of the nanocomposites hot-pressed into thin films were evaluated. The deposition of a hydroxyapatite layer occurred upon immersion in simulated body fluid. Additionally, a cell culture study with human mesenchymal stem cells for six weeks allowed a primary assessment of the cytocompatibility. Viability assays (alamarBlue and lactate dehydrogenase detection) proved the absence of cytotoxic effects of the scaffolds. Microscopic images after hematoxylin and eosin staining confirmed typical growth and morphology. A preliminary experiment analyzed the alkaline phosphatase activity after two weeks. These findings motivate further investigations on bioactive HDPE in bone tissue engineering.

A Field-Portable Cell Analyzer without a Microscope and Reagents.

PubMed

Seo, Dongmin; Oh, Sangwoo; Lee, Moonjin; Hwang, Yongha; Seo, Sungkyu

2017-12-29

This paper demonstrates a commercial-level field-portable lens-free cell analyzer called the NaviCell (No-stain and Automated Versatile Innovative cell analyzer) capable of automatically analyzing cell count and viability without employing an optical microscope and reagents. Based on the lens-free shadow imaging technique, the NaviCell (162 × 135 × 138 mm³ and 1.02 kg) has the advantage of providing analysis results with improved standard deviation between measurement results, owing to its large field of view. Importantly, the cell counting and viability testing can be analyzed without the use of any reagent, thereby simplifying the measurement procedure and reducing potential errors during sample preparation. In this study, the performance of the NaviCell for cell counting and viability testing was demonstrated using 13 and six cell lines, respectively. Based on the results of the hemocytometer ( de facto standard), the error rate (ER) and coefficient of variation (CV) of the NaviCell are approximately 3.27 and 2.16 times better than the commercial cell counter, respectively. The cell viability testing of the NaviCell also showed an ER and CV performance improvement of 5.09 and 1.8 times, respectively, demonstrating sufficient potential in the field of cell analysis.

Oxygen measurement in interstitially perfused cellularized constructs cultured in a miniaturized bioreactor.

PubMed

Raimondi, Manuela T; Giordano, Carmen; Pietrabissa, Riccardo

2015-12-18

The possibility of developing engineered tissue in vitro and maintaining the cell viability and functionality is primarily related to the possibility of controlling key culture parameters such as oxygen concentration and cell-specific oxygen consumption. We measured these parameters in a three-dimensional (3D) cellularized construct maintained under interstitially perfused culture in a miniaturized bioreactor. MG63 osteosarcoma cells were seeded at high density on a 3D polystyrene scaffold. The 3D scaffolds were sensorized with sensor foils made of a polymer, which fluoresce with intensity proportional to the local oxygen tension. Images of the sensor foil in contact with the cellularized construct were acquired with a video camera every four hours for six culture days and were elaborated with analytical imaging software to obtain oxygen concentration maps. The data collected indicate a globally decreasing oxygen concentration profile, with a total drop of 28% after six days of culture and an average drop of 10.5% between the inlet and outlet of the perfused construct. Moreover, by importing the measured oxygen concentration data and the cell counts in a model of mass transport, we calculated the cell-specific oxygen consumption over the whole culture period. The consumption increased with oxygen availability and ranged from 0.1 to 0.7 µmol/h/106 cells. The sensors used here allowed a non-invasive, contamination-free and non-destructive oxygen measurement over the whole culture period. This study is the basis for optimization of the culture parameters involved in oxygen supply, in order to guarantee maintenance of cell viability in our system.

Improved immunomagnetic enrichment of CD34(+) cells from umbilical cord blood using the CliniMACS cell separation system.

PubMed

Blake, Joseph M; Nicoud, Ian B; Weber, Daniel; Voorhies, Howard; Guthrie, Katherine A; Heimfeld, Shelly; Delaney, Colleen

2012-08-01

CD34(+) enrichment from cord blood units (CBU) is used increasingly in clinical applications involving ex vivo expansion. The CliniMACS instrument from Miltenyi Biotec is a current good manufacturing practice (cGMP) immunomagnetic selection system primarily designed for processing larger numbers of cells: a standard tubing set (TS) can process a maximum of 60 billion cells, while the larger capacity tubing set (LS) will handle 120 billion cells. In comparison, most CBU contain only 1-2 billion cells, raising a question regarding the optimal tubing set for CBU CD34(+) enrichment. We compared CD34(+) cell recovery and overall viability after CliniMACS processing of fresh CBU with either TS or LS. Forty-six freshly collected CBU (≤ 36 h) were processed for CD34(+) enrichment; 22 consecutive units were selected using TS and a subsequent 24 processed with LS. Cell counts and immunophenotyping were performed pre- and post-selection to assess total nucleated cells (TNC), viability and CD34(+) cell content. Two-sample t-tests of mean CD34(+) recovery and viability revealed significant differences in favor of LS (CD34(+) recovery, LS = 56%, TS = 45%, P = 0.003; viability, LS = 74%, TS = 59%, P = 0.011). Stepwise linear regression, considering pre-processing unit age, viability, TNC and CD34(+) purity, demonstrated statistically significant correlations only with the tubing set used and age of unit. For CD34(+) enrichment from fresh CBU, LS provided higher post-selection viability and more efficient recovery. In this case, a lower maximum TNC specification of TS was not predictive of better performance. The same may hold for smaller scale enrichment of other cell types with the CliniMACS instrument.

Zoledronic Acid Inhibits Aromatase Activity and Phosphorylation: Potential Mechanism for Additive Zoledronic Acid and Letrozole Drug Interaction

PubMed Central

Schech, Amanda J.; Nemieboka, Brandon E.; Brodie, Angela H.

2012-01-01

Zoledronic acid (ZA), a bisphosphonate originally indicated for use in osteoporosis, has been reported to exert a direct effect on breast cancer cells, although the mechanism of this effect is currently unknown. Data from the ABCSG-12 and ZO-FAST clinical trials suggest that treatment with the combination of ZA and aromatase inhibitors (AI) result in increased disease free survival in breast cancer patients over AI alone. To determine whether the mechanism of this combination involved inhibition of aromatase, AC-1 cells (MCF-7 human breast cancer cells transfected with an aromatase construct) were treated simultaneously with combinations of ZA and AI letrozole for 72 hours. This combination significantly increased inhibition of aromatase activity of AC-1 cells by compared to letrozole alone. Combination treatment of 1nM letrozole and 1μM and 10μM zoledronic acid resulted in an additive drug interaction on inhibiting cell viability, as measured by MTT assay. Treatment with ZA was found to inhibit phosphorylation of aromatase on serine 473. Zoledronic acid was also shown to be more effective in inhibiting cell viability in aromatase transfected AC-1 cells when compared to inhibition of cell viability observed in non-transfected MCF-7. Estradiol was able to partially rescue the effect of 1μM and 10μM ZA on cell viability following treatment for 72 hours, as shown by a shift to the right in the estradiol dose response curve. In conclusion, these results indicate that the combination of ZA and letrozole results in an additive inhibition of cell viability. Furthermore, ZA alone can inhibit aromatase activity through inhibition of serine phosphorylation events important for aromatase enzymatic activity and contributes to inhibition of cell viability. PMID:22659283

Analysis of Required Supporting Systems for the Supercritical CO(2) Power Conversion System

DTIC Science & Technology

2007-09-01

been drawn to the viability of using S-C02 as a working fluid in modern reactor designs. Near the critical point, C02 has a rapid rise in density...viability of using S-CO2 as a working fluid in modern reactor designs. Near the critical point, CO2 has a rapid rise in density allowing a significant...32 Figure 2.2.3 Effect on Mass Transferred of Changing ICV Initial Temperature for emptying PCS ...................32 Figure 2.2.4 Effect

Monosodium urate monohydrate crystals inhibit osteoblast viability and function: implications for development of bone erosion in gout.

PubMed

Chhana, Ashika; Callon, Karen E; Pool, Bregina; Naot, Dorit; Watson, Maureen; Gamble, Greg D; McQueen, Fiona M; Cornish, Jillian; Dalbeth, Nicola

2011-09-01

Bone erosion is a common manifestation of chronic tophaceous gout. To investigate the effects of monosodium urate monohydrate (MSU) crystals on osteoblast viability and function. The MTT assay and flow cytometry were used to assess osteoblast cell viability in the MC3T3-E1 and ST2 osteoblast-like cell lines, and primary rat and primary human osteoblasts cultured with MSU crystals. Quantitative real-time PCR and von Kossa stained mineralised bone formation assays were used to assess the effects of MSU crystals on osteoblast differentiation using MC3T3-E1 cells. The numbers of osteoblasts and bone lining cells were quantified in bone samples from patients with gout. MSU crystals rapidly reduced viability in all cell types in a dose-dependent manner. The inhibitory effect on cell viability was independent of crystal phagocytosis and was not influenced by differing crystal length or addition of serum. Long-term culture of MC3T3-E1 cells with MSU crystals showed a reduction in mineralisation and decreased mRNA expression of genes related to osteoblast differentiation such as Runx2, Sp7 (osterix), Ibsp (bone sialoprotein), and Bglap (osteocalcin). Fewer osteoblast and lining cells were present on bone directly adjacent to gouty tophus than bone unaffected by tophus in patients with gout. MSU crystals have profound inhibitory effects on osteoblast viability and differentiation. These data suggest that bone erosion in gout occurs at the tophus-bone interface through alteration of physiological bone turnover, with both excessive osteoclast formation, and reduced osteoblast differentiation from mesenchymal stem cells.

Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matveeva, V. G., E-mail: matveeva-vg@mail.ru; Antonova, L. V., E-mail: antonova.la@mail.ru; Velikanova, E. A.

We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwovenmore » scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.« less

Comparative evaluation of the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin scaffold

PubMed Central

Khurana, Rohit; Kudva, Praveen Bhasker; Husain, Syed Yawer

2017-01-01

Background: The present study aims to comparatively evaluate the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin (PRF) scaffold. Materials and Methods: A total of 15 systemically healthy individuals between the age group of 15–25 years requiring third molar or orthodontic premolar extractions. Teeth were extracted atraumatically and transported to the laboratory. Stem cells were isolated from dental pulp and periodontal ligament. After attaining more than 90% confluency by the 7th day, these cells were tested for their viability and characterization. Stem cells were also incubated with PRF and viability was assessed on the 7th day. Results: The mean number of cell for dental pulp stem cells (DPSCs) and periodontal ligament stem cell (PDLSC) was statistically insignificant (P > 0.05). The mean live cell viability was compared between DPSC (98.07%) and PDLSC (98%). Both DPSC and PDLSC showed a high percentage of expression of CD73 markers, 30.40% and 29.80%, respectively. However, DPSCs and PDLSCs lacked expression of CD34 expressing only 3.47% and 3.53%, respectively. PRF membrane as a scaffold exhibited no cytotoxic effects on DPCS's or PDLSC's. The cell viability of cells cultured with PRF was statistically insignificant (P > 0.05) when compared to the cells cultured with culture media. Conclusion: The study thus indicates that dental pulp and periodontal ligament are both rich sources of mesenchymal stem cells and can be successfully used for obtaining stem cells. PRF exhibits no cytotoxic effects on the cells and can be used in conjunction with dental stem cells. PMID:29386795

Agglomeration, sedimentation, and cellular toxicity of alumina nanoparticles in cell culture medium

NASA Astrophysics Data System (ADS)

Yoon, Dokyung; Woo, Daekwang; Kim, Jung Heon; Kim, Moon Ki; Kim, Taesung; Hwang, Eung-Soo; Baik, Seunghyun

2011-06-01

The cytotoxicity of alumina nanoparticles (NPs) was investigated for a wide range of concentration (25-200 μg/mL) and incubation time (0-72 h) using floating cells (THP-1) and adherent cells (J774A.1, A549, and 293). Alumina NPs were gradually agglomerated over time although a significant portion of sedimentation occurred at the early stage within 6 h. A decrease of the viability was found in floating (THP-1) and adherent (J774A.1 and A549) cells in a dose-dependent manner. However, the time-dependent decrease in cell viability was observed only in adherent cells (J774A.1 and A549), which is predominantly related with the sedimentation of alumina NPs in cell culture medium. The uptake of alumina NPs in macrophages and an increased cell-to-cell adhesion in adherent cells were observed. There was no significant change in the viability of 293 cells. This in vitro test suggests that the agglomeration and sedimentation of alumina NPs affected cellular viability depending on cell types such as monocytes (THP-1), macrophages (J774A.1), lung carcinoma cells (A549), and embryonic kidney cells (293).

Effect of fluoride on the cell viability, cell organelle potential, and photosynthetic capacity of freshwater and soil algae.

PubMed

Chae, Yooeun; Kim, Dokyung; An, Youn-Joo

2016-12-01

Although fluoride occurs naturally in the environment, excessive amounts of fluoride in freshwater and terrestrial ecosystems can be harmful. We evaluated the toxicity of fluoride compounds on the growth, viability, and photosynthetic capacity of freshwater (Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata) and terrestrial (Chlorococcum infusionum) algae. To measure algal growth inhibition, a flow cytometric method was adopted (i.e., cell size, granularity, and auto-fluorescence measurements), and algal yield was calculated to assess cell viability. Rhodamine123 and fluorescein diacetate were used to evaluate mitochondrial membrane potential (MMA, ΔΨ m ) and cell permeability. Nine parameters related to the photosynthetic capacity of algae were also evaluated. The results indicated that high concentrations of fluoride compounds affected cell viability, cell organelle potential, and photosynthetic functions. The cell viability measurements of the three algal species decreased, but apoptosis was only observed in C. infusionum. The MMA (ΔΨ m ) of cells exposed to fluoride varied among species, and the cell permeability of the three species generally decreased. The decrease in the photosynthetic activity of algae may be attributable to the combination of fluoride ions (F - ) with magnesium ions (Mg 2+ ) in chlorophyll. Our results therefore provide strong evidence for the potential risks of fluoride compounds to microflora and microfauna in freshwater and terrestrial ecosystems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Changes in localization and density of CD63-positive exosome-like substances in the hen oviduct with artificial insemination and their effect on sperm viability.

PubMed

Huang, A; Isobe, N; Yoshimura, Y

2017-10-01

Avian sperm are stored in the sperm storage tubules (SSTs) of the hen oviduct for a prolonged period. However, the precise mechanisms by which sperm are kept alive in the SSTs are still not fully understood. The aim of this study was to determine whether exosomes are secreted by SST cells and play a role in the survival of sperm. Utero-vaginal junction (UVJ) tissue from approximately 50 wk old White Leghorn hens was collected before (control group) and after intravaginal insemination with seminal plasma (SP group) or semen (AI group). The samples were used to prepare frozen sections and total protein extraction. The localization of the CD63, an exosome marker, was determined by immunohistochemistry and its protein level in the UVJ mucosal tissues was examined by Western blot. Exosomes were isolated from the culture media of UVJ and vaginal mucosa cells by ultracentrifugation and characterized by SDS-PAGE and Western blot. The viability and motility of sperm incubated with exosomes were also examined. CD63 was localized in the apical region of UVJ mucosal epithelium cells and SST cells of control, SP, and AI groups. The CD63 protein decreased in SST cells surrounding resident sperm and tended to appear in the SST lumen in the AI group. The protein level of CD63 in UVJ mucosal tissues was significantly higher in the AI group than control. The CD63 protein (approximately 75 kDa) was detected in ultracentrifugation pellets from the culture medium of UVJ and vagina cells. The viability of sperm incubated with 1 μg/μl vaginal exosomes was significantly decreased but was not affected by UVJ exosomes. These results suggest that exosomes were synthesized by SST cells and may be secreted into SST lumen when sperm were stored in SSTs. The role of SST exosomes in sperm storage needs to be examined further. Copyright © 2017 Elsevier Inc. All rights reserved.

Viability and Virulence of Experimentally Stressed Nonculturable Salmonella typhimurium

PubMed Central

Caro, Audrey; Got, Patrice; Lesne, Jean; Binard, Sylvie; Baleux, Bernard

1999-01-01

Maintenance of pathogenicity of viable but nonculturable Salmonella typhimurium cells experimentally stressed with UV-C and seawater, was investigated relative to the viability level of the cellular population. Pathogenicity, tested in a mouse model, was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by respiratory activity and cytoplasmic membrane and genomic integrities. PMID:10388726

Curcumin and Viscum album Extract Decrease Proliferation and Cell Viability of Soft-Tissue Sarcoma Cells: An In Vitro Analysis of Eight Cell Lines Using Real-Time Monitoring and Colorimetric Assays.

PubMed

Harati, K; Behr, B; Daigeler, A; Hirsch, T; Jacobsen, F; Renner, M; Harati, A; Wallner, C; Lehnhardt, M; Becerikli, M

2017-01-01

The cytostatic effects of the polyphenol curcumin and Viscum album extract (VAE) were assessed in soft-tissue sarcoma (STS) cells. Eight human STS cell lines were used: fibrosarcoma (HT1080), liposarcoma (SW872, T778, MLS-402), synovial sarcoma (SW982, SYO1, 1273), and malignant fibrous histiocytoma (U2197). Primary human fibroblasts served as control cells. Cell proliferation, viability, and cell index (CI) were analyzed by BrdU assay, MTT assay, and real-time cell analysis (RTCA). As indicated by BrdU and MTT, curcumin significantly decreased the cell proliferation of five cell lines (HT1080, SW872, SYO1, 1273, and U2197) and the viability of two cell lines (SW872 and SW982). VAE led to significant decreases of proliferation in eight cell lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, 1293, and U2197) and reduced viability in seven STS lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, and 1273). As indicated by RTCA for 160 h, curcumin decreased the CI of all synovial sarcoma cell lines as well as T778 and HT1080. VAE diminished the CI in most of the synovial sarcoma (SW982, SYO1) and liposarcoma (SW872, T778) cell lines as well as HT1080. Primary fibroblasts were not affected adversely by the two compounds in RTCA. Curcumin and VAE can inhibit the proliferation and viability of STS cells.

Dragon's Blood Sap (Croton Lechleri) As Storage Medium For Avulsed Teeth: In Vitro Study Of Cell Viability.

PubMed

Martins, Christine Men; Hamanaka, Elizane Ferreira; Hoshida, Thayse Yumi; Sell, Ana Maria; Hidalgo, Mirian Marubayashi; Silveira, Catarina Soares; Poi, Wilson Roberto

2016-01-01

Tooth replantation success depends on the condition of cementum periodontal ligament after tooth avulsion; which is influenced by storage medium. The dragon's blood (Croton lechleri) sap has been suggested as a promising medium because it supports collagen formation and exhibits healing, anti-inflammatory and antimicrobial properties. Thus, the aim of this study was to evaluate the efficacy of dragon's blood sap as a storage medium for avulsed teeth through evaluation of functional and metabolic cell viability. This in vitro study compared the efficacy of different storage media to maintain the viability of human peripheral blood mononuclear and periodontal ligament cells. A 10% dragon's blood sap was tested while PBS was selected as its control. Ultra pasteurized whole milk was used for comparison as a commonly used storage medium. DMEM and distilled water were the positive and negative controls, respectively. The viability was assessed through trypan blue exclusion test and colorimetric MTT assay after 1, 3, 6, 10 and 24 h of incubation. The dragon's blood sap showed promising results due to its considerable maintenance of cell viability. For trypan blue test, the dragon's blood sap was similar to milk (p<0.05) and both presented the highest viability values. For MTT, the dragon's blood sap showed better results than all storage media, even better than milk (p<0.05). It was concluded that the dragon's blood sap was as effective as milk, the gold standard for storage medium. The experimental sap preserved the membrane of all cells and the functional viability of periodontal ligament cells.

The Effects of Oxygen Level and Glucose Concentration on the Metabolism of Porcine TMJ Disc Cells

PubMed Central

Cisewski, Sarah E.; Zhang, Lixia; Kuo, Jonathan; Wright, Gregory J.; Wu, Yongren; Kern, Michael J.; Yao, Hai

2015-01-01

Objective To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells. Design TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 hours with 0, 1.5, 5, or 25mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-3H]proline and [35S]sulfate into the cells, respectively. Results TMJ disc cell viability significantly decreased (P<0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P<0.0001), while a decrease in glucose concentration significantly decreased viability (P<0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P<0.0001) and matrix synthesis (P<0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P<0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P<0.0001), ATP production (P=0.00015), and collagen (P=0.0002) and proteoglycan synthesis (P<0.0001). Conclusions Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply. PMID:26033165

The effects of oxygen level and glucose concentration on the metabolism of porcine TMJ disc cells.

PubMed

Cisewski, S E; Zhang, L; Kuo, J; Wright, G J; Wu, Y; Kern, M J; Yao, H

2015-10-01

To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells. TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 h with 0, 1.5, 5, or 25 mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-(3)H] proline and [(35)S] sulfate into the cells, respectively. TMJ disc cell viability significantly decreased (P < 0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P < 0.0001), while a decrease in glucose concentration significantly decreased viability (P < 0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P < 0.0001) and matrix synthesis (P < 0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P < 0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P < 0.0001), ATP production (P = 0.00015), and collagen (P = 0.0002) and proteoglycan synthesis (P < 0.0001). Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Molecular Viability Testing of UV-Inactivated Bacteria.

PubMed

Weigel, Kris M; Nguyen, Felicia K; Kearney, Moira R; Meschke, John S; Cangelosi, Gerard A

2017-05-15

PCR is effective in detecting bacterial DNA in samples, but it is unable to differentiate viable bacteria from inactivated cells or free DNA fragments. New PCR-based analytical strategies have been developed to address this limitation. Molecular viability testing (MVT) correlates bacterial viability with the ability to rapidly synthesize species-specific rRNA precursors (pre-rRNA) in response to brief nutritional stimulation. Previous studies demonstrated that MVT can assess bacterial inactivation by chlorine, serum, and low-temperature pasteurization. Here, we demonstrate that MVT can detect inactivation of Escherichia coli , Aeromonas hydrophila , and Enterococcus faecalis cells by UV irradiation. Some UV-inactivated E. coli cells transiently retained the ability to synthesize pre-rRNA postirradiation (generating false-positive MVT results), but this activity ceased within 1 h following UV exposure. Viable but transiently undetectable (by culture) E. coli cells were consistently detected by MVT. An alternative viability testing method, viability PCR (vPCR), correlates viability with cell envelope integrity. This method did not distinguish viable bacteria from UV-inactivated bacteria under some conditions, indicating that the inactivated cells retained intact cell envelopes. MVT holds promise as a means to rapidly assess microbial inactivation by UV treatment. IMPORTANCE UV irradiation is increasingly being used to disinfect water, food, and other materials for human use. Confirming the effectiveness of UV disinfection remains a challenging task. In particular, microbiological methods that rely on rapid detection of microbial DNA can yield misleading results, due to the detection of remnant DNA associated with dead microbial cells. This report describes a novel method that rapidly distinguishes living microbial cells from dead microbial cells after UV disinfection. Copyright © 2017 American Society for Microbiology.

MiR-34a Inhibits Viability and Invasion of Human Papillomavirus-Positive Cervical Cancer Cells by Targeting E2F3 and Regulating Survivin.

PubMed

Geng, Dianzhong; Song, Xiaohua; Ning, Fangling; Song, Qianhua; Yin, Honghua

2015-05-01

Previous studies confirmed that high-risk human papillomavirus (HR-HPV) infection is a risk factor of cervical cancer, and the infection was associated with significantly reduced miR-34a expression during carcinogenesis. However, the downstream targets of miR-34a and their roles are still not well understood. This study explored the regulative role of miR-34a on E2F3 and survivin expression and the viability and invasion of HPV-positive cervical cancer cells. MiR-34a and survivin expression in 56 cases of HR-HPV-positive patients, 28 cases of HR-HPV-negative patients, and 28 normal cases without HR-HPV infections were measured. Human papillomavirus-18-positive HeLa cervical cancer cells and HPV-16-positive SiHa cells were used to explore the effect of miR-34a on cell viability and invasion. The molecular target of miR-34a was also explored in cervical cancer cells. The results showed that miR-34a overexpression could inhibit HPV-positive cancer cell viability, whereas its downregulation promoted cell viability. E2F3 is a direct target of miR-34a in HPV-positive cervical cancer cells. By targeting E2F3, miR-34a could regulate the expression of survivin. Thus, through regulating E2F3 and survivin, miR-34a could reduce the viability and invasion of HPV-positive cervical cancer cells. This study confirmed a novel miR-34a-E2F3-survivin axis in the tumor suppressor role of miR-34a in cervical cancer.

The type and composition of alginate and hyaluronic-based hydrogels influence the viability of stem cells of the apical papilla.

PubMed

Lambricht, Laure; De Berdt, Pauline; Vanacker, Julie; Leprince, Julian; Diogenes, Anibal; Goldansaz, Hadi; Bouzin, Caroline; Préat, Véronique; Dupont-Gillain, Christine; des Rieux, Anne

2014-12-01

The goal of the present work was to evaluate in vitro and in vivo the influence of various types and compositions of natural hydrogels on the viability and metabolic activity of SCAPs. Two alginate, three hyaluronic-based (Corgel™) hydrogel formulations and Matrigel were characterized for their mechanical, surface and microstructure properties using rheology, X-ray photoelectron spectroscopy and scanning electron microscopy, respectively. A characterized SCAP cell line (RP89 cells) was encapsulated in the different experimental hydrogel formulations. Cells were cultured in vitro, or implanted in cyclosporine treated mice. In vitro cell viability was evaluated using a Live/Dead assay and in vitro cellular metabolic activity was evaluated with a MTS assay. In vivo cell apoptosis was evaluated by a TUNEL test and RP89 cells were identified by human mitochondria immunostaining. Hydrogel composition influenced their mechanical and surface properties, and their microstructure. In vitro cell viability was above 80% after 2 days but decreased significantly after 7 days (60-40%). Viability at day 7 was the highest in Matrigel (70%) and then in Corgel 1.5 (60%). Metabolic activity increased over time in all the hydrogels, excepted in alginate SLM. SCAPs survived after 1 week in vivo with low apoptosis (<1%). The highest number of RP89 cells was found in Corgel 5.5 (140cells/mm(2)). Collectively, these data demonstrate that SCAP viability was directly modulated by hydrogel composition and suggest that a commercially available hyaluronic acid-based formulation might be a suitable delivery vehicle for SCAP-based dental pulp regeneration strategies. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Study of the stability of packaging and storage conditions of human mesenchymal stem cell for intra-arterial clinical application in patient with critical limb ischemia.

PubMed

Gálvez-Martín, Patricia; Hmadcha, Abdelkrim; Soria, Bernat; Calpena-Campmany, Ana C; Clares-Naveros, Beatriz

2014-04-01

Critical limb ischemia (CLI) is associated with significant morbidity and mortality. In this study, we developed and characterized an intra-arterial cell suspension containing human mesenchymal stem cells (hMSCs) for the treatment of CLI. Equally, the stability of cells was studied in order to evaluate the optimal conditions of storage that guarantee the viability from cell processing to the administration phase. Effects of various factors, including excipients, storage temperature and time were evaluated to analyze the survival of hMSCs in the finished medicinal product. The viability of hMSCs in different packaging media was studied for 60 h at 4 °C. The best medium to maintain hMSCs viability was then selected to test storage conditions (4, 8, 25 and 37 °C; 60 h). The results showed that at 4 °C the viability was maintained above 80% for 48 h, at 8 °C decreased slightly, whereas at room temperature and 37 °C decreased drastically. Its biocompatibility was assessed by cell morphology and cell viability assays. During stability study, the stored cells did not show any change in their phenotypic or genotypic characteristics and physicochemical properties remained constant, the ability to differentiate into adipocytes and osteocytes and sterility requirements were also unaltered. Finally, our paper proposes a packing media composed of albumin 20%, glucose 5% and Ringer's lactate at a concentration of 1×10(6) cells/mL, which must be stored at 4 °C as the most suitable to maintain cell viability (>80%) and without altering their characteristics for more than 48 h. Copyright © 2013 Elsevier B.V. All rights reserved.

Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function.

PubMed

Germann, Anja; Oh, Young-Joo; Schmidt, Tomm; Schön, Uwe; Zimmermann, Heiko; von Briesen, Hagen

2013-10-01

The ability to analyze cryopreserved peripheral blood mononuclear cell (PBMC) from biobanks for antigen-specific immunity is necessary to evaluate response to immune-based therapies. To ensure comparable assay results, collaborative research in multicenter trials needs reliable and reproducible cryopreservation that maintains cell viability and functionality. A standardized cryopreservation procedure is comprised of not only sample collection, preparation and freezing but also low temperature storage in liquid nitrogen without any temperature fluctuations, to avoid cell damage. Therefore, we have developed a storage approach to minimize suboptimal storage conditions in order to maximize cell viability, recovery and T-cell functionality. We compared the influence of repeated temperature fluctuations on cell health from sample storage, sample sorting and removal in comparison to sample storage without temperature rises. We found that cyclical temperature shifts during low temperature storage reduce cell viability, recovery and immune response against specific-antigens. We showed that samples handled under a protective hood system, to avoid or minimize such repeated temperature rises, have comparable cell viability and cell recovery rates to samples stored without any temperature fluctuations. Also T-cell functionality could be considerably increased with the use of the protective hood system compared to sample handling without such a protection system. This data suggests that the impact of temperature fluctuation on cell integrity should be carefully considered in future clinical vaccine trials and consideration should be given to optimal sample storage conditions. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

PubMed

Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

2016-07-01

Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.

Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic

PubMed Central

Mulye, Minal; Samanta, Dhritiman; Winfree, Seth; Heinzen, Robert A.

2017-01-01

ABSTRACT Coxiella burnetii is an intracellular bacterial pathogen and a significant cause of culture-negative endocarditis in the United States. Upon infection, the nascent Coxiella phagosome fuses with the host endocytic pathway to form a large lysosome-like vacuole called the parasitophorous vacuole (PV). The PV membrane is rich in sterols, and drugs perturbing host cell cholesterol homeostasis inhibit PV formation and bacterial growth. Using cholesterol supplementation of a cholesterol-free cell model system, we found smaller PVs and reduced Coxiella growth as cellular cholesterol concentration increased. Further, we observed in cells with cholesterol a significant number of nonfusogenic PVs that contained degraded bacteria, a phenotype not observed in cholesterol-free cells. Cholesterol had no effect on axenic Coxiella cultures, indicating that only intracellular bacteria are sensitive to cholesterol. Live-cell microscopy revealed that both plasma membrane-derived cholesterol and the exogenous cholesterol carrier protein low-density lipoprotein (LDL) traffic to the PV. To test the possibility that increasing PV cholesterol levels affects bacterial survival, infected cells were treated with U18666A, a drug that traps cholesterol in lysosomes and PVs. U18666A treatment led to PVs containing degraded bacteria and a significant loss in bacterial viability. The PV pH was significantly more acidic in cells with cholesterol or cells treated with U18666A, and the vacuolar ATPase inhibitor bafilomycin blocked cholesterol-induced PV acidification and bacterial death. Additionally, treatment of infected HeLa cells with several FDA-approved cholesterol-altering drugs led to a loss of bacterial viability, a phenotype also rescued by bafilomycin. Collectively, these data suggest that increasing PV cholesterol further acidifies the PV, leading to Coxiella death. PMID:28246364

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans.

PubMed

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K

2008-03-01

BACKGROUND: A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO(R)13, SYTO(R)24 and SYBR(R)14 as possible alternatives to FDA. RESULTS: We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO(R)13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. CONCLUSIONS: From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability.

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans

PubMed Central

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K.

2010-01-01

Background A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO®13, SYTO®24 and SYBR®14 as possible alternatives to FDA. Results We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO®13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. Conclusions From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability. PMID:20814586

Effects of Fluid Shear Stress on Cancer Stem Cell Viability

NASA Astrophysics Data System (ADS)

Sunday, Brittney; Triantafillu, Ursula; Domier, Ria; Kim, Yonghyun

2014-11-01

Cancer stem cells (CSCs), which are believed to be the source of tumor formation, are exposed to fluid shear stress as a result of blood flow within the blood vessels. It was theorized that CSCs would be less susceptible to cell death than non-CSCs after both types of cell were exposed to a fluid shear stress, and that higher levels of fluid shear stress would result in lower levels of cell viability for both cell types. To test this hypothesis, U87 glioblastoma cells were cultured adherently (containing smaller populations of CSCs) and spherically (containing larger populations of CSCs). They were exposed to fluid shear stress in a simulated blood flow through a 125-micrometer diameter polyetheretherketone (PEEK) tubing using a syringe pump. After exposure, cell viability data was collected using a BioRad TC20 Automated Cell Counter. Each cell type was tested at three physiological shear stress values: 5, 20, and 60 dynes per centimeter squared. In general, it was found that the CSC-enriched U87 sphere cells had higher cell viability than the CSC-depleted U87 adherent cancer cells. Interestingly, it was also observed that the cell viability was not negatively affected by the higher fluid shear stress values in the tested range. In future follow-up studies, higher shear stresses will be tested. Furthermore, CSCs from different tumor origins (e.g. breast tumor, prostate tumor) will be tested to determine cell-specific shear sensitivity. National Science Foundation Grant #1358991 supported the first author as an REU student.

The in vitro viability and growth of fibroblasts cultured in the presence of different bone grafting materials (NanoBone and Straumann Bone Ceramic).

PubMed

Kauschke, E; Rumpel, E; Fanghänel, J; Bayerlein, T; Gedrange, T; Proff, P

2006-02-01

Different clinical applications, including dentistry, are making increasing demands on bone grafting material. In the present study we have analysed the viability, proliferation and growth characteristics of fibroblasts cultured in vitro together with two different bone grafting materials, NanoBone and Straumann Bone Ceramic, over a period of 24 and 28 days respectively. Viability was measured at least every 72 hours by using the alamarBlue assay, a test that measures quantitatively cell proliferation and viability but does not require cell fixation or extraction. After one week of culture fibroblast viability was as high as in controls for both grafting materials and remained high (> 90%) for the duration of the experiment. Cell growth was evaluated microscopically. Scanning electron microscopy revealed a dense fibroblast growth at the surface of both bone grafting materials after three weeks of in vitro culture. Generally, our in vitro analyses contribute to further insights into cell - scaffold interactions.

Effects of voluntary exercise on the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups born from morphine- dependent mothers during pregnancy.

PubMed

Haydari, Sakineh; Safari, Manouchehr; Zarbakhsh, Sam; Bandegi, Ahmad Reza; Miladi-Gorji, Hossein

2016-11-10

This study was designed to investigate whether free access to a running wheel during pregnancy in morphine-dependent mothers would influence the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups. Pregnant rats were made dependent by chronic administration of morphine in drinking water simultaneously with free access to a running wheel. Male pups are weaned at 21days of birth and their bones marrows were aspirated from the femurs and tibias and also the bone marrow stromal cells (BMSCs) cultured. MTT assay was used to determine cell viability and proliferation rate. The level of BDNF was measured in the supernant of BMSCs culture by ELISA. The sedentary morphine-dependent mothers' pups showed a significant increase in the percentage cell viability and proliferation rate and also a significant decrease in the BDNF protein levels in BMSCs. The rat pups borne from exercising the control and morphine-dependent mothers exhibited an increase in the percentage viability, proliferation rate and BDNF levels of the BMSCs. This study showed that maternal exercise during pregnancy in morphine-dependent and non-dependent mothers, with increasing of BDNF levels increased the proliferation and viability of BMSCs in the rat pups. Also, chronic administration of morphine during pregnancy was able to increase the proliferation and viability of BMSCs in the rat pups. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effect of doping in carbon nanotubes on the viability of biomimetic chitosan-carbon nanotubes-hydroxyapatite scaffolds.

PubMed

Fonseca-García, Abril; Mota-Morales, Josué D; Quintero-Ortega, Iraís A; García-Carvajal, Zaira Y; Martínez-López, V; Ruvalcaba, Erika; Landa-Solís, Carlos; Solis, Lilia; Ibarra, Clemente; Gutiérrez, María C; Terrones, Mauricio; Sanchez, Isaac C; del Monte, Francisco; Velasquillo, María C; Luna-Bárcenas, G

2014-10-01

This work describes the preparation and characterization of biomimetic chitosan/multiwall carbon nanotubes/nano-hydroxyapatite (CTS/MWCNT/nHAp) scaffolds and their viability for bone tissue engineering applications. The cryogenic process ice segregation-induced self-assembly (ISISA) was used to fabricate 3D biomimetic CTS scaffolds. Proper combination of cryogenics, freeze-drying, nature and molecular ratio of solutes give rise to 3D porous interconnected scaffolds with clusters of nHAp distributed along the scaffold surface. The effect of doping in CNT (e.g. with oxygen and nitrogen atoms) on cell viability was tested. Under the same processing conditions, pore size was in the range of 20-150 μm and irrespective on the type of CNT. Studies on cell viability with scaffolds were carried out using human cells from periosteum biopsy. Prior to cell seeding, the immunophenotype of mesenchymal periosteum or periosteum-derived stem cells (MSCs-PCs) was characterized by flow cytometric analysis using fluorescence-activated and characteristic cell surface markers for MSCs-PCs. The characterized MSCs-PCs maintained their periosteal potential in cell cultures until the 2nd passage from primary cell culture. Thus, the biomimetic CTS/MWCNT/nHAp scaffolds demonstrated good biocompatibility and cell viability in all cases such that it can be considered as promising biomaterials for bone tissue engineering. © 2013 Wiley Periodicals, Inc.

[Impact of cryopreservation duration of 605 units umbilical cord blood on quality of hematopoietic stem cell and outcome of clinical transplantation].

PubMed

Zhang, Yi; Zhu, Hua; Jin, Huanying; Wang, Yinting; Shao, Xiayan; Kong, Jingsi; Huang, Wenhao; Hong, Yan; Li, Chunli; Gao, Feng; Chen, Liang; Wang, Feng; Lu, Yao

2015-01-01

To investigate the impact of cryopreservation duration of umbilical cord blood (UCB) on quality of hematopoietic stem cell and outcome of clinical transplantation. 605 units of UCB which had been used in clinical transplantation were previously cryopreserved for 820 (88-2651) days in average. UCB was detected for total nucleated cell count, CD34+ cells count, cell recovery rate, cell viability and CFU-GM after thawing. No statistical correlation was found between cryopreservation duration and cell recovery rate, cell viability. CFU-GM decreased along with the extension of cryopreservation duration (P=0.011), ranging between 109.6 and 105.7/1 × 10⁵. There was no significant difference on hematopoietic reconstitution time, graft failure, acute GVHD and overall survival among groups with different cryopreservation duration. Cryopreservation duration has no significant effect on cell recovery rate, cell viability and clinical transplantation outcome. Extension of cryopreservation duration may reduce CFU-GM of stem cells with fluctaion still in normal range. UCB could maintain cell viability and function to achieve satisfactory clinical transplantation outcome even when thawed after 3 to 7 years' cryopreservation.

Dasatinib and Doxorubicin Treatment of Sarcoma Initiating Cells: A Possible New Treatment Strategy.

PubMed

Aggerholm-Pedersen, Ninna; Demuth, Christina; Safwat, Akmal; Meldgaard, Peter; Kassem, Moustapha; Sandahl Sorensen, Boe

2016-01-01

Background. One of the major challenges affecting sarcoma treatment outcome, particularly that of metastatic disease, is resistance to chemotherapy. Cancer-initiating cells are considered a major contributor to this resistance. Methods. An immortalised nontransformed human stromal (mesenchymal) stem cell line hMSC-TERT4 and a transformed cell line hMSC-TERT20-CE8, known to form sarcoma-like tumours when implanted in immune-deficient mice, were used as models. Receptor tyrosine kinase (RTK) activation was analysed by RTK arrays and cellular viability after tyrosine kinases inhibitor (TKI) treatment with or without doxorubicin was assessed by MTS assay. Results. Initial results showed that the hMSC-TERT4 was more doxorubicin-sensitive while hMSC-TERT20-CE8 was less doxorubicin-sensitive evidenced by monitoring cell viability in the presence of doxorubicin at different doses. The epidermal growth factor receptor (EGFR) was activated in both cell lines. However hMSC-TERT20-CE8 exhibited significantly higher expression of the EGFR ligands. EGFR inhibitors such as erlotinib and afatinib alone or in combination with doxorubicin failed to further decrease cell viability of hMSC-TERT20-CE8. However, inhibition with the TKI dasatinib in combination with doxorubicin decreased cell viability of the hMSC-TERT20-CE8 cell line. Conclusion. Our results demonstrate that dasatinib, but not EGFR-directed treatment, can decrease cell viability of stromal cancer stem cells less sensitive to doxorubicin.

Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

PubMed

Cao, Ting-Ting; Zhang, Yu-Qing

2015-09-01

Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later.

Mechanism of H₂O₂-induced oxidative stress regulating viability and biocontrol ability of Rhodotorula glutinis.

PubMed

Chen, Jian; Li, Boqiang; Qin, Guozheng; Tian, Shiping

2015-01-16

The use of antagonistic yeasts to control postharvest pathogens is a promising alternative to fungicides. The effectiveness of the antagonists against fungal pathogens is greatly dependent on their viability, which is usually mediated by reactive oxygen species (ROS). Here, we investigated the effects of H₂O₂-induced oxidative stress on the viability and biocontrol efficacy of Rhodotorula glutinis and, using flow cytometric analysis, observed the changes of ROS accumulation and apoptosis in the yeast cells with or without H₂O₂ treatment. We found that the viability of R. glutinis decreased in a time- and dose-dependent manner under H₂O₂-induced oxidative stress. Compared to the control, yeast cells exposed to oxidative stress exhibited more accumulation of ROS and higher levels of protein oxidative damage, but showed lower efficacy for biocontrol of Penicillium expansum causing blue mold rot on peach fruit. The results indicate that apoptosis is a main cause of the cell viability loss in R. glutinis, which is attributed to ROS accumulation under oxidative stress. These findings offer a plausible explanation that oxidative stress affects biocontrol efficacy of R. glutinis via regulating its viability and cell apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

A cell transportation solution that preserves live circulating tumor cells in patient blood samples.

PubMed

Stefansson, Steingrimur; Adams, Daniel L; Ershler, William B; Le, Huyen; Ho, David H

2016-05-06

Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90% viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs after days of storage. Therefore, we suggest an effective and economical transportation of cancer patient blood samples containing live CTCs can be achieved.

Microwave decoration of Pt nanoparticles on entangled 3D carbon nanotube architectures as PEM fuel cell cathode.

PubMed

Sherrell, Peter C; Zhang, Weimin; Zhao, Jie; Wallace, Gordon G; Chen, Jun; Minett, Andrew I

2012-07-01

Proton-exchange membrane fuel cells (PEMFCs) are expected to provide a complementary power supply to fossil fuels in the near future. The current reliance of fuel cells on platinum catalysts is undesirable. However, even the best-performing non-noble metal catalysts are not as efficient. To drive commercial viability of fuel cells forward in the short term, increased utilization of Pt catalysts is paramount. We have demonstrated improved power and energy densities in a single PEMFC using a designed cathode with a Pt loading of 0.1 mg cm(-2) on a mesoporous conductive entangled carbon nanotube (CNT)-based architecture. This electrode allows for rapid transfer of both fuel and waste to and from the electrode, respectively. Pt particles are bound tightly, directly to CNT sidewalls by a microwave-reduction technique, which provided increased charge transport at this interface. The Pt entangled CNT cathode, in combination with an E-TEK 0.2 mg cm(-2) anode, has a maximum power and energy density of 940 mW cm(-2) and 2700 mA cm(-2), respectively, and a power and energy density of 4.01 W mg(Pt)(-1) and 6.35 A mg(Pt)(-1) at 0.65 V. These power densities correspond to a specific mass activity of 0.81 g Pt per kW for the combined mass of both anode and cathode electrodes, approaching the current US Department of Energy efficiency target. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Manganese-Enhanced Magnetic Resonance Imaging Enables In Vivo Confirmation of Peri-Infarct Restoration Following Stem Cell Therapy in a Porcine Ischemia-Reperfusion Model.

PubMed

Dash, Rajesh; Kim, Paul J; Matsuura, Yuka; Ikeno, Fumiaki; Metzler, Scott; Huang, Ngan F; Lyons, Jennifer K; Nguyen, Patricia K; Ge, Xiaohu; Foo, Cheryl Wong Po; McConnell, Michael V; Wu, Joseph C; Yeung, Alan C; Harnish, Phillip; Yang, Phillip C

2015-07-27

The exact mechanism of stem cell therapy in augmenting the function of ischemic cardiomyopathy is unclear. In this study, we hypothesized that increased viability of the peri-infarct region (PIR) produces restorative benefits after stem cell engraftment. A novel multimodality imaging approach simultaneously assessed myocardial viability (manganese-enhanced magnetic resonance imaging [MEMRI]), myocardial scar (delayed gadolinium enhancement MRI), and transplanted stem cell engraftment (positron emission tomography reporter gene) in the injured porcine hearts. Twelve adult swine underwent ischemia-reperfusion injury. Digital subtraction of MEMRI-negative myocardium (intrainfarct region) from delayed gadolinium enhancement MRI-positive myocardium (PIR and intrainfarct region) clearly delineated the PIR in which the MEMRI-positive signal reflected PIR viability. Human amniotic mesenchymal stem cells (hAMSCs) represent a unique population of immunomodulatory mesodermal stem cells that restored the murine PIR. Immediately following hAMSC delivery, MEMRI demonstrated an increased PIR viability signal compared with control. Direct PIR viability remained higher in hAMSC-treated hearts for >6 weeks. Increased PIR viability correlated with improved regional contractility, left ventricular ejection fraction, infarct size, and hAMSC engraftment, as confirmed by immunocytochemistry. Increased MEMRI and positron emission tomography reporter gene signal in the intrainfarct region and the PIR correlated with sustained functional augmentation (global and regional) within the hAMSC group (mean change, left ventricular ejection fraction: hAMSC 85±60%, control 8±10%; P<0.05) and reduced chamber dilatation (left ventricular end-diastole volume increase: hAMSC 24±8%, control 110±30%; P<0.05). The positron emission tomography reporter gene signal of hAMSC engraftment correlates with the improved MEMRI signal in the PIR. The increased MEMRI signal represents PIR viability and the restorative potential of the injured heart. This in vivo multimodality imaging platform represents a novel, real-time method of tracking PIR viability and stem cell engraftment while providing a mechanistic explanation of the therapeutic efficacy of cardiovascular stem cells. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Analytical Research to Determine the effects of the Components of ONGABO on the Viability of HepG2 Cancer Cells by Using the Sovereign, Minister, Assistant and Courier Principle (君臣佐使論)

PubMed Central

Shin, Jeong-Hun; Jun, Seung-lyul; Hwang, Sung-Yeoun; Ahn, Seong-Hun

2012-01-01

Objectives: This study used the basic principle of Oriental medicine, the sovereign, minister, assistant and courier principle (君臣佐使論) to investigate the effects of the component of ONGABO, which is composed of Ginseng Radix (Red Ginseng), Angelica Gigantis Radix, Schisandrae Fructus, Cuscuta Semen and Curcumae tuber on the viability of HepG2 cells. Methods: Single and mixed extracts of the component of ONGABO were prepared by lypohilizing powder of Red Ginseng (6-year root from Kanghwa), Angelica Gigantis Radix, Schisandrae Fructus, Cuscuta Semen, Curcumae Tuber (from Omniherb Co., Ltd., Korea) at the laboratory of herbal medicine in Woosuk University and were eluted after being macerated with 100% ethanol for three days. The cell viability of HepG2 was determined by using an absorptiometric analysis with PrestoBlue (Invitrogen) reagent after the plate had been incubated for 48 hours. All of the experiments were repeated three times to obtain the average value and standard deviation. The statistical analysis was done and the correlation factor was obtained by using Microsoft Office Excel 2007 and Origin 6.0 software. Results: Although Ginseng Radix (Red Ginseng) and Schisandrae Fructus did not enhance the viability of HepG2 cells, they were shown to provide protection of those cells. On the other hand, Angelica Gigantis Radix decreased the viability of HepG2 cells significantly, Cuscuta Semen and Curcumae Tuber had a small or no effect on the viability of HepG2 cells. Conclusions: In the sovereign, minister, assistant and courier principle (君臣佐使論), Ginseng Radix (Red Ginseng) corresponds to the sovereign component because it provides cell protection effects, Angelica Gigantis Radix corresponds to minister medicinal because it kills cells, Schisandrae Fructus corresponds to the assistant medicinal to help red ginseng having cell protect effects. Cuscuta Semen and Curcumae Tuber correspond to the courier medicinal having no effect in cell viability in HepG2. We hope this study provides motivation for advanced research on the sovereign, minister, assistant and courier principle. PMID:25780653

Analytical Research to Determine the effects of the Components of ONGABO on the Viability of HepG2 Cancer Cells by Using the Sovereign, Minister, Assistant and Courier Principle ().

PubMed

Shin, Jeong-Hun; Jun, Seung-Lyul; Hwang, Sung-Yeoun; Ahn, Seong-Hun

2012-12-01

This study used the basic principle of Oriental medicine, the sovereign, minister, assistant and courier principle () to investigate the effects of the component of ONGABO, which is composed of Ginseng Radix (Red Ginseng), Angelica Gigantis Radix, Schisandrae Fructus, Cuscuta Semen and Curcumae tuber on the viability of HepG2 cells. Single and mixed extracts of the component of ONGABO were prepared by lypohilizing powder of Red Ginseng (6-year root from Kanghwa), Angelica Gigantis Radix, Schisandrae Fructus, Cuscuta Semen, Curcumae Tuber (from Omniherb Co., Ltd., Korea) at the laboratory of herbal medicine in Woosuk University and were eluted after being macerated with 100% ethanol for three days. The cell viability of HepG2 was determined by using an absorptiometric analysis with PrestoBlue (Invitrogen) reagent after the plate had been incubated for 48 hours. All of the experiments were repeated three times to obtain the average value and standard deviation. The statistical analysis was done and the correlation factor was obtained by using Microsoft Office Excel 2007 and Origin 6.0 software. Although Ginseng Radix (Red Ginseng) and Schisandrae Fructus did not enhance the viability of HepG2 cells, they were shown to provide protection of those cells. On the other hand, Angelica Gigantis Radix decreased the viability of HepG2 cells significantly, Cuscuta Semen and Curcumae Tuber had a small or no effect on the viability of HepG2 cells. In the sovereign, minister, assistant and courier principle (), Ginseng Radix (Red Ginseng) corresponds to the sovereign component because it provides cell protection effects, Angelica Gigantis Radix corresponds to minister medicinal because it kills cells, Schisandrae Fructus corresponds to the assistant medicinal to help red ginseng having cell protect effects. Cuscuta Semen and Curcumae Tuber correspond to the courier medicinal having no effect in cell viability in HepG2. We hope this study provides motivation for advanced research on the sovereign, minister, assistant and courier principle.

A Field-Portable Cell Analyzer without a Microscope and Reagents

PubMed Central

Oh, Sangwoo; Lee, Moonjin; Hwang, Yongha

2017-01-01

This paper demonstrates a commercial-level field-portable lens-free cell analyzer called the NaviCell (No-stain and Automated Versatile Innovative cell analyzer) capable of automatically analyzing cell count and viability without employing an optical microscope and reagents. Based on the lens-free shadow imaging technique, the NaviCell (162 × 135 × 138 mm3 and 1.02 kg) has the advantage of providing analysis results with improved standard deviation between measurement results, owing to its large field of view. Importantly, the cell counting and viability testing can be analyzed without the use of any reagent, thereby simplifying the measurement procedure and reducing potential errors during sample preparation. In this study, the performance of the NaviCell for cell counting and viability testing was demonstrated using 13 and six cell lines, respectively. Based on the results of the hemocytometer (de facto standard), the error rate (ER) and coefficient of variation (CV) of the NaviCell are approximately 3.27 and 2.16 times better than the commercial cell counter, respectively. The cell viability testing of the NaviCell also showed an ER and CV performance improvement of 5.09 and 1.8 times, respectively, demonstrating sufficient potential in the field of cell analysis. PMID:29286336

Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp. bulgaricus.

PubMed

Shao, Yuyu; Wang, Zhaoxia; Bao, Qiuhua; Zhang, Heping

2016-12-01

In this study, a combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) was used to develop a method to determine the viability of cells of Lactobacillus delbrueckii ssp. bulgaricus ND02 (L. bulgaricus) that may have entered into a viable but nonculturable state. This can happen due to its susceptibility to cold shock during lyophilization and storage. Propidium monoazide concentration, PMA incubation time, and light exposure time were optimized to fully exploit the PMA-qPCR approach to accurately assess the total number of living L. bulgaricus ND02. Although PMA has little influence on living cells, when concentrations of PMA were higher than 30μg/mL the number of PCR-positive living bacteria decreased from 10 6 to 10 5 cfu/mL in comparison with qPCR enumeration. Mixtures of living and dead cells were used as method verification samples for enumeration by PMA-qPCR, demonstrating that this method was feasible and effective for distinguishing living cells of L. bulgaricus when mixed with a known number of dead cells. We suggest that several conditions need to be studied further before PMA-qPCR methods can be accurately used to distinguish living from dead cells for enumeration under more realistic sampling situations. However, this research provides a rapid way to enumerate living cells of L. bulgaricus and could be used to optimize selection of cryoprotectants in the lyophilization process and develop technologies for high cell density cultivation and optimal freeze-drying processes. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Chrysin, Abundant in Morinda citrifolia Fruit Water-EtOAc Extracts, Combined with Apigenin Synergistically Induced Apoptosis and Inhibited Migration in Human Breast and Liver Cancer Cells.

PubMed

Huang, Cheng; Wei, Yu-Xuan; Shen, Ma-Ching; Tu, Yu-Hsuan; Wang, Chia-Chi; Huang, Hsiu-Chen

2016-06-01

The composition of Morinda citrifolia (M. citrifolia) was determined using high-performance liquid chromatography (HPLC), and the anticancer effects of M. citrifolia extract evaluated in HepG2, Huh7, and MDA-MB-231 cancer cells. M. citrifolia fruit extracts were obtained by using five different organic solvents, including hexane (Hex), methanol (MeOH), ethyl acetate (EtOAc), chloroform (CHCl3), and ethanol (EtOH). The water-EtOAc extracts from M. citrifolia fruits was found to have the highest anticancer activity. HPLC data revealed the predominance of chrysin in water-EtOAc extracts of M. citrifolia fruit. Furthermore, the combined effects of cotreatment with apigenin and chrysin on liver and breast cancer were investigated. Treatment with apigenin plus chrysin for 72-96 h reduced HepG2 and MDA-MB-231 cell viability and induced apoptosis through down-regulation of S-phase kinase-associated protein-2 (Skp2) and low-density lipoprotein receptor-related protein 6 (LRP6) expression. However, the combination treatment for 36 h synergistically decreased MDA-MB-231 cell motility but not cell viability through down-regulation of MMP2, MMP9, fibronectin, and snail in MDA-MB-231 cells. Additionally, chrysin combined with apigenin also suppressed tumor growth in human MDA-MB-231 breast cancer cells xenograft through down-regulation of ki-67 and Skp2 protein. The experimental results showed that chrysin combined with apigenin can reduce HepG2 and MDA-MB-231 proliferation and cell motility and induce apoptosis. It also offers opportunities for exploring new drug targets, and further investigations are underway in this regard.

Synthesis of stiffness-tunable and cell-responsive Gelatin-poly(ethylene glycol) hydrogel for three-dimensional cell encapsulation.

PubMed

Cao, Ye; Lee, Bae Hoon; Peled, Havazelet Bianco; Venkatraman, Subbu S

2016-10-01

Biosynthetic poly(ethylene glycol) (PEG)-based hydrogels have been extensively investigated as extracellular matrix (ECM) mimicking gels as they retain the benefits of both ECM (biological cues) and synthetic hydrogels (tunable mechanical properties). In this article, we developed and characterized a new gelatin-PEG (GP) hydrogel that retains the benefits of gelatin and synthetic hydrogels. In this strategy, the thiolation of gelatin was accomplished by reacting with Traut's reagent; the thiolated gelatin was then conjugated to one end of PEG diacrylate (PEGDA) by Michael-type addition reaction. Two kinds of GP precursors, GP30 and GP60, were synthesized by changing the amount of Traut's reagent, while the weight ratio between thiolated-gelatin and PEGDA of GP30 and GP60 was 1.451:1 and 0.785:1, respectively. Finally, neonatal human dermal fibroblasts were encapsulated into the hydrogel by cross-linking the remaining double bonds of precursor under ultraviolet light. These GP hydrogels can encapsulate the fibroblasts in situ with high cell viability. Moreover, the behaviors of cells within the GP hydrogels can be modulated by varying the cross-linking density of GP hydrogel (storage modulus from 40 to 2000 Pa). In particular, this article showed that a minimum amount of cell-binding motifs (gelatin >2.30 wt/vol % and 44.0% dry weight percentage) are required for attachment; and appropriate initial rheological and structural properties (storage modulus <∼100 Pa and mesh size >∼150 nm) can accelerate the attachment of cells and improve cell viability. Hence, this mixed-hydrogel platform allows an easily control hydrogel structure and modulates cell behavior to reconstruct new tissue in the three-dimensional microenvironments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2401-2411, 2016. © 2016 Wiley Periodicals, Inc.

The effect of CO2 laser beam welded AISI 316L austenitic stainless steel on the viability of fibroblast cells, in vitro.

PubMed

Köse, Ceyhun; Kaçar, Ramazan; Zorba, Aslı Pınar; Bağırova, Melahat; Allahverdiyev, Adil M

2016-03-01

It has been determined by the literature research that there is no clinical study on the in vivo and in vitro interaction of the cells with the laser beam welded joints of AISI 316L biomaterial. It is used as a prosthesis and implant material and that has adequate mechanical properties and corrosion resistance characteristics. Therefore, the interaction of the CO2 laser beam welded samples and samples of the base metal of AISI 316L austenitic stainless steel with L929 fibroblast cells as an element of connective tissue under in vitro conditions has been studied. To study the effect of the base metal and the laser welded test specimens on the viability of the fibroblast cells that act as an element of connective tissues in the body, they were kept in DMEMF-12 medium for 7, 14, 28 days and 18 months. The viability study was experimentally studied using the MTT method for 7, 14, 28 days. In addition, the direct interaction of the fibroblast cells seeded on 6 different plates with the samples was examined with an inverted microscope. The MTT cell viability experiment was repeated on the cells that were in contact with the samples. The statistical relationship was analyzed using a Tukey test for the variance with the GraphPad statistics software. The data regarding metallic ion release were identified with the ICP-MS method after the laser welded and main material samples were kept in cell culture medium for 18 months. The cell viability of the laser welded sample has been detected to be higher than that of the base metal and the control based on 7th day data. However, the laser welded sample's viability of the fibroblast cells has diminished by time during the test period of 14 and 28 days and base metal shows better viability when compared to the laser welded samples. On the other hand, the base metal and the laser welded sample show better cell viability effect when compared to the control group. According to the ICP-MS results of the main material and laser welded samples which were kept in the cell culture medium for 18 months, it was determined that the Fe, Ni and Cr ion concentration released to the cell culture medium from the laser welded test sample was less than that of the main material. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultraporous, Compressible, Wettable Polylactide/Polycaprolactone Sponges for Tissue Engineering.

PubMed

Mader, Michael; Jérôme, Valérie; Freitag, Ruth; Agarwal, Seema; Greiner, Andreas

2018-05-14

Ultraporous, degradable sponges made of either polylactide or of blends of polylactide/poly(ε-caprolactone) are prepared by freeze-drying of dispersions of short electrospun fibers and subsequent thermal annealing. The sponges feature ultrahigh porosity (99.6%), a hierarchical cellular structure, and high reversible compressibility with fast recovery from deformation in the dry as well as in the wet state. The sponge properties depend on the fiber dispersion concentration and the annealing temperature. Sponge characteristics like fiber density (2.5-20 mg/cm 3 ), size, shape, crystallinity, mechanical strength, wetability, and structural integrity are user adjustable. Cell culture experiments were successfully performed with Jurkat cells with Confocal Laser Scanning Microscopy and MTT staining showing rapid cell proliferation. Live/Dead staining demonstrated high viability of the seeded cells. The sponge characteristics and modifications investigated and presented here reveal that these sponges are highly promising for tissue engineering applications.

Evaluation of goat milk as storage media to preserve viability of human periodontal ligament cells in vitro.

PubMed

Ulusoy, Ayça Tuba; Kalyoncuoglu, Elif; Kaya, Senay; Cehreli, Zafer Cavit

2016-08-01

The purpose of this study was to evaluate the effectiveness of goat milk as a storage media for maintenance of periodontal ligament (PDL) cell viability of avulsed teeth and compare it with commonly used and/or investigated storage media. PDL cells were obtained from the root surface of healthy premolars and were cultured in Eagle's maintenance medium (EMM). Cell cultures were treated with the following storage media: tap water (negative control); EMM (positive control); Hank's balanced salt solution; ultra high temperature (UHT) long-shelf-life lactose-free cow milk; UHT long-shelf-life whole cow milk; UHT long-shelf-life skimmed cow milk; UHT long-shelf-life soy milk; UHT long-shelf-life goat milk, UHT long-shelf-life follow on milk with probiotic, 20% propolis, and egg white. Culture plates were incubated with experimental media at 20°C for 1, 3, 6, 12, and 24 h. PDL cell viability was assessed by tetrazolium salt-based colorimetric (MTT) assay at each test period. One-way anova was used to evaluate the effects of storage solutions at each time point, followed by post hoc Duncan's multiple comparison test (P = 0.05). A dendrogram was constructed to show the arrangement of hierarchical clustering. Goat milk displayed the highest capacity to maintain cell viability at all test intervals (P < 0.001). Between 3 and 24 h, milk with the probiotic showed the lowest time-dependent PDL cell viability among all test media (P < 0.001). Compared with all milks, HBSS performed significantly less effectively in maintaining PDL cell viability during the entire test period (P < 0.001). Based on PDL viability, goat milk can be recommended as a suitable storage medium for avulsed teeth. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Storage effect on viability and biofunctionality of human adipose tissue-derived stromal cells.

PubMed

Falah, Mizied; Rayan, Anwar; Srouji, Samer

2015-09-01

In our recent studies, the transplantation of human adipose tissue-derived stromal cells (ASCs) has shown promise for treatment of diseases related to bone and joint disorders. For the current clinical applications, ASCs were formulated and suspended in PlasmaLyte A supplemented with heparin, glucose and human serum albumin, balanced to pH 7.4 with sodium bicarbonate. This cell solution constitutes 20% of the overall transplanted mixture and is supplemented with hyaluronic acid (60%) and OraGraft particles (20%). We intended to investigate the effect of this transplantation mixture on the viability and biofunctionality of ASCs in bone formation. Freshly harvested cells were resuspended and incubated in the indicated mixture for up to 48 h at 4°C. Cell viability was assessed using trypan blue and AlamarBlue, and cell functionality was determined by quantifying their adhesion rate in vitro and bone formation in an ectopic mouse model. More than 80% of the ASCs stored in the transplantation mixture were viable for up to 24 h. Cell viability beyond 24 h in storage decreased to approximately 50%. In addition, an equal degree of bone formation was observed between the cells transplanted following incubation in transplantation mixture for up to 24 h and zero-time non-incubated cells (control). The viability and functionality of ASCs stored in the presented formulation will make such cell therapy accessible to larger and more remote populations. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Fabrication of scalable and structured tissue engineering scaffolds using water dissolvable sacrificial 3D printed moulds.

PubMed

Mohanty, Soumyaranjan; Larsen, Layla Bashir; Trifol, Jon; Szabo, Peter; Burri, Harsha Vardhan Reddy; Canali, Chiara; Dufva, Marin; Emnéus, Jenny; Wolff, Anders

2015-10-01

One of the major challenges in producing large scale engineered tissue is the lack of ability to create large highly perfused scaffolds in which cells can grow at a high cell density and viability. Here, we explore 3D printed polyvinyl alcohol (PVA) as a sacrificial mould in a polymer casting process. The PVA mould network defines the channels and is dissolved after curing the polymer casted around it. The printing parameters determined the PVA filament density in the sacrificial structure and this density resulted in different stiffness of the corresponding elastomer replica. It was possible to achieve 80% porosity corresponding to about 150 cm(2)/cm(3) surface to volume ratio. The process is easily scalable as demonstrated by fabricating a 75 cm(3) scaffold with about 16,000 interconnected channels (about 1m(2) surface area) and with a channel to channel distance of only 78 μm. To our knowledge this is the largest scaffold ever to be produced with such small feature sizes and with so many structured channels. The fabricated scaffolds were applied for in-vitro culturing of hepatocytes over a 12-day culture period. Smaller scaffolds (6×4 mm) were tested for cell culturing and could support homogeneous cell growth throughout the scaffold. Presumably, the diffusion of oxygen and nutrient throughout the channel network is rapid enough to support cell growth. In conclusion, the described process is scalable, compatible with cell culture, rapid, and inexpensive. Copyright © 2015. Published by Elsevier B.V.

Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal-Limbal and Human Conjunctival Epithelial Cells.

PubMed

Schuerer, Nadine; Stein, Elisabeth; Inic-Kanada, Aleksandra; Pucher, Marion; Hohenadl, Christine; Bintner, Nora; Ghasemian, Ehsan; Montanaro, Jacqueline; Barisani-Asenbauer, Talin

2017-06-01

To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal-limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model. HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT-based assay. The morphology of cells was also investigated. HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers. We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface.

Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal–Limbal and Human Conjunctival Epithelial Cells

PubMed Central

Schuerer, Nadine; Stein, Elisabeth; Inic-Kanada, Aleksandra; Pucher, Marion; Hohenadl, Christine; Bintner, Nora; Ghasemian, Ehsan; Montanaro, Jacqueline

2017-01-01

Purpose: To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal–limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model. Methods: HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT–based assay. The morphology of cells was also investigated. Results: HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers. Conclusions: We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface. PMID:28399036

Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device.

PubMed

Germain, Todd; Ansari, Megan; Pappas, Dimitri

2016-09-14

Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. Copyright © 2016 Elsevier B.V. All rights reserved.

A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy.

PubMed

Gorokhova, Elena; Mattsson, Lisa; Sundström, Annica M

2012-06-01

Two fluorescent dyes, TO-PRO-1 iodide and 5-CFDA-AM, were evaluated for LIVE/DEAD assessment of unicellular marine algae Brachiomonas submarina and Tetraselmis suecica. Epifluorescence microscopy was used to estimate cell viability in predetermined mixtures of viable and non-viable algal cells and validated using microplate growth assay as reference measurements. On average, 5-CFDA-AM underestimated live cell abundance by ~25% compared with viability estimated by the growth assay, whereas TO-PRO-1 iodide provided accurate viability estimates. Furthermore, viability estimates based on staining with TO-PRO-1 iodide were not affected by a storage period of up to one month in -80°C, making the assay a good candidate for routine assessment of phytoplankton populations in field and laboratory studies. Copyright © 2012 Elsevier B.V. All rights reserved.

The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion, but Not Permeability, and Controls Vascular Development and Embryonic Viability*

PubMed Central

Souza, Cleiton Martins; Davidson, Dominique; Rhee, Inmoo; Gratton, Jean-Philippe; Davis, Elaine C.; Veillette, André

2012-01-01

Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability. PMID:23105101

Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions

PubMed Central

Tanti, N.C.; Jones, L.; Sheardown, H.

2010-01-01

Purpose Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. Methods An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate  (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Results Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of β1 and α3 integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis. Conclusions Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells. PMID:20169012

Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions.

PubMed

Gorbet, M B; Tanti, N C; Jones, L; Sheardown, H

2010-02-19

Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of beta(1) and alpha(3) integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis. Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells.

Chondrotoxicity of Liposomal Bupivacaine in Articular Chondrocytes: Preliminary Findings.

PubMed

Shaw, K Aaron; Johnson, Peter C; Zumbrun, Steve; Chuang, Augustine H; Cameron, Craig D

2017-03-01

The chondrotoxicity of local anesthetics has been previously recognized. Recent introduction of a liposomal formulation of bupivacaine has been found to significantly improve postoperative pain control but its effect on chondrocyte viability has yet to be investigated with this new formulation. We sought to assess the in vitro chondrotoxicity of liposomal bupivacaine. Chondrocytes were isolated from articular cartilage from fresh stifle joints and grown in culture medium. Cultured chondrocyte-derived cells (CDCs) were treated with 0.9% normal saline solution, 0.5%, 0.25%, and 0.13% bupivacaine and ropivacaine, 1.3% liposomal bupivacaine for 1 hour. Following treatment, cells were washed and incubated in media for 23 hours. The CDCs were then harvested and viability was assessed by flow cytometry using SYTOX green dead cell stain. Treated CDCs demonstrated a dose-response effect for chondrocyte viability when treated with bupivacaine, ropivacaine, and liposomal bupivacaine. Liposomal bupivacaine demonstrated the highest chondrocyte viability following treatment. Ropivacaine demonstrated higher chondrocyte viability than bupivacaine. Following 1 hour of treatment, liposomal bupivacaine demonstrated the highest chondrocyte viability. Chondrocyte viability was inversely proportional to anesthetic concentration. Reprint & Copyright © 2017 Association of Military Surgeons of the U.S.

Antiproliferative and cytotoxic effects of green coffee and yerba mate extracts, their main hydroxycinnamic acids, methylxanthine and metabolites in different human cell lines.

PubMed

Amigo-Benavent, M; Wang, S; Mateos, R; Sarriá, B; Bravo, L

2017-08-01

This work aimed at studying the effects of green coffee bean (GCBE) and yerba mate (YME) extracts, their main phenolic components (5-caffeoylquinic acid, 5-CQA; 3,5-dicaffeoylquinic acid, 3,5-DCQA) and metabolites (ferulic acid, FA; caffeic acid, CA; dihydrocaffeic acid, DHCA; and dihydroferulic acid, DHFA) along with caffeine (CAF) on the viability and proliferation of different human cell lines. Extracts (10-1000 μg/mL) and standards (10-1000 μM) were assayed in colon (Caco-2), lung (A549), oesophageal (OE-33), urinary bladder (T24) human carcinoma cells, and a non-cancer cell line (CCD-18Co). YME significantly reduced viability of cancer cells at all assayed concentrations, the higher doses also reducing cell proliferation. GCBE effects on cell viability were more effective at 100 and 1000 μg/mL, showing modest effects on cell proliferation. The highest doses of 5-CQA and 3,5-DCQA reduced cell viability and proliferation in all cell lines, whereas FA, DHCA and DHFA had lower and variable effects. Caffeine had no effect. Dietary-attainable concentrations (0.1, 1 and 10 μg/mL) of YME were tested for cytotoxicity and reactive oxygen species generation, showing no cytotoxic effect. Low concentrations of all tested compounds were non-cytotoxic to CCD-18Co cells. YME and to a lower degree GCBE, their phenolic components and metabolites may decrease cancer cell viability and proliferation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

PubMed

de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

2013-12-31

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination. Copyright © 2013 Elsevier B.V. All rights reserved.

Viability and Isolation of Marine Bacteria by Dilution Culture: Theory, Procedures, and Initial Results

PubMed Central

Button, D. K.; Schut, Frits; Quang, Pham; Martin, Ravonna; Robertson, Betsy R.

1993-01-01

Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 104 or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60% and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 104 cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms. PMID:16348896

Pregnenolone protects the PC-12 cell line against amyloid beta peptide toxicity but its sulfate ester does not.

PubMed

Akan, Pinar; Kizildag, Servet; Ormen, Murat; Genc, Sermin; Oktem, Mehmet Ali; Fadiloglu, Meral

2009-01-15

Pregnenolone (P), the main precursor of the steroids, and its sulfate ester, pregnenolone sulfate (PS), are the major neurosteroids produced in the neural tissue. Many neuroendocrinological studies stressed the neuroprotective role of neurosteroids although it has been suggested that the inhibition of P and PS synthesis can delay neuronal cell death. The potential roles of P and PS in vital neuronal functions and in amyloid beta peptide (Abeta) toxicity are not clearly identified. This work aims to investigate the effects of P and PS on cell viability and Abeta peptide toxicity in a concentration and exposure time-dependent manner in rat PC-12 cells. The cells were treated with 20muM Abeta peptide 25-35 and variable concentrations of P and PS ranging from 0.5muM to 100muM. To examine the effects of steroid treatment on Abeta peptide toxicity, 0.5muM (low) and 50muM (high) neurosteroids were used. The cell viability and lactate dehydrogenase release of cells were evaluated after 24, 48 and 72h. Morphological changes of cells were also examined. The treatment with higher than 1muM concentrations of P and PS significantly decreased the cell viability comparing to untreated cells. At lower concentrations, P and PS had no toxic actions until 72h. The Abeta treatment resulted in a significant decrease in cell viability comparing to untreated cells. P showed a dose-dependent protective effect against Abeta peptide in PC-12 cells. But its sulfate ester did not have the same effect on Abeta peptide toxicity, even it significantly decreased cell viability in Abeta-treated cells. Consequently, the discrepant effects of P and PS on Abeta peptide toxicity may provide insight on the pathogenesis of Alzheimer's disease.

Effect of all-trans retinoic acid (ATRA) on viability, proliferation, activation and lineage-specific transcription factors of CD4+ T cells.

PubMed

Bidad, Katayoon; Salehi, Eisa; Oraei, Mona; Saboor-Yaraghi, Ali-Akbar; Nicknam, Mohammad Hossein

2011-12-01

All-trans retinoic acid (ATRA), as an active metabolite of vitamin A, has been shown to affect immune cells. This study was performed to evaluate the effect of ATRA on viability, proliferation, activation and lineage-specific transcription factors of CD4+ T cells. CD4+ T cells were separated from heparinized blood of healthy donors and were cultured in conditions, some with, some without ATRA. Viability was assessed by PI flowcytometry and proliferation was measured by MTT assay. CD69 expression was determined by flowcytometry as a measure of cell activation. Lineage-specific transcription factors (FOXP3, RORγt and T-bet) were examined by intracellular staining and flowcytometry. High doses of ATRA (0.1-1 mM) caused extensive cell death in both PBMCs and CD4+ T cells. Doses of ATRA equal to or lower than 10 µM did not adversely affect cell viability and proliferation in comparison to culture medium without ATRA. Doses of ATRA between 10 µM and 1nM significantly increased cell activation when compared to culture medium without ATRA. ATRA could increase FOXP3+ and also FOXP3+RORγt+ T cells while it decreased RORγt+ and T-bet+ T cells. This study showed that doses of ATRA up to 10 µM are safe when using with CD4+ T cells in terms of cell viability, proliferation and activation. We could also show that ATRA diverts the human immune response in neutral conditions (without adding polarizing cytokines) by increasing FOXP3+ cells and decreasing RORγt+ cells. ATRA could be regarded as a potential therapy in inflammatory conditions and autoimmunities.

Serum-free cryopreservation of human amniotic epithelial cells before and after isolation from their natural scaffold.

PubMed

Niknejad, Hassan; Deihim, Tina; Peirovi, Habibollah; Abolghasemi, Hassan

2013-08-01

Amniotic epithelial cells are a promising source for stem cell-based therapy through their potential capacity to differentiate into the cell lineages of all three germ layers. Long-term preservation is necessary to have a ready-to-use source of stem cells, when required. Reduced differentiation capability, decrease of viability and use of fetal bovine serum (FBS) are three drawbacks of clinical application of cryopreserved stem cells. In this study, we used human amniotic fluid instead of animal serum, and evaluated viability and multipotency of amniotic epithelial cells after cryopreservation in suspension and compared with those cryopreserved on their natural scaffold (in situ cryopreservation). There was no significant difference in viability of the cells cryopreserved in amniotic fluid and FBS. Also, the same results were achieved for expression of pluripotency marker OCT-4 when FBS was replaced by amniotic fluid in the samples with the same cryoprotectant. The cells cryopreserved in presence of scaffold had a higher level of viability compared to the cells cryopreserved in suspension. Although, the number of the cells expressed OCT-4 significantly decreased within cryopreservation in suspension, no decrease in expression of OCT-4 was observed when the cells cryopreserved with their natural scaffold. Upon culturing of post-thawed cells in specific lineage differentiating mediums, the markers of neuronal, hepatic, cardiomyocytic and pancreatic were found in differentiated cells. These results show that replacement of FBS by amniotic fluid and in situ cryopreservation of amniotic epithelial cells is an effective approach to overcome limitations related to long-term preservation including differentiation during cryopreservation and decrease of viability. Copyright © 2013 Elsevier Inc. All rights reserved.

Nanodiamonds on tetrahedral amorphous carbon significantly enhance dopamine detection and cell viability.

PubMed

Peltola, Emilia; Wester, Niklas; Holt, Katherine B; Johansson, Leena-Sisko; Koskinen, Jari; Myllymäki, Vesa; Laurila, Tomi

2017-02-15

We hypothesize that by using integrated carbon nanostructures on tetrahedral amorphous carbon (ta-C), it is possible to take the performance and characteristics of these bioelectrodes to a completely new level. The integrated carbon electrodes were realized by combining nanodiamonds (NDs) with ta-C thin films coated on Ti-coated Si-substrates. NDs were functionalized with mixture of carboxyl and amine groups ND andante or amine ND amine , carboxyl ND vox or hydroxyl groups ND H and drop-casted or spray-coated onto substrate. By utilizing these novel structures we show that (i) the detection limit for dopamine can be improved by two orders of magnitude [from 10µM to 50nM] in comparison to ta-C thin film electrodes and (ii) the coating method significantly affects electrochemical properties of NDs and (iii) the ND coatings selectively promote cell viability. ND andante and ND H showed most promising electrochemical properties. The viability of human mesenchymal stem cells and osteoblastic SaOS-2 cells was increased on all ND surfaces, whereas the viability of mouse neural stem cells and rat neuroblastic cells was improved on ND andante and ND H and reduced on ND amine and ND vox. The viability of C6 cells remained unchanged, indicating that these surfaces will not cause excess gliosis. In summary, we demonstrated here that by using functionalized NDs on ta-C thin films we can significantly improve sensitivity towards dopamine as well as selectively promote cell viability. Thus, these novel carbon nanostructures provide an interesting concept for development of various in vivo targeted sensor solutions. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion.

PubMed

Smeringaiova, Ingrida; Trosan, Peter; Mrstinova, Miluse Berka; Matecha, Jan; Burkert, Jan; Bednar, Jan; Jirsova, Katerina

2017-09-01

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.

Comparison of the effect of three autogenous bone harvesting methods on cell viability in rabbits

PubMed Central

Moradi Haghgoo, Janet; Arabi, Seyed Reza; Hosseinipanah, Seyyed Mohammad; Solgi, Ghasem; Rastegarfard, Neda; Farhadian, Maryam

2017-01-01

Background. This study was designed to compare the viability of autogenous bone grafts, harvested using different methods, in order to determine the best harvesting technique with respect to more viable cells. Methods. In this animal experimental study, three harvesting methods, including manual instrument (chisel), rotary device and piezosurgery, were used for harvesting bone grafts from the lateral body of the mandible on the left and right sides of 10 rabbits. In each group, 20 bone samples were collected and their viability was assessed using MTS kit. Statistical analyses, including ANOVA and post hoc Tukey tests, were used for evaluating significant differences between the groups. Results. One-way ANOVA showed significant differences between all the groups (P=0.000). Data analysis using post hoc Tukey tests indicated that manual instrument and piezosurgery had no significant differences with regard to cell viability (P=0.749) and the cell viability in both groups was higher than that with the use of a rotary instrument (P=0.000). Conclusion. Autogenous bone grafts harvested with a manual instrument and piezosurgery had more viable cells in comparison to the bone chips harvested with a rotary device. PMID:28748046

Withagulatin A inhibits hepatic stellate cell viability and procollagen I production through Akt and Smad signaling pathways

PubMed Central

Liu, Qiong; Chen, Jing; Wang, Xu; Yu, Liang; Hu, Li-hong; Shen, Xu

2010-01-01

Aim: To investigate the effects of the natural product Withagulatin A on hepatic stellate cell (HSC) viability and type I procollagen production. The potential mechanism underlying the pharmacological actions was also explored. Methods: The effect of Withagulatin A on cell viability was evaluated in HSC and LX-2 cells using a sulforhodamine B (SRB) assay. Cell cycle distribution was analyzed using flow cytometry. Type I procollagen gene expression was determined using real-time PCR. Regulation of signaling molecules by Withagulatin A was detected using Western blotting. Results: Primary rat HSCs and the human hepatic stellate cell line LX-2 treated with Withagulatin A (0.625-20 μmol/L) underwent a dose-dependent decrease in cell viability, which was associated with S phase arrest and the induction of cell apoptosis. In addition, the natural product decreased phosphorylation of the Akt/mTOR/p70S6K pathway that controls cell proliferation and survival. Furthermore, Withagulatin A (1, 2 μmol/L) inhibited transforming growth factor-β (TGF-β) stimulated type I procollagen gene expression, which was attributable to the suppression of TGF-β stimulated Smad2 and Smad3 phosphorylation. Conclusion: Our results demonstrated that Withagulatin A potently inhibited HSC viability and type I procollagen production, thereby implying that this natural product has potential use in the development of anti-fibrogenic reagents for the treatment of hepatic fibrosis. PMID:20644552

Enhanced resveratrol production in Vitis vinifera cell suspension cultures by heavy metals without loss of cell viability.

PubMed

Cai, Zhenzhen; Kastell, Anja; Speiser, Claire; Smetanska, Iryna

2013-09-01

The effects of heavy metal ions (Co(2+), Ag(+), Cd(2+)) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50 μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15 % of the control level, 24 h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.

Serum-dependent effects of tamoxifen and cannabinoids upon C6 glioma cell viability.

PubMed

Jacobsson, S O; Rongård, E; Stridh, M; Tiger, G; Fowler, C J

2000-12-15

In the present study, the effects of the combination of tamoxifen ((Z)-2[p-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylamine citrate) and three cannabinoids (Delta(9)-tetrahydrocannabinol [Delta(9)-THC], cannabidiol, and anandamide [AEA]) upon the viability of C6 rat glioma cells was assessed at different incubation times and using different culturing concentrations of foetal bovine serum (FBS). Consistent with previous data for human glioblastoma cells, the tamoxifen sensitivity of the cells was increased as the FBS content of the culture medium was reduced from 10 to 0.4 and 0%. The cells expressed protein kinase C alpha and calmodulin (the concentration of which did not change significantly as the FBS concentration was reduced), but did not express estrogen receptors. Delta(9)-THC and cannabidiol, but not AEA, produced a modest reduction in cell viability after 6 days of incubation in serum-free medium, whereas no effects were seen in 10% FBS-containing medium. There was no observed synergy between the effects of tamoxifen and the cannabinoids upon cell viability.

Effects of cryopreservation and hypothermic storage on cell viability and enzyme activity in recombinant encapsulated cells overexpressing alpha-L-iduronidase.

PubMed

Mayer, Fabiana Quoos; Baldo, Guilherme; de Carvalho, Talita Giacomet; Lagranha, Valeska Lizzi; Giugliani, Roberto; Matte, Ursula

2010-05-01

Here, we show the effects of cryopreservation and hypothermic storage upon cell viability and enzyme release in alginate beads containing baby hamster kidney cells overexpressing alpha-L-iduronidase (IDUA), the enzyme deficient in mucopolysaccharidosis type I. In addition, we compared two different concentrations of alginate gel (1% and 1.5%) in respect to enzyme release from the beads and their shape and integrity. Our results indicate that in both alginate concentrations, the enzyme is released in lower amounts compared with nonencapsulated cells. Alginate 1% beads presented increased levels of IDUA release, although this group presented more deformities when compared with alginate 1.5% beads. Importantly, both encapsulated groups presented higher cell viability after long cryopreservation period and hypothermic storage. In addition, alginate 1.5% beads presented higher enzyme release after freezing protocols. Taken together, our findings suggest a benefic effect of alginate upon cell viability and functionality. These results may have important application for treatment of both genetic and nongenetic diseases using microencapsulation-based artificial organs.

Antagonism of serotonin receptor 1B decreases viability and promotes apoptosis in the COS canine osteosarcoma cell line.

PubMed

Viall, A K; Goodall, C P; Stang, B; Marley, K; Chappell, P E; Bracha, S

2016-06-01

Serotonin receptor 1B (5HTR1B) traditionally exhibits anti-proliferative activity in osteoblasts. We examined the expression and function of 5HTR1B in the COS canine osteosarcoma cell line and normal canine osteoblasts. Equal levels of 5HTR1B gene and protein expression were found between normal and malignant osteoblasts. Treatment with serotonin enhanced viability of osteosarcoma cells but not normal osteoblasts. Challenge with the 5HTR1B agonist anpirtoline caused no change in cell viability. Rather incubation with the specific receptor antagonist SB224289 caused reduction in osteoblast viability, with this effect more substantial in osteosarcoma cells. Investigation of this inhibitory activity showed 5HTR1B antagonism induces apoptosis in malignant cells. Evaluation of phosphorylated levels of CREB and ERK, transcriptional regulators associated with serotonin receptor signalling in osteoblasts, revealed aberrant 5HTR1B signalling in COS. Our results confirm the presence of 5HTR1B in a canine osteosarcoma cell line and highlight this receptor as a possible novel therapeutic target. © 2014 John Wiley & Sons Ltd.

Long non-coding RNA HOTAIR promotes UVB-induced apoptosis and inflammatory injury by up-regulation of PKR in keratinocytes.

PubMed

Liu, Guo; Zhang, Wenhao

2018-06-11

Excessive exposure to ultraviolet (UV) rays can cause damage of the skin and may induce cancer, immunosuppression, photoaging, and inflammation. The long non-coding RNA (lncRNA) HOX antisense intergenic RNA (HOTAIR) is involved in multiple human biological processes. However, its role in UVB-induced keratinocyte injury is unclear. This study was performed to investigate the effects of HOTAIR in UVB-induced apoptosis and inflammatory injury in human keratinocytes (HaCaT cells). Quantitative real-time polymerase chain reaction was performed to analyze the expression levels of HOTAIR, PKR, TNF-α, and IL-6. Cell viability was measured using trypan blue exclusion method and cell apoptosis using flow cytometry and western blot. ELISA was used to measure the concentrations of TNF-α and IL-6. Western blot was used to measure the expression of PKR, apoptosis-related proteins, and PI3K/AKT and NF-κB pathway proteins. UVB induced HaCaT cell injury by inhibiting cell viability and promoting cell apoptosis and expressions of IL-6 and TNF-α. UVB also promoted the expression of HOTAIR. HOTAIR suppression increased cell viability and decreased apoptosis and expression of inflammatory factors in UVB-treated cells. HOTAIR also promoted the expression of PKR. Overexpression of HOTAIR decreased cell viability and increased cell apoptosis and expression of inflammatory factors in UVB-treated cells by upregulating PKR. Overexpression of PKR decreased cell viability and promoted cell apoptosis in UVB-treated cells. Overexpression of PKR activated PI3K/AKT and NF-κB pathways. Our findings identified an essential role of HOTAIR in promoting UVB-induced apoptosis and inflammatory injury by up-regulating PKR in keratinocytes.

Influence of Waveform on Cell Viability during Ultrasound Exposure

NASA Astrophysics Data System (ADS)

Saliev, Timur; Feril, Loreto B.; McLean, Donald A.; Tachibana, Katsuro; Campbell, Paul A.

2011-09-01

We examined the role of ultrasound standing waves, and their travelling wave counterparts, on cell viability in an in-vitro insonation apparatus. Furthermore, the effect of distinct waveforms (sine and top-hat) was also explored, together with the role of microbubble presence. Measurements of cell viability in standing wave scenarios demonstrated a relatively higher rate of lysis (63.13±10.89% remaining viable) compared with the travelling wave data, where 96.22±4.0% remained viable. Significant differences were also seen as a function of waveform, where insonations employing top-hat wave shapes resulted in an average end stage viability of 30.31±5.71% compared with 61.94±14.28% in the sinusoidal counterparts.

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation.

PubMed

Chaytor, Jennifer L; Tokarew, Jacqueline M; Wu, Luke K; Leclère, Mathieu; Tam, Roger Y; Capicciotti, Chantelle J; Guolla, Louise; von Moos, Elisabeth; Findlay, C Scott; Allan, David S; Ben, Robert N

2012-01-01

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.

Droplet size influences division of mammalian cell factories in droplet microfluidic cultivation.

PubMed

Periyannan Rajeswari, Prem Kumar; Joensson, Haakan N; Andersson-Svahn, Helene

2017-01-01

The potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous cell division during cultivation in droplets. Here, we report the influence of droplet size on mammalian cell division and viability during cultivation in droplets. Chinese Hamster Ovary (CHO) cells, the most widely used mammalian host cells for biopharmaceuticals production were encapsulated and cultivated in 33, 180 and 320 pL droplets for 3 days. Periodic monitoring of the droplets during incubation showed that the cell divisions in 33 pL droplets stopped after 24 h, whereas continuous cell division was observed in 180 and 320 pL droplets for 72 h. The viability of the cells cultivated in the 33 pL droplets also dropped to about 50% in 72 h. In contrast, the viability of the cells in the larger droplets was above 90% even after 72 h of cultivation, making them a more suitable droplet size for 72-h cultivation. This study shows a direct correlation of microfluidic droplet size to the division and viability of mammalian cells. This highlights the importance of selecting suitable droplet size for mammalian cell factory screening assays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Octanol preconditioning alleviates mouse cardiomyocyte swelling induced by simulated ischemia/reperfusion challenge in vitro].

PubMed

Luo, Yukun; Fang, Jun; Fan, Lin; Lin, Chaogui; Chen, Zhaoyang; Chen, Lianglong

2012-10-01

To investigate the role of connexin 43-formed hemichannels in cell volume regulation induced by simulated ischemia/reperfusion (SI/R). Mouse cardiomyocytes isolated on a Langendorff apparatus with enzyme solution were aliquoted into control, SI/R and SI/R +octanol groups. Calcein-AM was used to stain the cells and the cell volume was measured with confocal microscope by stack scanning. Trypan blue was used to measure the cell viability after the treatments. Calcein-AM staining and cofocal microscopy yielded stable and reproducible results for cell volume measurement. Mouse cardiomyocytes subjected to simulated SI/R showed obvious cell swelling as compared with the control cells [(126∓6)% vs 100%, P<0.05], and octanol preconditioning significantly attenuated the cell swelling [(113∓6)%, P<0.05]. SI/R caused a significant reduction of the cell viability compared to the control cells [(19∓2)% vs (45∓3)%, P<0.01], and octanol preconditioning obviously reduced the viability of the cells with SI/R challenge [(31∓2)%, P<0.01]. Connexin 43-formed hemichannels are involved in the regulation of cardiomyocyte volumes induced by SI/R challenge, and octanol can alleviate the cell swelling to enhance the viability of the cardiomyocytes following SI/R.

Measurement of cell viability in in vitro cultures.

PubMed

Castro-Concha, Lizbeth A; Escobedo, Rosa María; Miranda-Ham, María de Lourdes

2006-01-01

An overview of the methods for assessing cell viability in in vitro cultures is presented. The protocols of four of the most commonly used assays are described in detail, so the readers may be able to determine which assay is suitable for their own projects using plant cell cultures.

Hydrogen Supplementation of Preservation Solution Improves Viability of Osteochondral Grafts

PubMed Central

Yamada, Takuya; Onuma, Kenji; Kuzuno, Jun; Ujihira, Masanobu; Kurokawa, Ryosuke; Sakai, Rina; Takaso, Masashi

2014-01-01

Allogenic osteochondral tissue (OCT) is used for the treatment of large cartilage defects. Typically, OCTs collected during the disease-screening period are preserved at 4°C; however, the gradual reduction in cell viability during cold preservation adversely affects transplantation outcomes. Therefore, improved storage methods that maintain the cell viability of OCTs are needed to increase the availability of high-quality OCTs and improve treatment outcomes. Here, we evaluated whether long-term hydrogen delivery to preservation solution improved the viability of rat OCTs during cold preservation. Hydrogen-supplemented Dulbecco's Modified Eagles Medium (DMEM) and University of Wisconsin (UW) solution both significantly improved the cell viability of OCTs during preservation at 4°C for 21 days compared to nonsupplemented media. However, the long-term cold preservation of OCTs in DMEM containing hydrogen was associated with the most optimal maintenance of chondrocytes with respect to viability and morphology. Our findings demonstrate that OCTs preserved in DMEM supplemented with hydrogen are a promising material for the repair of large cartilage defects in the clinical setting. PMID:25506061

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

PubMed Central

2009-01-01

Background Saccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain. Results Our results show that the concentrations of ACA in the feeding solution, corresponding to those routinely used in the literature, are limiting for the growth of S. cerevisiae BY4741 [PIR4-IL1β] in fed-batch reactor. Even in the presence of a proper ACA supplementation, S. cerevisiae BY4741 [PIR4-IL1β] did not achieve a high cell density. The Δyca1 deletion did not have a beneficial effect on the overall performance of the strain, but it had a clear effect on its viability, which was not impaired during fed-batch operations, as shown by the kd value (0.0045 h-1), negligible if compared to that of the parental strain (0.028 h-1). However, independently of their robustness, both the parental and the Δyca1 mutant ceased to grow early during fed-batch runs, both strains using most of the available carbon source for maintenance, rather than for further proliferation. The mathematical model used evidenced that the energy demand for maintenance was even higher in the case of the Δyca1 mutant, accounting for the growth arrest observed despite the fact that cell viability remained comparatively high. Conclusions The paper points out the relevance of a proper ACA formulation for the outcome of a fed-batch reactor growth carried out with S. cerevisiae BY4741 [PIR4-IL1β] strain and shows the sensitivity of this commonly used auxotrophic strain to aerated fed-batch operations. A Δyca1 disruption was able to reduce the loss of viability, but not to improve the overall performance of the process. A mathematical model has been developed that is able to describe the behaviour of both the parental and mutant producer strain during fed-batch runs, and evidence the role played by the energy demand for maintenance in the outcome of the process. PMID:20042083

Comparison of different particles and methods for magnetic isolation of circulating tumor cells

NASA Astrophysics Data System (ADS)

Sieben, S.; Bergemann, C.; Lübbe, A.; Brockmann, B.; Rescheleit, D.

2001-01-01

A more effective method for tumor cell separation from peripheral blood was established. The results of optimized magnetic particles verified by analyzing yield, purity and viability of isolated epithelial tumor cells were compared with a commercial kit for immunomagnetic cell separation. Porous silica particles of 230 nm were found to give best recovery rates and high viability of extracted cells.

Long Non-Coding RNA HOXA-AS2 Regulates Malignant Glioma Behaviors and Vasculogenic Mimicry Formation via the MiR-373/EGFR Axis.

PubMed

Gao, Yana; Yu, Hai; Liu, Yunhui; Liu, Xiaobai; Zheng, Jian; Ma, Jun; Gong, Wei; Chen, Jiajia; Zhao, Lini; Tian, Yu; Xue, Yixue

2018-01-01

Vasculogenic mimicry (VM) has been reported to be a novel glioma neovascularization process. Anti-VM therapy provides new insight into glioma clinical management. In this study, we revealed the role of the long non-coding RNA HOXA cluster antisense RNA 2 (HOXA-AS2) in malignant glioma behaviors and VM formation. Quantitative real-time PCR was performed to determine the expression levels of HOXA-AS2 in glioma samples and glioblastoma cell lines. CD34-periodic acid-Schiff dual-staining was performed to assess VM in glioma samples. CCK-8, transwell, and Matrigel tube formation assays were performed to measure the effects of HOXA-AS2 knockdown on cell viability, migration, invasion, and VM tube formation, respectively. RNA immunoprecipitation, dual-luciferase reporter and Western blot assays were performed to explore the molecular mechanisms underlying the functions of HOXS-AS2 in glioblastoma cells. A nude mouse xenograft model was used to investigate the role of HOXA-AS2 in xenograft glioma growth and VM density. Student's t-tests, one-way ANOVAs followed by Bonferroni posthoc tests, and chi-square tests were used for the statistical analyses. HOXA-AS2 was upregulated in glioma samples and cell lines and was positively correlated with VM. HOXA-AS2 knockdown attenuated cell viability, migration, invasion, and VM formation in glioma cells and inhibited the expression of vascular endothelial-cadherin (VE-cadherin), as well as the expression and activity of matrix metalloproteinase matrix metalloproteinase (MMP)-2 and MMP-9. miR-373 was downregulated in glioma samples and cell lines and suppressed malignancy in glioblastoma cells. HOXA-AS2 bound to miR-373 and negatively regulated its expression. Epidermal growth factor receptor (EGFR), a target of miR-373, increased the expression levels of VE-cadherin, as well as the expression and activity levels of MMP-2 and MMP-9, via activating phosphatidylinositol 3-kinase/serine/threonine kinase pathways. HOXA-AS2 knockdown combined with miR-373 overexpression yielded optimal tumor suppressive effects and the lowest VM density in vivo. HOXA-AS2 knockdown inhibited malignant glioma behaviors and VM formation via the miR-373/EGFR axis. © 2018 The Author(s). Published by S. Karger AG, Basel.

Comparison of liposomal and 2-hydroxypropyl-β-cyclodextrin-lidocaine on cell viability and inflammatory response in human keratinocytes and gingival fibroblasts.

PubMed

Ferreira, Luiz Eduardo Nunes; Muniz, Bruno Vilela; Dos Santos, Cleiton Pita; Volpato, Maria Cristina; de Paula, Eneida; Groppo, Francisco Carlos

2016-06-01

The aim of this study was to observe the effect multilamellar liposomes (MLV) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in the in-vitro effects of lidocaine in cell viability, pro-inflammatory cytokines and prostaglandin E2 release of both human keratinocytes (HaCaT) and gingival fibroblasts (HGF) cells. HaCaT and HGF cells were exposed to lidocaine 100-1 μm in plain, MLV and HP-β-CD formulations for 6 h or 24 h. The formulation effects in cell viability were measured by XTT assay and by fluorescent labelling. Cytokines (IL-8, IL-6 and TNF-α) and PGE2 release were quantified by ELISA. MLV and HP-β-CD formulations did not affect the HaCaT viability, which was significantly decreased by plain lidocaine after 24 h of exposure. Both drug carriers increased all cytokines released by HGF after 24-h exposure, and none of the carriers was able to reduce the PGE2 release induced by lidocaine. The effect of drug carrier in the lidocaine effects was dependent on the cell type, concentration and time of exposure. MLV and HP-β-CD showed benefits in improving cell viability; however, both of them showed a tendency to increase cytokine release when compared to the plain solution. © 2016 Royal Pharmaceutical Society.

Protective effect of salidroside against bone loss via hypoxia-inducible factor-1α pathway-induced angiogenesis

PubMed Central

Li, Ling; Qu, Ye; Jin, Xin; Guo, Xiao Qin; Wang, Yue; Qi, Lin; Yang, Jing; Zhang, Peng; Li, Ling Zhi

2016-01-01

Hypoxia-inducible factor (HIF)-1α plays a critical role in coupling angiogenesis with osteogenesis during bone development and regeneration. Salidroside (SAL) has shown anti-hypoxic effects in vitro and in vivo. However, the possible roles of SAL in the prevention of hypoxia-induced osteoporosis have remained unknown. Two osteoblast cell lines, MG-63 and ROB, were employed to evaluate the effects of SAL on cell viability, apoptosis, differentiation and mineralization in vitro. Rats subjected to ovariectomy-induced bone loss were treated with SAL in vivo. Our results showed that pre-treatment with SAL markedly attenuated the hypoxia-induced reductions in cell viability, apoptosis, differentiation and mineralization. SAL down-regulated HIF-1α expression and inhibited its translocation; however, SAL increased its transcriptional activity and, consequently, up-regulated vascular endothelial growth factor (VEGF). In vivo studies further demonstrated that SAL caused decreases in the mineral, alkaline phosphatase (ALP), and BGP concentrations in the blood of ovariectomized (OVX) rats. Moreover, SAL improved the trabecular bone microarchitecture and increased bone mineral density in the distal femur. Additionally, SAL administration partially ameliorated this hypoxia via the HIF-1α-VEGF signalling pathway. Our results indicate that SAL prevents bone loss by enhancing angiogenesis and osteogenesis and that these effects are associated with the activation of HIF-1α signalling. PMID:27558909

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix

PubMed Central

1989-01-01

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern- regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM. PMID:2473081

Effect of nano-hydroxyapatite coating on the osteoinductivity of porous biphasic calcium phosphate ceramics.

PubMed

Hu, Jianzhong; Zhou, Yongchun; Huang, Lihua; Liu, Jun; Lu, Hongbin

2014-04-01

Porous biphasic calcium phosphate (BCP) ceramics exhibit good biocompatibility and bone conduction but are not inherently osteoinductive. To overcome this disadvantage, we coated conventional porous BCP ceramics with nano-hydroxyapatite (nHA). nHA was chosen as a coating material due to its high osteoinductive potential. We used a hydrothermal deposition method to coat conventional porous BCP ceramics with nHA and assessed the effects of the coating on the physical and mechanical properties of the underlying BCP. Next, its effects on mesenchymal stem cell (MSC) attachment, proliferation, viability, and osteogenic differentiation were investigated. nHA formed a deposited layer on the BCP surface, and synthesized nHA had a rod-like shape with lengths ranging from ~50-200 nm and diameters from ~15-30 mm. The nHA coating did not significantly affect the density, porosity, flexural strength, or compressive strength of the underlying BCP (P > 0.1). Scanning electron microscopy showed MSC attachment to the scaffolds, with a healthy morphology and anchorage to nHA crystals via cytoplasmic processes. The densities of MSCs attached on BCP and nHA-coated BCP scaffolds were 62 ± 26 cells/mm2 and 63 ± 27 cells/mm2 (P > 0.1), respectively, after 1 day and 415 ± 62 cells/mm2 and 541 ± 35 cells/mm2 (P < 0.05) respectively, after 14 days. According to an MTT assay, MSC viability was higher on nHA-coated BCP scaffolds than on BCP scaffolds (P < 0.05). In addition, MSCs on nHA-coated BCP scaffolds produced more alkaline phosphatase, collagen type I, and osteocalcin than MSCs on BCP scaffolds (P < 0.05). Our results demonstrate that BCP scaffolds coated with nHA were more conducive for MSC adhesion, proliferation, and osteogenic differentiation than conventional, uncoated BCP scaffolds, indicating that nHA coating can enhance the osteoinductive potential of BCP ceramics, making this material more suitable for applications in bone tissue engineering.

Effect of nano-hydroxyapatite coating on the osteoinductivity of porous biphasic calcium phosphate ceramics

PubMed Central

2014-01-01

Background Porous biphasic calcium phosphate (BCP) ceramics exhibit good biocompatibility and bone conduction but are not inherently osteoinductive. To overcome this disadvantage, we coated conventional porous BCP ceramics with nano-hydroxyapatite (nHA). nHA was chosen as a coating material due to its high osteoinductive potential. Methods We used a hydrothermal deposition method to coat conventional porous BCP ceramics with nHA and assessed the effects of the coating on the physical and mechanical properties of the underlying BCP. Next, its effects on mesenchymal stem cell (MSC) attachment, proliferation, viability, and osteogenic differentiation were investigated. Results nHA formed a deposited layer on the BCP surface, and synthesized nHA had a rod-like shape with lengths ranging from ~50–200 nm and diameters from ~15–30 mm. The nHA coating did not significantly affect the density, porosity, flexural strength, or compressive strength of the underlying BCP (P > 0.1). Scanning electron microscopy showed MSC attachment to the scaffolds, with a healthy morphology and anchorage to nHA crystals via cytoplasmic processes. The densities of MSCs attached on BCP and nHA-coated BCP scaffolds were 62 ± 26 cells/mm2 and 63 ± 27 cells/mm2 (P > 0.1), respectively, after 1 day and 415 ± 62 cells/mm2 and 541 ± 35 cells/mm2 (P < 0.05) respectively, after 14 days. According to an MTT assay, MSC viability was higher on nHA-coated BCP scaffolds than on BCP scaffolds (P < 0.05). In addition, MSCs on nHA-coated BCP scaffolds produced more alkaline phosphatase, collagen type I, and osteocalcin than MSCs on BCP scaffolds (P < 0.05). Conclusions Our results demonstrate that BCP scaffolds coated with nHA were more conducive for MSC adhesion, proliferation, and osteogenic differentiation than conventional, uncoated BCP scaffolds, indicating that nHA coating can enhance the osteoinductive potential of BCP ceramics, making this material more suitable for applications in bone tissue engineering. PMID:24690170

Differential eosinophil and mast cell regulation: Mast cell viability and accumulation in inflammatory tissue are independent of proton-sensing receptor GPR65

PubMed Central

Zhu, Xiang; Mose, Eucabeth; Hogan, Simon P.

2014-01-01

Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor G protein-coupled receptor 65 (GPR65) regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present study, we employed in vitro experiments and an intestinal anaphylaxis model in which both mastocytosis and eosinophilia can be observed, particularly in the gastrointestinal tract, to enable us to directly compare the effect of GPR65 expression on these two cell types. We identified GPR65 expression on mast cells; however, unlike eosinophil viability, mast cell viability in vitro is not affected by acidification or GPR65 expression. Mechanistically, we determined that mast cells do not respond to extracellular acidification with increased cAMP levels. Furthermore, in the intestinal anaphylaxis model, we observed a significant reduction of eosinophils (59.1 ± 9.2% decrease) in the jejunum of allergen-challenged GPR65-deficient mice compared with allergen-challenged wild-type mice, despite the degree of antigen sensitization and the expression levels of Th2 cytokines (Il4, Il13) and eosinophil chemokines (Ccl11, Ccl24) in the jejunum being comparable. In contrast, the accumulation of mast cells in allergen-challenged mice was not affected by GPR65 deficiency. In conclusion, our study demonstrates differential regulation of eosinophils and mast cells in inflammatory tissue, with mast cell viability and accumulation being independent of GPR65. PMID:24742990

Long non-coding RNA HULC promotes UVB-induced injury by up-regulation of BNIP3 in keratinocytes.

PubMed

Zhao, Li; Man, Yigang; Liu, Shumei

2018-08-01

Ultraviolet radiation b (UVB) is a common high-energy radiation which can lead to cell damage and even skin cancer. The mechanisms of lncRNAs in various diseases have attracted much attention of researchers. Herein, we investigated the effects and regulations of lncRNA highly up-regulated in liver cancer (HULC) on UVB-induced injury in HaCaT cells. The HaCaT cells were exposed to UVB at a wavelength of 280-320 nm. Cell viability was detected at different times (0, 3, 6, 12 and 24 h) after UVB treatment. Cells were transfected with sh-HULC, pc-HULC, sh-BNIP3 (Bcl-2 interacting protein 3) or pc-BNIP3, respectively. ZM 39,923 HCl was used as JAK/STAT(1/3) inhibitor. Cell viability and apoptosis were tested by trypan blue dye and flow cytometry analysis, respectively. The expression levels of autophagy-related factors were analyzed by Western blot assay. The expression changes of HULC and BNIP3 were measured by qRT-PCR. We found that UVB decreased cell viability, increased apoptosis and autophagy, and up-regulated the expression of HULC in HaCaT cells. Overexpression of HULC reduced cell viability, enhanced apoptosis and autophagy, and up-regulated BNIP3 expression by activating JAK/STAT(1/3) signaling pathway. Finally, BNIP3 suppression increased cell viability, reduced apoptosis and autophagy via the deactivation of mTOR signaling pathway. The results demonstrated that lncRNA HULC up-regulated BNIP3 and activated JAK/STAT(1/3) signaling pathway to accelerate UVB-induced cell damage in HaCaT cells. This study provides a possible target for the clinical treatment of UVB-induced keratinocyte injury. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Effect of histone deacetylase inhibitor in combination with 5-fluorouracil on pancreas cancer and cholangiocarcinoma cell lines.

PubMed

Iwahashi, Shuichi; Ishibashi, Hiroki; Utsunomiya, Tohru; Morine, Yuji; Ochir, Tovuu Lkhaguva; Hanaoka, Jun; Mori, Hiroki; Ikemoto, Tetsuya; Imura, Satoru; Shimada, Mitsuo

2011-02-01

Histone deacetylase (HDAC) is well known to be associated with tumorigenesis through epigenetic regulation, and its inhibitors (HDACIs) induce differentiation and apoptosis of tumor cells. We examined the therapeutic effects of valproic acid (VPA, a HDACI) with a combination of 5-fluorouracil (5-FU) in vitro. A human pancreas cancer cell line (SUIT-2) and a cholangiocarcinoma cell line (HuCCT1) were used. Cell viabilities were evaluated by a cell proliferation assay. We determined the anticancer effects of VPA combined with 5-FU in these cell lines. Pancreas cancer (SUIT-2): No effect of 5-FU (1.0 µM) was observed, but 17% and 30% of proliferation-inhibitory effects were recognized in a dose of 2.5 or 5.0 µM, respectively. Cell viability was only weakly reduced by VPA (0.5 mM). However, in combination of 5-FU (1.0 µM) with VPA (0.5 mM), 19% of inhibitory effect was observed. Cholangiocarcinoma (HuCCT1): 5-FU (1.0 µM) did not suppress the cell viability, but 5-FU (2.5 µM) suppressed by 23%. VPA (0.5 mM) did not suppress the cell viability, while VPA (1.0 mM) weakly decreased it by 11%. Combination of 5-FU (1.0 µM) and VPA (0.5 mM) markedly reduced the cell viability by 30%. VPA augmented the anti-tumor effects of 5-FU in cancer cell lines. Therefore, a combination therapy of 5-FU plus VPA may be a promising therapeutic option for patients with pancreas cancer and cholangiocarcinoma.

siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

PubMed

Rebelo, Thalia M; Vania, Leila; Ferreira, Eloise; Weiss, Stefan F T

2018-07-01

The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. Copyright © 2018. Published by Elsevier Inc.

Micro-RNA-181a suppresses progestin-promoted breast cancer cell growth.

PubMed

Gu, Muqing; Wang, Lijuan; Yang, Chun; Li, Xue; Jia, Chanwei; Croteau, Stephane; Ruan, Xiangyan; Hardy, Pierre

2018-08-01

Recent investigations have indicated that hormone therapy may increase the risk of breast cancer (BC), and the addition of synthetic progestins may play a critical role in this. Several studies have pointed out the important role of progesterone receptor membrane component 1 (PGRMC1) in the development of BC, especially with hormone therapy using progestins. Although the deregulation of microRNA-181a (miR-181a) is often associated with human BC, the effect of miR-181a on PGRMC1 expression during hormone therapy has not been investigated. Cell viability assay and apoptosis assay were performed to investigate the pro-BC effect of progestin (norethisterone, NET) and anti-BC effect of miR-181a on MCF-7 cells. Quantitative RT-PCR and Western blot analysis were used to evaluate gene expressions in the NET-treated MCF-7 cells. NET dose-dependently increased BC cell viability and this effect was accompanied by increased expression of PGRMC1. Overexpression of miR-181a strongly reduced the cell viability of MCF-7 cells, mainly through increased apoptosis, which was evidenced by substantially increased gene expression of pro-apoptosis factors such as BAX and CASPASE 9 in miR-181a overexpressed cells. Importantly, miR-181a abrogated NET-stimulated cell viability and PGRMC1 expression. We provide evidence that miR-181a promotes MCF-7 cell apoptosis. Moreover, miR-181a suppressed NET-provoked cell viability and PGRMC1 expression in MCF-7 cells. These data may suggest a therapeutic strategy of using miR-181a to reduce BC risk in progestin hormone replacement therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

[Simultaneous staining with fluorescein diacetate-propidium iodide to determine isolated cochlear outer hair cell viability of guinea pig].

PubMed

Yu, Q; Shi, H; Wang, J

1995-01-01

A simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is discribed for use in the determination of isolated cochlear outer hair cell viability. With exciter light, viable cells fluoresce bright green, while nonviable cells are bright red. In cell culture and cytotoxicity studies, double-staining with FDA-PI is a accurate method to discriminate between live and nonviable cells.

Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses

PubMed Central

Espina, Miguel; Jülke, Henriette; Brehm, Walter; Ribitsch, Iris; Winter, Karsten

2016-01-01

Background. Mesenchymal stromal cells (MSCs) are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I) five different transport containers (cryotube, two types of plastic syringes, glass syringe, CellSeal), (II) seven different transport media, four temperatures (4 °C vs. room temperature; −20 °C vs. −80 °C), four time frames (24 h vs. 48 h; 48 h vs. 72 h), and (III) three MSC concentrations (5 × 106, 10 × 106, 20 × 106 MSC/ml). Cell viability (Trypan Blue exclusion; percent and total number viable cell), proliferation and trilineage differentiation capacity were assessed for each test condition. Further, the recovered volume of the suspension was determined in part I. Each condition was evaluated using samples of six horses (n = 6) and differentiation protocols were performed in duplicates. Results. In part I of the study, no significant differences in any of the parameters were found when comparing transport containers at room temperature. The glass syringe was selected for all subsequent evaluations (highest recoverable volume of cell suspension and cell viability). In part II, media, temperatures, or time frames had also no significant influence on cell viability, likely due to the large number of comparisons and small sample size. Highest cell viability was observed using autologous bone marrow supernatant as transport medium, and “transport” at 4 °C for 24 h (70.6% vs. control group 75.3%); this was not significant. Contrary, viability was unacceptably low (<40%) for all freezing protocols at −20 °C or −80 °C, particularly with bone marrow supernatant or plasma and DMSO. In part III, various cell concentrations also had no significant influence on any of the evaluated parameters. Chondrogenic differentiation showed a trend towards being decreased for all transport conditions, compared to control cells. Discussion. In this study, transport conditions were not found to impact viability, proliferation or ability for trilineage differentiation of MSCs, most likely due to the small sample size and large number of comparisons. The unusual low viability after all freezing protocols is in contrast to previous equine studies. Potential causes are differences in the freezing, but also in thawing method. Also, the selected container (glass syringe) may have impacted viability. Future research may be warranted into the possibly negative effect of transport on chondrogenic differentiation. PMID:27019778

Direct-write Bioprinting of Cell-laden Methacrylated Gelatin Hydrogels

PubMed Central

Bertassoni, Luiz E.; Cardoso, Juliana C.; Manoharan, Vijayan; Cristino, Ana L.; Bhise, Nupura S.; Araujo, Wesleyan A.; Zorlutuna, Pinar; Vrana, Nihal E.; Ghaemmaghami, Amir M.

2014-01-01

Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least 8 days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms. PMID:24695367

A comparative study of the effects of different low-level lasers on the proliferation, viability, and migration of human melanocytes in vitro.

PubMed

AlGhamdi, Khalid M; Kumar, Ashok; Ashour, Abdelkader E; AlGhamdi, Attieh A

2015-07-01

The aim of this study was to investigate the effects of different low-level laser therapies (LLLTs) of various wavelengths and energies on normal cultured human melanocytes. Various studies have shown the effects of LLLs on various types of cultured cells. Presently, little is known about the biological effects of LLLTs on melanocytes. Melanocytes were exposed to LLLT at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 J/cm(2) using a blue (457 nm), red (635 nm), or ultraviolet (UV) (355 nm) laser. Melanocyte viability, proliferation, and migration were monitored at 72 h after irradiation. The blue (P < 0.001) and red (P < 0.001 and P < 0.01) lasers significantly enhanced viability at 0.5 to 2.0 J/cm(2), whereas the UV laser (P < 0.001) could significantly enhance viability only at 0.5 and 1.0 J/cm(2) compared with controls. The blue and red lasers also significantly enhanced the proliferation of the melanocytes at 0.5 to 2.0 J/cm(2) (P < 0.001), and the UV laser significantly enhanced proliferation at 0.5 to 1.5 J/cm(2) (P < 0.001 and P < 0.01) compared with controls. The blue laser significantly enhanced melanocyte migration at 0.5 to 4.0 J/cm(2) (P < 0.001 to P < 0.05), but the red (P < 0.001 and P < 0.01) and UV (P < 0.001 to P < 0.05) lasers could significantly enhance such migration at 0.5 to 1.0 J/cm(2) and 0.5 to 2.0 J/cm(2), respectively, compared with controls. LLLT at low energy densities is able to significantly increase melanocyte viability, proliferation, and migration in vitro, and at higher energy densities, it gives non-stimulatory results. Additionally, the blue laser was the best among the three lasers. These findings might have potential application in vitiligo treatment in future.

Effects of aluminum in red spruce (Picea rubens) cell cultures: Cell growth and viability, mitochondrial activity, ultrastructure and potential sites of intracellular aluminum accumulation

Treesearch

Rakesh Minocha; Carolyn McQuattie; Wayne Fagerberg; Stephanie Long; Eun Woon Noh

2001-01-01

The effects of Al on red spruce (Picea rubens Sarg.) cell suspension cultures were examined using biochemical, stereo-logical and microscopic methods. Exposure to Al for 24-48 h resulted in a loss of cell viability, inhibition of growth and a significant decrease in mitochondrial activity. Soluble protein content increased in cells treated with Al....

Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation.

PubMed

Kitzmann, J P; Karatzas, T; Mueller, K R; Avgoustiniatos, E S; Gruessner, A C; Balamurugan, A N; Bellin, M D; Hering, B J; Papas, K K

2014-01-01

Replacement of β-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36-72-hour culture period before IT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability. After standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C, whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IEQ)/cm(2) adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content [OCR/DNA] and membrane integrity). Results indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin in the culture media. Current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability before transplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations. Copyright © 2014 Elsevier Inc. All rights reserved.

Viability of fungal and actinomycetal spores after microwave radiation of building materials.

PubMed

Górny, Rafał L; Mainelis, Gediminas; Wlazło, Agnieszka; Niesler, Anna; Lis, Danuta O; Marzec, Stanisław; Siwińska, Ewa; Łudzeń-Izbińska, Beata; Harkawy, Aleksander; Kasznia-Kocot, Joanna

2007-01-01

The effects of microwave radiation on viability of fungal and actinomycetal spores growing on agar (medium optimal for growth) as well as on wooden panel and drywall (common building construction/finishing materials) were studied. All materials were incubated at high (97-99%) and low (32-33%) relative humidity to mimic "wet" and "dry" environmental conditions. Two microwave power densities (10 and 60 mW/cm2) and three times of exposure (5, 30, and 60 min) were tested to find the most effective parameters of radiation which could be applied to non-invasive reduction or cleaning of building materials from microbial contaminants. Additionally, a control of the surface temperature during the experiments allowed differentiation between thermal and microwave effect of such radiation. The results showed that the viability of studied microorganisms differed depending on their strains, growth conditions, power density of microwave radiation, time of exposure, and varied according to the applied combination of the two latter elements. The effect of radiation resulting in a decrease of spore viability on "wet" wooden panel and drywall was generally observed at 60 min exposure. Shorter exposure times decreased the viability of fungal spores only, while in actinomycetes colonizing the studied building materials, such radiation caused an opposite (supporting growth) effect.

Atomic force microscopic study of the influence of physical stresses on Saccharomyces cerevisiae and Schizosaccharomyces pombe.

PubMed

Adya, Ashok K; Canetta, Elisabetta; Walker, Graeme M

2006-01-01

Morphological changes in the cell surfaces of the budding yeast Saccharomyces cerevisiae (strain NCYC 1681), and the fission yeast Schizosaccharomyces pombe (strain DVPB 1354), in response to thermal and osmotic stresses, were investigated using an atomic force microscope. With this microscope imaging, together with measurements of culture viability and cell size, it was possible to relate topological changes of the cell surface at nanoscale with cellular stress physiology. As expected, when the yeasts were exposed to thermostress or osmostress, their viability together with the mean cell volume decreased in conjunction with the increase in thermal or osmotic shock. Nevertheless, the viability of cells stressed for up to 1 h remained relatively high. For example, viabilities were >50% and >90% for the thermostressed, and >60% and >70% for the osmostressed S. cerevisiae and Schiz. pombe, respectively. Mean cell volume measurements, and bearing and roughness analyses of atomic force microscope images of stressed yeasts indicate that Schiz. pombe may be more resistant to physical stresses than S. cerevisiae. Overall, this study has highlighted the usefulness of atomic force microscope in studies of yeast stress physiology.

Human Bone Marrow-Derived Mesenchymal Cell Reactions to 316L Stainless Steel: An in Vitro Study on Cell Viability and Interleukin-6 Expression.

PubMed

Anwar, Iwan Budiwan; Santoso, Asep; Saputra, Eko; Ismail, Rifky; Jamari, J; Van der Heide, Emile

2017-06-01

Purpose: Human bone marrow-derived mesenchymal cell (hBMC) reactions to 316L stainless steel (316L-SS) have never been evaluated. The objective of this study was to assess cell viability and interleukin-6 expression of hBMC cultures upon treatment with a 316L-SS implant. Methods: A cytotoxicity analysis was conducted with a 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium (MTT) assay after a period of 24, 48 and 72 hours of incubation. Expression of interleukin-6 was measured using enzyme-linked immunosorbent assay (ELISA). Results: Cell viability measurement was performed via IC50 formula. All treatment group showed a > 50 % cell viability with a range of 56,5 - 96,9 % at 24 hours, 51,8-77,3% at 48 hours and 70,1- 120 % at 72 hours. Interleukin-6 expression was downregulated subsequent to treatment with 316L-SS compared to the control group. Conclusion: We found that 316L-SS did not exhibit toxicity towards hBMC culture.

Human Bone Marrow-Derived Mesenchymal Cell Reactions to 316L Stainless Steel: An in Vitro Study on Cell Viability and Interleukin-6 Expression

PubMed Central

Anwar, Iwan Budiwan; Santoso, Asep; Saputra, Eko; Ismail, Rifky; Jamari, J.; Van der Heide, Emile

2017-01-01

Purpose: Human bone marrow-derived mesenchymal cell (hBMC) reactions to 316L stainless steel (316L-SS) have never been evaluated. The objective of this study was to assess cell viability and interleukin-6 expression of hBMC cultures upon treatment with a 316L-SS implant. Methods: A cytotoxicity analysis was conducted with a 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium (MTT) assay after a period of 24, 48 and 72 hours of incubation. Expression of interleukin-6 was measured using enzyme-linked immunosorbent assay (ELISA). Results: Cell viability measurement was performed via IC50 formula. All treatment group showed a > 50 % cell viability with a range of 56,5 - 96,9 % at 24 hours, 51,8-77,3% at 48 hours and 70,1- 120 % at 72 hours. Interleukin-6 expression was downregulated subsequent to treatment with 316L-SS compared to the control group. Conclusion: We found that 316L-SS did not exhibit toxicity towards hBMC culture. PMID:28761837

Sustaining fermentation in high-gravity ethanol production by feeding yeast to a temperature-profiled multifeed simultaneous saccharification and co-fermentation of wheat straw.

PubMed

Westman, Johan O; Wang, Ruifei; Novy, Vera; Franzén, Carl Johan

2017-01-01

Considerable progress is being made in ethanol production from lignocellulosic feedstocks by fermentation, but negative effects of inhibitors on fermenting microorganisms are still challenging. Feeding preadapted cells has shown positive effects by sustaining fermentation in high-gravity simultaneous saccharification and co-fermentation (SSCF). Loss of cell viability has been reported in several SSCF studies on different substrates and seems to be the main reason for the declining ethanol production toward the end of the process. Here, we investigate how the combination of yeast preadaptation and feeding, cell flocculation, and temperature reduction improves the cell viability in SSCF of steam pretreated wheat straw. More than 50% cell viability was lost during the first 24 h of high-gravity SSCF. No beneficial effects of adding selected nutrients were observed in shake flask SSCF. Ethanol concentrations greater than 50 g L -1 led to significant loss of viability and prevented further fermentation in SSCF. The benefits of feeding preadapted yeast cells were marginal at later stages of SSCF. Yeast flocculation did not improve the viability but simplified cell harvest and improved the feasibility of the cell feeding strategy in demo scale. Cultivation at 30 °C instead of 35 °C increased cell survival significantly on solid media containing ethanol and inhibitors. Similarly, in multifeed SSCF, cells maintained the viability and fermentation capacity when the temperature was reduced from 35 to 30 °C during the process, but hydrolysis yields were compromised. By combining the yeast feeding and temperature change, an ethanol concentration of 65 g L -1 , equivalent to 70% of the theoretical yield, was obtained in multifeed SSCF on pretreated wheat straw. In demo scale, the process with flocculating yeast and temperature profile resulted in 5% (w/w) ethanol, equivalent to 53% of the theoretical yield. Multifeed SSCF was further developed by means of a flocculating yeast and a temperature-reduction profile. Ethanol toxicity is intensified in the presence of lignocellulosic inhibitors at temperatures that are beneficial to hydrolysis in high-gravity SSCF. The counteracting effects of temperature on cell viability and hydrolysis call for more tolerant microorganisms, enzyme systems with lower temperature optimum, or full optimization of the multifeed strategy with temperature profile.

Ganoderma lucidum (Reishi) inhibits cancer cell growth and expression of key molecules in inflammatory breast cancer.

PubMed

Martínez-Montemayor, Michelle M; Acevedo, Raysa Rosario; Otero-Franqui, Elisa; Cubano, Luis A; Dharmawardhane, Suranganie F

2011-01-01

Inflammatory breast cancer (IBC) is the most lethal and least understood form of advanced breast cancer. Its lethality originates from its nature of invading the lymphatic system and absence of a palpable tumor mass. Different from other metastatic breast cancer cells, IBC cells invade by forming tumor spheroids that retain E-cadherin-based cell-cell adhesions. Herein we describe the potential of the medicinal mushroom Ganoderma lucidum (Reishi) as an attractive candidate for anti-IBC therapy. Reishi contains biological compounds that are cytotoxic against cancer cells. We report the effects of Reishi on viability, apoptosis, invasion, and its mechanism of action in IBC cells (SUM-149). Results show that Reishi selectively inhibits cancer cell viability although it does not affect the viability of noncancerous mammary epithelial cells. Apoptosis induction is consistent with decreased cell viability. Reishi inhibits cell invasion and disrupts the cell spheroids that are characteristic of the IBC invasive pathology. Reishi decreases the expression of genes involved in cancer cell survival and proliferation (BCL-2, TERT, PDGFB), and invasion and metastasis (MMP-9), whereas it increases the expression of IL8. Reishi reduces BCL-2, BCL-XL, E-cadherin, eIF4G, p120-catenin, and c-Myc protein expression and gelatinase activity. These findings suggest that Reishi is an effective anti-IBC therapeutic.

Three-Dimensional Cell Printing of Large-Volume Tissues: Application to Ear Regeneration.

PubMed

Lee, Jung-Seob; Kim, Byoung Soo; Seo, Donghwan; Park, Jeong Hun; Cho, Dong-Woo

2017-03-01

The three-dimensional (3D) printing of large-volume cells, printed in a clinically relevant size, is one of the most important challenges in the field of tissue engineering. However, few studies have reported the fabrication of large-volume cell-printed constructs (LCCs). To create LCCs, appropriate fabrication conditions should be established: Factors involved include fabrication time, residence time, and temperature control of the cell-laden hydrogel in the syringe to ensure high cell viability and functionality. The prolonged time required for 3D printing of LCCs can reduce cell viability and result in insufficient functionality of the construct, because the cells are exposed to a harsh environment during the printing process. In this regard, we present an advanced 3D cell-printing system composed of a clean air workstation, a humidifier, and a Peltier system, which provides a suitable printing environment for the production of LCCs with high cell viability. We confirmed that the advanced 3D cell-printing system was capable of providing enhanced printability of hydrogels and fabricating an ear-shaped LCC with high cell viability. In vivo results for the ear-shaped LCC also showed that printed chondrocytes proliferated sufficiently and differentiated into cartilage tissue. Thus, we conclude that the advanced 3D cell-printing system is a versatile tool to create cell-printed constructs for the generation of large-volume tissues.

CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions.

PubMed

McVey, Duncan; Aronov, Michael; Rizzi, Giovanni; Cowan, Alexis; Scott, Charo; Megill, John; Russell, Reb; Tirosh, Boaz

2016-09-01

The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Effect of bioactive extruded PLA/HA composite films on focal adhesion formation of preosteoblastic cells.

PubMed

Persson, Maria; Lorite, Gabriela S; Kokkonen, Hanna E; Cho, Sung-Woo; Lehenkari, Petri P; Skrifvars, Mikael; Tuukkanen, Juha

2014-09-01

The quality of the initial cell attachment to a biomaterial will influence any further cell function, including spreading, proliferation, differentiation and viability. Cell attachment is influenced by the material's ability to adsorb proteins, which is related to the surface chemistry and topography of the material. In this study, we incorporated hydroxyapatite (HA) particles into a poly(lactic acid) (PLA) composite and evaluated the surface structure and the effects of HA density on the initial cell attachment in vitro of murine calvarial preosteoblasts (MC3T3-EI). Scanning electron microscopy (SEM), atomic force microscopy (AFM) and infrared spectroscopy (FTIR) showed that the HA particles were successfully incorporated into the PLA matrix and located at the surface which is of importance in order to maintain the bioactive effect of the HA particles. SEM and AFM investigation revealed that the HA density (particles/area) as well as surface roughness increased with HA loading concentration (i.e. 5, 10, 15 and 20wt%), which promoted protein adsorption. Furthermore, the presence of HA on the surface enhanced cell spreading, increased the formation of actin stress fibers and significantly improved the expression of vinculin in MC3T3-E1 cells which is a key player in the regulation of cell adhesion. These results suggest the potential utility of PLA/HA composites as biomaterials for use as a bone substitute material and in tissue engineering applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing

PubMed Central

Ng, Wei Long; Yeong, Wai Yee; Naing, May Win

2017-01-01

Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%–3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process. PMID:28772551

Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing.

PubMed

Ng, Wei Long; Yeong, Wai Yee; Naing, May Win

2017-02-16

Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%-3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process.

Thermosensitive nanospheres with a gold layer revealed as low-cytotoxic drug vehicles.

PubMed

Qin, Jian; Jo, Yun Suk; Ihm, Jong Eun; Kim, Do Kyung; Muhammed, Mamoun

2005-09-27

In this paper, the positive effect of a gold layer on cell viability is demonstrated by examining the results given by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfop henyl)-2H-tetrazolium (MTS) assay and two-color cell fluorescence viability (TCCV) assay. These cytotoxicity tests were performed with human cervical adenocarcinoma cells (HeLa cell line) and transformed African green monkey kidney fibroblast cells (Cos-7 cell line). To fabricate the nanostructures as drug vehicles, first, poly(l,l-lactide-co-ethylene glycol) (PLLA-PEG) and poly(N-isopropylacrylamide-co-D,D-lactide) (PNIPAAm-PDLA) were synthesized, and then two kinds of thermosensitive nanospheres comprising "shell-in-shell" structures without a gold layer (PLLA-PEG@PNIPAAm-PDLA) and with a gold layer (Au@PLLA-PEG@PNIPAAm-PDLA) were constructed by a modified double-emulsion method (MDEM). Both of them displayed a unique thermosensitive character exhibiting the lower critical solubility temperature (LCST) at 36.7 degrees C which was confirmed by UV-vis spectroscopy and differential scanning calorimetry (DSC). The release profiles of entrapped bovine serum albumin (BSA) were monitored at 22 and 37 degrees C, respectively, to reveal the thermal dependence on the release rate. In cell viability tests, both PLLA-PEG@PNIPAAm-PDLA and Au@PLLA-PEG@PNIPAAm-PDLA showed excellent cell viability, and furthermore, Au@PLLA-PEG@PNIPAAm-PDLA, particularly at high doses, exhibited more enhanced cell viability than PLLA-PEG@PNIPAAm-PDLA. This effect is mainly attributed to the gold layer which binds the protein molecules first and consequently facilitates transmembrane uptake of essential nutrients in the cell media, resulting in favorable cell proliferation.

Investigations on gel forming media use in low gravity bioseparations research

NASA Technical Reports Server (NTRS)

Todd, Paul; Szlag, David C.; Plank, Lindsay D.; Delcourt, Scott G.; Kunze, M. Elaine

1989-01-01

Research on gelling media and conditions suitable for the preservation of the spatial configuration of cell suspensions and macromolecular solutions after separation in free fluid during low gravity experiments is presented. The examples studied included free electrophoresis of cells in a cylindrical column and two-phase aqueous polymer separation. Microgravity electrophoresis experiments were simulated by separating model cell types (animal or human) in a vertical density gradient containing low-conductivity buffer, 1.7-6.5 percent Ficoll, 6.8-5.0 percent sucrose, and 1 percent SeaPrep low-melting temperature agarose. Upon cooling, a gel formed in the column and cells could be captured at the forming locations. Two-phase extraction experiments were simulated using two-polymer solutions in which phase separation occurs in normal saline at temperatures compatible with cell viability and in which one or both phases form a gel upon cooling. Suitable polymers included commercial agaroses (1-2 percent), maltodextrin (5-7 percent), and gelatin (5-20 percent).

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

PubMed

Karayazi Atici, Ödül; Urbanska, Anna; Gopinathan, Sesha Gopal; Boutillon, Florence; Goffin, Vincent; Shemanko, Carrie S

2018-02-01

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents. Copyright © 2018 Endocrine Society.

Evaluation of Commercial-off-the-Shelf Materials for the Preservation of Bacillus anthracis Vegetative Cells for Forensic Analysis.

PubMed

Angelini, Daniel J; Harris, Jacquelyn V; Burton, Laura L; Rastogi, Pooja R; Smith, Lisa S; Rastogi, Vipin K

2018-03-01

Environmental surface sampling is crucial in determining the zones of contamination and overall threat assessment. Viability retention of sampled material is central to such assessments. A systematic study was completed to determine viability of vegetative cells under nonpermissive storage conditions. Despite major gains in nucleic acid sequencing technologies, initial positive identification of threats must be made through direct culture of the sampled material using classical microbiological methods. Solutions have been developed to preserve the viability of pathogens contained within clinical samples, but many have not been examined for their ability to preserve biological agents. The purpose of this study was to systematically examine existing preservation materials that can retain the viability of Bacillus anthracis vegetative cells stored under nonpermissive temperatures. The results show effectiveness of five of seventeen solutions, which are capable of retaining viability of a sporulation deficient strain of B. anthracis Sterne when stored under nonrefrigerated conditions. © 2017 American Academy of Forensic Sciences.

Biocompatibility of hydroxyapatite scaffolds processed by lithography-based additive manufacturing.

PubMed

Tesavibul, Passakorn; Chantaweroad, Surapol; Laohaprapanon, Apinya; Channasanon, Somruethai; Uppanan, Paweena; Tanodekaew, Siriporn; Chalermkarnnon, Prasert; Sitthiseripratip, Kriskrai

2015-01-01

The fabrication of hydroxyapatite scaffolds for bone tissue engineering applications by using lithography-based additive manufacturing techniques has been introduced due to the abilities to control porous structures with suitable resolutions. In this research, the use of hydroxyapatite cellular structures, which are processed by lithography-based additive manufacturing machine, as a bone tissue engineering scaffold was investigated. The utilization of digital light processing system for additive manufacturing machine in laboratory scale was performed in order to fabricate the hydroxyapatite scaffold, of which biocompatibilities were eventually evaluated by direct contact and cell-culturing tests. In addition, the density and compressive strength of the scaffolds were also characterized. The results show that the hydroxyapatite scaffold at 77% of porosity with 91% of theoretical density and 0.36 MPa of the compressive strength are able to be processed. In comparison with a conventionally sintered hydroxyapatite, the scaffold did not present any cytotoxic signs while the viability of cells at 95.1% was reported. After 14 days of cell-culturing tests, the scaffold was able to be attached by pre-osteoblasts (MC3T3-E1) leading to cell proliferation and differentiation. The hydroxyapatite scaffold for bone tissue engineering was able to be processed by the lithography-based additive manufacturing machine while the biocompatibilities were also confirmed.

Collagen scaffolds derived from a marine source and their biocompatibility.

PubMed

Song, Eun; Yeon Kim, So; Chun, Taehoon; Byun, Hyun-Jung; Lee, Young Moo

2006-05-01

The primary sources of industrial collagens are calf skin and bone. However, these carry a high risk of bovine spongiform encephalopathy or transmissible spongiform encephalopathy. In this study, a novel form of acid-soluble collagen was extracted from jellyfish in an effort to obtain an alternative and safer collagen. Porous scaffolds composed of jellyfish collagen were prepared by freeze-drying and cross-linking with 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide to be used in tissue engineering applications. Enzymatic degradation kinetics of jellyfish collagen scaffolds were controlled by EDC/NHS-cross-linking density. Results from an MTT assay indicated that jellyfish collagen exhibited higher cell viability than other naturally derived biomaterials, including bovine collagen, gelatin, hyaluronic acid, and glucan. Jellyfish collagen scaffolds also had a highly porous and interconnected pore structure, which is useful for an high-density cell seeding, an efficient nutrient and an oxygen supply to the cells cultured in the three-dimensional matrices. To determine whether jellyfish collagen evokes any specific inflammatory response compared to that induced by bovine collagen or gelatin, we measured the levels of pro-inflammatory cytokines and antibody secretions and monitored the population changes of immune cells after in vivo implantation. Jellyfish collagen was found to induce an immune response at least comparable to those caused by bovine collagen and gelatin.

Effects of tocotrienols on cell viability and apoptosis in normal murine liver cells (BNL CL.2) and liver cancer cells (BNL 1ME A.7R.1), in vitro.

PubMed

Har, Chan Hooi; Keong, Chan Kok

2005-01-01

The effects of tocotrienols on murine liver cell viability and their apoptotic events were studied over a dose range of 0-32 microg mL(-1). Normal murine liver cells (BNL CL.2) and murine liver cancer cells (BNL 1ME A.7R.1) were treated with tocotrienols (T(3)), alpha tocopherol (alpha-T) and the chemo drug, Doxorubicin (Doxo, as a positive control). Cell viability assay showed that T(3) significantly (P < or = 0.05) lowered the percentage of BNL 1ME A.7R.1 cell viability in a dose-responsive manner (8-16 microg mL(-1)), whereas T did not show any significant (P>0.05) inhibition in cell viability with increasing treatment doses of 0-16 microg mL(-1). The IC(50) for tocotrienols were 9.8, 8.9, 8.1, 9.7, 8.1 and 9.3 microg mL(-1) at 12, 24, 36, 48, 60 and 72 hours respectively. Early apoptosis was detected 6 hours following T(3) treatment of BNL 1ME A.7R.1 liver cancer cells, using Annexin V-FITC fluorescence microscopy assay for apoptosis, but none were observed for the non-treated liver cancer cells at the average IC(50) of 8.98 microg mL(-1) tocotrienols for liver cancer cells. Several apoptotic bodies were detected in BNL 1ME A.7R.1 liver cancer cells at 6 hours post-treatment with tocotrienols (8.98 microg mL(-1)) using Acridine Orange/Propidium Iodide fluorescence assay. However, only a couple of apoptotic bodies were seen in the non-treated liver cancer cells and the BNL CL.2 normal liver cells. Some mitotic bodies were also observed in the T(3)-treated BNL 1ME A.7R.1 liver cancer cells but were not seen in the untreated BNL 1ME A.7R.1 cells and the BNL CL.2 liver cells. Following T(3)-treatment (8.98 microg mL(-1)) of the BNL 1ME A.7R.1 liver cancer cells, 24.62%, 25.53% and 44.90% of the cells showed elevated active caspase 3 activity at 9, 12 and 24 hours treatment period, respectively. DNA laddering studies indicated DNA fragmentation occurred in the T(3)-treated liver cancer cells, BNL 1ME A.7R.1 but not in non-treated liver cancer cells and the T(3)-treated and non-treated normal liver cells. These results suggest that tocotrienols were able to reduce the cell viability in the murine liver cancer cells at a dose of 8-32 microg mL(-1) and that this decrease in percentage cell viability may be due to apoptosis.

Involvement of TRPV1 and AQP2 in hypertonic stress by xylitol in odontoblast cells.

PubMed

Tokuda, M; Fujisawa, M; Miyashita, K; Kawakami, Y; Morimoto-Yamashita, Y; Torii, M

2015-02-01

To examine the responses of mouse odontoblast-lineage cell line (OLC) cultures to xylitol-induced hypertonic stress. OLCs were treated with xylitol, sucrose, sorbitol, mannitol, arabinose and lyxose. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The expression of transient receptor potential vanilloids (TRPV) 1, 3 and 4 was detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The expression of aquaporin (AQP) 2 was detected using immunofluorescence and Western blotting analysis. The expression of interleukin-6 (IL-6) under xylitol-induced hypertonic stress was assessed using an enzyme-linked immunosorbent assay (ELISA). Small interfering ribonucleic acid (siRNA) for AQP-2 was used to inhibition assay. Xylitol-induced hypertonic stress did not decrease OLC viability, unlike the other sugars tested. OLCs expressed TRPV1, 3 and 4 as well as AQP2. Xylitol inhibited lipopolysaccharide (LPS)-induced IL-6 expression after 3 h of hypertonic stress. TRPV1 mRNA expression was upregulated by xylitol. Costimulation with HgCl2 (AQP inhibitor) and Ruthenium red (TRPV1 inhibitor) decreased cell viability with xylitol stimulation. OLCs treated with siRNA against TRPV1 exhibited decreased cell viability with xylitol stimulation. OLCs have high-cell viability under xylitol-induced hypertonic stress, which may be associated with TRPV1 and AQP2 expressions.

Hydrogenated amorphous silicon coatings may modulate gingival cell response

NASA Astrophysics Data System (ADS)

Mussano, F.; Genova, T.; Laurenti, M.; Munaron, L.; Pirri, C. F.; Rivolo, P.; Carossa, S.; Mandracci, P.

2018-04-01

Silicon-based materials present a high potential for dental implant applications, since silicon has been proven necessary for the correct bone formation in animals and humans. Notably, the addition of silicon is effective to enhance the bioactivity of hydroxyapatite and other biomaterials. The present work aims to expand the knowledge of the role exerted by hydrogen in the biological interaction of silicon-based materials, comparing two hydrogenated amorphous silicon coatings, with different hydrogen content, as means to enhance soft tissue cell adhesion. To accomplish this task, the films were produced by plasma enhanced chemical vapor deposition (PECVD) on titanium substrates and their surface composition and hydrogen content were analyzed by means of X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectrophotometry (FTIR) respectively. The surface energy and roughness were measured through optical contact angle analysis (OCA) and high-resolution mechanical profilometry respectively. Coated surfaces showed a slightly lower roughness, compared to bare titanium samples, regardless of the hydrogen content. The early cell responses of human keratinocytes and fibroblasts were tested on the above mentioned surface modifications, in terms of cell adhesion, viability and morphometrical assessment. Films with lower hydrogen content were endowed with a surface energy comparable to the titanium surfaces. Films with higher hydrogen incorporation displayed a lower surface oxidation and a considerably lower surface energy, compared to the less hydrogenated samples. As regards mean cell area and focal adhesion density, both a-Si coatings influenced fibroblasts, but had no significant effects on keratinocytes. On the contrary, hydrogen-rich films increased manifolds the adhesion and viability of keratinocytes, but not of fibroblasts, suggesting a selective biological effect on these cells.

Neuroprotective effect of astaxanthin against rat retinal ganglion cell death under various stresses that induce apoptosis and necrosis.

PubMed

Yamagishi, Reiko; Aihara, Makoto

2014-01-01

Astaxanthin is a type of carotenoid known to have strong antioxidant effects. The purpose of this study was to investigate whether astaxanthin confers a neuroprotective effect against glutamate stress, oxidative stress, and hypoxia-induced apoptotic or necrotic cell death in primary cultures of rat retinal ganglion cells (RGCs). Purified rat RGCs were exposed to three kinds of stressors induced by 25 μM glutamate for 72 h, B27 medium without an antioxidant for 4 h, and a reduced oxygen level of 5% for 12 h. Each assay was repeated 12 times, with or without 1 nM, 10 nM, and 100 nM astaxanthin. The number of live RGCs was then counted using a cell viability assay. RGC viability in each condition was evaluated and compared with controls. In addition, we measured apoptosis and DNA damage. We found that under glutamate stress, RGC viability was reduced to 58%. Cultures with 1 nM, 10 nM, and 100 nM astaxanthin showed an increase in RGC viability of 63%, 74%, and 84%, respectively. Under oxidative stress, RGC viability was reduced to 40%, and astaxanthin administration resulted in increased viability of 43%, 50%, and 67%, respectively. Under hypoxia, RGC viability was reduced to 66%, and astaxanthin administration resulted in a significant increase in viability to 67%, 77%, and 93%, respectively. These results indicate that 100 nM astaxanthin leads to a statistically significant increase in RGC viability under the three kinds of stressors tested, compared to controls (Dunnett's test, p<0.05). The apoptotic activity of RGCs under glutamate stress increased to 32%, but was reduced to 15% with 100 nM astaxanthin administration. Glutamate stress led to a 58% increase in DNA damage, which was reduced to 43% when cultured with 100 nM astaxanthin. Thus, 100 nM astaxanthin showed a statistically significant reduction in apoptosis and DNA damage in RGCs (Wilcoxon rank-sum test, p<0.05). Our results suggest that astaxanthin has a neuroprotective effect against RGC death induced by glutamate stress, oxidative stress, and hypoxia, which induce apoptotic and necrotic cell death.

Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes

PubMed Central

Usta, O. Berk; Kim, Yeonhee; Ozer, Sinan; Bruinsma, Bote G.; Lee, Jungwoo; Demir, Esin; Berendsen, Tim A.; Puts, Catheleyne F.; Izamis, Maria-Louisa; Uygun, Korkut; Uygun, Basak E.; Yarmush, Martin L.

2013-01-01

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials. PMID:23874947

1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

PubMed Central

Xing, Fuqiang; Zhan, Qiuqiang; He, Yiduo; Cui, Jiesheng; He, Sailing; Wang, Guanyu

2016-01-01

Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis. PMID:27689798

Ganoderma lucidum (Reishi) Inhibits Cancer Cell Growth and Expression of Key Molecules in Inflammatory Breast Cancer

PubMed Central

Martínez-Montemayor, Michelle M.; Acevedo, Raysa Rosario; Otero-Franqui, Elisa; Cubano, Luis. A.; Dharmawardhane, Suranganie F.

2011-01-01

Inflammatory breast cancer (IBC) is the most lethal and least understood form of advanced breast cancer. Its lethality originates from its nature of invading the lymphatic system and absence of a palpable tumor mass. Different from other metastatic breast cancer cells, IBC cells invade by forming tumor spheroids that retain E-cadherin-based cell–cell adhesions. Herein we describe the potential of the medicinal mushroom Ganoderma lucidum (Reishi) as an attractive candidate for anti-IBC therapy. Reishi contains biological compounds that are cytotoxic against cancer cells. We report the effects of Reishi on viability, apoptosis, invasion, and its mechanism of action in IBC cells (SUM-149). Results show that Reishi selectively inhibits cancer cell viability although it does not affect the viability of noncancerous mammary epithelial cells. Apoptosis induction is consistent with decreased cell viability. Reishi inhibits cell invasion and disrupts the cell spheroids that are characteristic of the IBC invasive pathology. Reishi decreases the expression of genes involved in cancer cell survival and proliferation (BCL-2, TERT, PDGFB), and invasion and metastasis (MMP-9), whereas it increases the expression of IL8. Reishi reduces BCL-2, BCL-XL, E-cadherin, eIF4G, p120-catenin, and c-Myc protein expression and gelatinase activity. These findings suggest that Reishi is an effective anti-IBC therapeutic. PMID:21888505

Successful isolation of viable adipose-derived stem cells from human adipose tissue subject to long-term cryopreservation: positive implications for adult stem cell-based therapeutics in patients of advanced age.

PubMed

Devitt, Sean M; Carter, Cynthia M; Dierov, Raia; Weiss, Scott; Gersch, Robert P; Percec, Ivona

2015-01-01

We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2-1159 days) from patients of varying ages (26-62 years). Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved <1 year than from tissue cryopreserved >2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages.

Two-dimensional and three-dimensional viability measurements of adult stem cells with optical coherence phase microscopy

NASA Astrophysics Data System (ADS)

Bagnaninchi, Pierre O.; Holmes, Christina; Drummond, Nicola; Daoud, Jamal; Tabrizian, Maryam

2011-08-01

Cell viability assays are essential tools for cell biology. They assess healthy cells in a sample and enable the quantification of cellular responses to reagents of interest. Noninvasive and label-free assays are desirable in two-dimensional (2D) and three-dimensional (3D) cell culture to facilitate time-course viability studies. Cellular micromotion, emanating from cell to substrate distance variations, has been demonstrated as a marker of cell viability with electric cell-substrate impedance sensing (ECIS). In this study we investigated if optical coherence phase microscopy (OCPM) was able to report phase fluctuations of adult stem cells in 2D and 3D that could be associated with cellular micromotion. An OCPM has been developed around a Thorlabs engine (λo = 930 nm) and integrated in an inverted microscope with a custom scanning head. Human adipose derived stem cells (ADSCs, Invitrogen) were cultured in Mesenpro RS medium and seeded either on ECIS arrays, 2D cell culture dishes, or in 3D highly porous microplotted polymeric scaffolds. ADSC micromotion was confirmed by ECIS analysis. Live and fixed ADSCs were then investigated in 2D and 3D with OCPM. Significant differences were found in phase fluctuations between the different conditions. This study indicated that OCPM could potentially assess cell vitality in 2D and in 3D microstructures.

Effect of tributyltin (TBT) in the metabolic activity of TBT-resistant and sensitive estuarine bacteria.

PubMed

Cruz, Andreia; Oliveira, Vanessa; Baptista, Inês; Almeida, Adelaide; Cunha, Angela; Suzuki, Satoru; Mendo, Sónia

2012-01-01

The effect of tributyltin (TBT) on growth and metabolic activity of three estuarine bacteria with different TBT resistance profiles was investigated in an organic-rich culture medium (TSB) and in phosphate buffered saline (PBS) buffer. Exposure to TBT was assessed by determining its effect on growth (OD(600 nm) measurement), bacterial productivity (leucine incorporation), viability (CFU counts), aggregation and cell size (from Live/Dead analysis), ATP and NADH concentrations. TBT exposure resulted in decrease of bacterial density, cell size, and metabolic activity. In addition, cell aggregates were observed in the TBT-treated cultures. TBT strongly affected bacterial cell metabolism and seemed to exert an effect on its equilibrium, interfering with cell activity. Also, TBT toxicity was lower when cells were grown in TSB than in PBS, suggesting that a nutrient-rich growth medium can protect cells from TBT toxicity. This study contributes to our understanding of the TBT-resistant cell behavior reflected in its physiology and metabolic activity. This information is of utmost importance for further studies of TBT bioremediation. Copyright © 2010 Wiley Periodicals, Inc.

Engineering muscle cell alignment through 3D bioprinting.

PubMed

Mozetic, Pamela; Giannitelli, Sara Maria; Gori, Manuele; Trombetta, Marcella; Rainer, Alberto

2017-09-01

Processing of hydrogels represents a main challenge for the prospective application of additive manufacturing (AM) to soft tissue engineering. Furthermore, direct manufacturing of tissue precursors with a cell density similar to native tissues has the potential to overcome the extensive in vitro culture required for conventional cell-seeded scaffolds seeking to fabricate constructs with tailored structural and functional properties. In this work, we present a simple AM methodology that exploits the thermoresponsive behavior of a block copolymer (Pluronic ® ) as a means to obtain good shape retention at physiological conditions and to induce cellular alignment. Pluronic/alginate blends have been investigated as a model system for the processing of C2C12 murine myoblast cell line. Interestingly, C2C12 cell model demonstrated cell alignment along the deposition direction, potentially representing a new avenue to tailor the resulting cell histoarchitecture during AM process. Furthermore, the fabricated constructs exhibited high cell viability, as well as a significantly improved expression of myogenic genes vs. conventional 2D cultures. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2582-2588, 2017. © 2017 Wiley Periodicals, Inc.

The reducibility of heLa cell viability by Sargassum polycystum extracts

NASA Astrophysics Data System (ADS)

Firdaus, M.; Setijawati, D.; Islam, I.; Nursyam, H.; Kartikaningsih, H.; Yufidasari, H. S.; Prihanto, A. A.; Nurdiani, R.; Jaziri, A. A.

2018-04-01

Cervical cancer is the second largest cause of death-related cancer in women. The efficacy of cancer drugs is still low. Bioactive of brown seaweed has been studied by in vitro and in vivo as anticancer. The aim of this study was to evaluate the cytotoxicity of Sargassum polycystum extracts on HeLa cell, to recognize bioactive on extract and estimate the interaction between the bioactive and target protein. S. polycystum was found from Talango Island waters and HeLa cell was obtained from Indonesian Science Institute. Sample was extracted by ethanol, ethyl acetate and hexane, concentrated and finally, extracts were assayed on HeLa cell. The viability of this cell was quantified on ELISA-Reader. The bioactive compounds of the extract were elucidated by GC-MS. The interaction between bioactive and target protein was evaluated by using in silico method. The result showed that the lowest viability of HeLa cell on n-hexane extracts treatment. The n-hexane extract of this seaweed contained benzenepropanoic acid. This compound reduced HeLa cell viability by reducing of thrombin concentration. In conclusion, the benzene propanoic acid of S. polycystum was the cytotoxic agent and it is potential agent for anti-cervical cancer.

Cross-link regulation of precursor N-cadherin and FGFR1 by GDNF increases U251MG cell viability.

PubMed

Tang, Chuan-Xi; Gu, Yan-Xia; Liu, Xin-Feng; Tong, Shu-Yan; Ayanlaja, Abiola A; Gao, Yue; Ji, Guang-Quan; Xiong, Ye; Huang, Lin-Yan; Gao, Dian-Shuai

2018-07-01

Glial cell line-derived neurotrophic factor (GDNF) is considered to be involved in the development of glioma. However, uncovering the underlying mechanism of the proliferation of glioma cells is a challenging work in progress. We have identified the binding of the precursor of N-cadherin (proN-cadherin) and GDNF on the cell membrane in previous studies. In the present study, we observed increased U251 Malignant glioma (U251MG) cell viability by exogenous GDNF (50 ng/ml). We also confirmed that the high expression of the proN-cadherin was stimulated by exogenous GDNF. Concurrently, we affirmed that lower expression of proN-cadherin correlated with reduced glioma cell viability. Additionally, we observed glioma cell U251MG viability as the phosphorylation level of FGFR1 at Y653 and Y654 was increased after exogenous GDNF treatment, which led to increased interaction between proN-cadherin and FGFR1 (pY653+Y654). Our experiments presented a new mechanism adopted by GDNF supporting glioma development and indicated a possible therapeutic potential via the inhibition of proN-cadherin/FGFR1 interaction.

Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells.

PubMed

Poma, Anna; Colafarina, Sabrina; Aruffo, Eleonora; Zarivi, Osvaldo; Bonfigli, Antonella; Di Bucchianico, Sebastiano; Di Carlo, Piero

2017-01-01

Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn't differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication.

Effects of size and surface of zinc oxide and aluminum-doped zinc oxide nanoparticles on cell viability inferred by proteomic analyses.

PubMed

Pan, Chih-Hong; Liu, Wen-Te; Bien, Mauo-Ying; Lin, I-Chan; Hsiao, Ta-Chih; Ma, Chih-Ming; Lai, Ching-Huang; Chen, Mei-Chieh; Chuang, Kai-Jen; Chuang, Hsiao-Chi

2014-01-01

Although the health effects of zinc oxide nanoparticles (ZnONPs) on the respiratory system have been reported, the fate, potential toxicity, and mechanisms in biological cells of these particles, as related to particle size and surface characteristics, have not been well elucidated. To determine the physicochemical properties of ZnONPs that govern cytotoxicity, we investigated the effects of size, electronic properties, zinc concentration, and pH on cell viability using human alveolar-basal epithelial A549 cells as a model. We observed that a 2-hour or longer exposure to ZnONPs induced changes in cell viability. The alteration in cell viability was associated with the zeta potentials and pH values of the ZnONPs. Proteomic profiling of A549 exposed to ZnONPs for 2 and 4 hours was used to determine the biological mechanisms of ZnONP toxicity. p53-pathway activation was the core mechanism regulating cell viability in response to particle size. Activation of the Wnt and TGFβ signaling pathways was also important in the cellular response to ZnONPs of different sizes. The cadherin and Wnt signaling pathways were important cellular mechanisms triggered by surface differences. These results suggested that the size and surface characteristics of ZnONPs might play an important role in their observed cytotoxicity. This approach facilitates the design of more comprehensive systems for the evaluation of nanoparticles.

The Use of Multidimensional Image-Based Analysis to Accurately Monitor Cell Growth in 3D Bioreactor Culture

PubMed Central

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells. PMID:22028809

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture.

PubMed

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.

Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells.

PubMed

Tsai, J C; Jain, M; Hsieh, C M; Lee, W S; Yoshizumi, M; Patterson, C; Perrella, M A; Cooke, C; Wang, H; Haber, E; Schlegel, R; Lee, M E

1996-02-16

Pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) have been used as antioxidants to prevent apoptosis in lymphocytes, neurons, and vascular endothelial cells. We report here that PDTC and NAC induce apoptosis in rat and human smooth muscle cells. In rat aortic smooth muscle cells, PDTC induced cell shrinkage, chromatin condensation, and DNA strand breaks consistent with apoptosis. In addition, overexpression of Bcl-2 suppressed vascular smooth muscle cell death caused by PDTC and NAC. The viability of rat aortic smooth muscle cells decreased within 3 h of treatment with PDTC and was reduced to 30% at 12 h. The effect of PDTC and NAC on smooth muscle cells was not species specific because PDTC and NAC both caused dose-dependent reductions in viability in rat and human aortic smooth muscle cells. In contrast, neither PDTC nor NAC reduced viability in human aortic endothelial cells. The use of antioxidants to induce apoptosis in vascular smooth muscle cells may help prevent their proliferation in arteriosclerotic lesions.

Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating.

PubMed

Heidari, Banafsheh; Gifani, Minoo; Shirazi, Abolfazl; Zarnani, Amir-Hassan; Baradaran, Behzad; Naderi, Mohammad Mehdi; Behzadi, Bahareh; Borjian-Boroujeni, Sara; Sarvari, Ali; Lakpour, Niknam; Akhondi, Mohammad Mehdi

2014-04-01

The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes.

Enrichment of Undifferentiated Type A Spermatogonia from Goat Testis Using Discontinuous Percoll Density Gradient and Differential Plating

PubMed Central

Heidari, Banafsheh; Gifani, Minoo; Shirazi, Abolfazl; Zarnani, Amir-Hassan; Baradaran, Behzad; Naderi, Mohammad Mehdi; Behzadi, Bahareh; Borjian-Boroujeni, Sara; Sarvari, Ali; Lakpour, Niknam; Akhondi, Mohammad Mehdi

2014-01-01

Background The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Methods Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). Results The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). Conclusion Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes. PMID:24834311

A new method for long-term storage of titred microbial standard solutions suitable for microbiologic quality control activities of pharmaceutical companies.

PubMed

Chiellini, Carolina; Mocali, Stefano; Fani, Renato; Ferro, Iolanda; Bruschi, Serenella; Pinzani, Alessandro

2016-08-01

Commercially available lyophilized microbial standards are expensive and subject to reduction in cell viability due to freeze-drying stress. Here we introduce an inexpensive and straightforward method for in-house microbial standard preparation and cryoconservation that preserves constant cell titre and cell viability over 14 months.

Inhibition of NFkappaB reduces cellular viability in GH3 pituitary adenoma cells.

PubMed

Vender, John R; Laird, Melissa D; Dhandapani, Krishnan M

2008-05-01

Adenomas of the pituitary gland are among the most common types of tumors of the adult brain. Although adenomas are histologically benign, they may be associated with significant morbidity and mortality, mostly because of their invasive growth pattern and hormone hypersecretion. Current medical therapies are suppressive, acting at a receptor level. Thus, there is a need to identify novel cellular and molecular targets for pituitary tumors. We investigated the possible role of the NFkappaB transcription factor in pituitary tumor cell growth. The effect of NFkappaB pathway inhibition on cellular viability was studied in the GH3 pituitary adenoma cell line, a well-characterized rat cell line that secretes growth hormone and prolactin. Cells were treated with mechanistically diverse pharmacological NFkappaB pathway inhibitors or with molecular inhibitors that were overexpressed in tumor cells before the assessment of cellular viability. NFkappaB activity was also assessed in GH3 cells using deoxyribonucleic acid binding assays. GH3 cells exhibited constitutive NFkappaB activity, which contributed to increased cellular proliferation. Treatment with wedelolactone, an IkappaB kinase inhibitor, or overexpression of an IkappaB super-repressor reduced cell viability, further implicating NFkappaB in pituitary tumor cell growth. Pharmacological or molecular inhibition of Akt similarly reduced GH3 viability and NFkappaB binding, suggesting that constitutive activation of NFkappaB may be, at least in part, mediated by Akt. Directed targeting of the Akt and NFkappaB signaling pathways may be a useful adjunct in the clinical management of pituitary tumors. Further elucidation of this pathway may yield novel information regarding the behavior of pituitary tumors in humans.

The biocompatibility of modified experimental Portland cements with potential for use in dentistry.

PubMed

Camilleri, J

2008-12-01

To evaluate the biocompatibility of a group of new potential dental materials and their eluants by assessing cell viability. Calcium sulpho-aluminate cement (CSA), calcium fluoro-aluminate cement (CFA) and glass-ionomer cement (GIC; Ketac Molar), used as the control, were tested for biocompatibility. Using a direct test method cell viability was measured quantitatively using alamarBluetrade mark dye, and an indirect test method where cells were grown on material elutions and cell viability was assessed using methyltetrazolium (MTT) assay as recommended by ISO 10 993-Part 5 for in vitro testing. Statistical analysis was performed by analysis of variance and Tukey multi-comparison test method. Elution collected from the prototype cements and the GIC cured for 1 and 7 days allowed high cell activity after 24 h cell exposure, which reduced after 48 h when compared to the nontoxic glass-ionomer control, but increased significantly after 72 h cell contact. Elutions collected after 28 days revealed reduced cell activity at all cell exposure times. Cells placed in direct contact with the prototype materials showed reduced cell activity when compared with the control. Cell growth was poor when seeded in direct contact with the prototype cements. GIC encouraged cell growth after 1 day of contact. The eluted species for all the cements tested exhibited adequate cell viability in the early ages with reduced cell activity at 28 days. Changes in the production of calcium hydroxide as a by-product of cement hydration affect the material biocompatibility adversely.

Effects of demethoxycurcumin on the viability and apoptosis of skin cancer cells.

PubMed

Wu, Yaoqun; Zhang, Pei; Yang, Hongyun; Ge, Yong; Xin, Yong

2017-07-01

The present study investigated the effects and mechanisms of demethoxycurcumin (DMC) on a human skin squamous cell carcinoma cell line, A431, and a human keratinocyte cell line, HaCaT. A431 and HaCaT cells were cultured in vitro. The effects of DMC treatment on cell viability were analyzed using the Cell Counting kit‑8 (CCK‑8) assay; cell cycle distribution was analyzed by flow cytometry; apoptosis was assessed by flow cytometry and Hoechst 33258 staining; and the protein expression levels of cytochrome c, B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (BAX), caspase‑9 and caspase‑3 were evaluated by western blotting. CCK‑8 assay results demonstrated that DMC treatment significantly inhibited viability of A431 and HaCaT cells in a dose‑dependent manner. Flow cytometric analysis confirmed that DMC treatment induced apoptosis in a dose‑dependent manner, and significantly increased the proportion of cells in G2/M phase. Western blot analysis indicated that the protein expression levels of Bcl‑2 were decreased, whereas the expression levels of BAX, caspase‑9, caspase‑3 and cytochrome c were increased following DMC treatment compared with in untreated cells. In conclusion, DMC treatment significantly inhibited viability of A431 and HaCaT cells, and induced cell cycle arrest in G2/M phase. The present study indicated that DMC may induce apoptosis of skin cancer cells through a caspase‑dependent pathway.

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro.

PubMed

Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Zhou, Ping; Zhang, Xiaolei; Zhang, Wei; Chen, Qingyu; Kou, Dongquan; Ying, Xiaozhou; Shen, Yue; Cheng, Xiaojie; Yu, Ziming; Peng, Lei; Lu, Chuanzhu

2013-09-01

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.

Comparative Evaluation of Cell Viability Immediately After Osteotomy for Implants With Drills and Piezosurgery: Immunohistochemistry Analysis.

PubMed

Pereira, Cassiano Costa Silva; Batista, Fábio Roberto de Souza; Jacob, Ricardo Garcia Mureb; Nogueira, Lamis Meorin; Carvalho, Abrahão Cavalcante Gomes de Souza; Gealh, Walter Cristiano; Garcia-Júnior, Idelmo Rangel; Okamoto, Roberta

2018-05-08

To evaluate the effect of reusing drills and piezosurgery tips during implant osteotomy on immediate bone cell viability through immunohistochemical analysis. Six male rabbits were divided into 2 groups and then divided into 5 subgroups-correspond to drills and tips used 10, 20, 30, 40, and 50 times, respectively. All animals received 10 osteotomies in each tibia, by use of the classic drilling procedure in one group (G1) and the piezosurgery device in the other group (G2). For immunohistochemical technique were utilized the osteoprotegerin, RANKL, osteocalcin, and caspase 3. Control procedures were performed by omitting the primary antibodies (negative control). Bone formation and resorption responses presented in more intense way during the piezosurgery. The expression of osteocalcin had become quite intense in piezosurgery groups, but with reduced immunostaining from the 30th osteotomy. The caspase 3 showed the viability of the osteoblast from the 20th osteotomy with piezosurgery and remained constant until the 50th. Piezosurgery provides greater osteoblastic cell viability than the system of conventional drilling. This study will provide data so that the authors can recycle the drills and tips for implant placement, thus enabling a better cell viability for osseointegration.

Force dependent internalization of magnetic nanoparticles results in highly loaded endothelial cells for use as potential therapy delivery vectors.

PubMed

MacDonald, Cristin; Barbee, Kenneth; Polyak, Boris

2012-05-01

To investigate the kinetics, mechanism and extent of MNP loading into endothelial cells and the effect of this loading on cell function. MNP uptake was examined under field on/off conditions, utilizing varying magnetite concentration MNPs. MNP-loaded cell viability and functional integrity was assessed using metabolic respiration, cell proliferation and migration assays. MNP uptake in endothelial cells significantly increased under the influence of a magnetic field versus non-magnetic conditions. Larger magnetite density of the MNPs led to a higher MNP internalization by cells under application of a magnetic field without compromising cellular respiration activity. Two-dimensional migration assays at no field showed that higher magnetite loading resulted in greater cell migration rates. In a three-dimensional migration assay under magnetic field, the migration rate of MNP-loaded cells was more than twice that of unloaded cells and was comparable to migration stimulated by a serum gradient. Our results suggest that endothelial cell uptake of MNPs is a force dependent process. The in vitro assays determined that cell health is not adversely affected by high MNP loadings, allowing these highly magnetically responsive cells to be potentially beneficial therapy (gene, drug or cell) delivery systems.

Seed viability detection using computerized false-color radiographic image enhancement

NASA Technical Reports Server (NTRS)

Vozzo, J. A.; Marko, Michael

1994-01-01

Seed radiographs are divided into density zones which are related to seed germination. The seeds which germinate have densities relating to false-color red. In turn, a seed sorter may be designed which rejects those seeds not having sufficient red to activate a gate along a moving belt containing the seed source. This results in separating only seeds with the preselected densities representing biological viability lending to germination. These selected seeds demand a higher market value. Actual false-coloring isn't required for a computer to distinguish the significant gray-zone range. This range can be predetermined and screened without the necessity of red imaging. Applying false-color enhancement is a means of emphasizing differences in densities of gray within any subject from photographic, radiographic, or video imaging. Within the 0-255 range of gray levels, colors can be assigned to any single level or group of gray levels. Densitometric values then become easily recognized colors which relate to the image density. Choosing a color to identify any given density allows separation by morphology or composition (form or function). Additionally, relative areas of each color are readily available for determining distribution of that density by comparison with other densities within the image.

Trehalose effectiveness as a cryoprotectant in 2D and 3D cell cultures of human embryonic kidney cells.

PubMed

Hara, Jared; Tottori, Jordan; Anders, Megan; Dadhwal, Smritee; Asuri, Prashanth; Mobed-Miremadi, Maryam

2017-05-01

Post cryopreservation viability of human embryonic kidney (HEK) cells under two-dimensional (2D) and three-dimensional (3D) culture conditions was studied using trehalose as the sole cryoprotective agent. An L 9 (3 4 ) Taguchi design was used to optimize the cryoprotection cocktail seeding process prior to slow-freezing with the specific aim of maximizing cell viability measured 7 days post thaw, using the combinatorial cell viability and in-vitro cytotoxicity WST assay. At low (200 mM) and medium (800 mM) levels of trehalose concentration, encapsulation in alginate offered a greater protection to cryopreservation. However, at the highest trehalose concentration (1200 mM) and in the absence of the pre-incubation step, there was no statistical difference at the 95% CI (p = 0.0212) between the viability of the HEK cells under 2D and 3D culture conditions estimated to be 17.9 ± 4.6% and 14.0 ± 3.6%, respectively. A parallel comparison between cryoprotective agents conducted at the optimal levels of the L 9 study, using trehalose, dimethylsulfoxide and glycerol in alginate microcapsules yielded a viability of 36.0 ± 7.4% for trehalose, in average 75% higher than the results associated with the other two cell membrane-permeating compounds. In summary, the effectiveness of trehalose has been demonstrated by the fact that 3D cell cultures can readily be equilibrated with trehalose before cryopreservation, thus mitigating the cytotoxic effects of glycerol and dimethylsulfoxide.

Effects of long-term cryopreservation on peripheral blood progenitor cells.

PubMed

Vosganian, Gregory S; Waalen, Jill; Kim, Kevin; Jhatakia, Sejal; Schram, Ethan; Lee, Tracey; Riddell, Dan; Mason, James R

2012-11-01

The long-term stability of cryopreserved peripheral blood progenitor cells is an important issue for patients experiencing disease relapse. However, there is no consensus on how to evaluate the long-term effects of cryopreservation. We describe the effect of cryopreservation on viability and progenitor colony activity from 87 individual samples processed at the Scripps Green Hospital Stem Cell Processing Center (La Jolla, CA, USA). We randomly selected 87 peripheral blood hematopoietic stem cell (PBHSC) samples from 60 patients and evaluated the effect of cryopreservation on sample viability and red and white cell colony activity after < 24 h and 7, 10 and 15 years of cryopreservation. Viability was assayed via trypan blue dye exclusion and activity was measured following 14 days of culture. An age at collection older than 50 years may result in suboptimal activity and viability following long-term cryopreservation, while gender and disease status had no effect. Cryopreservation did not significantly affect white or red cell activity following 10 years of cryopreservation. However, for samples stored longer than 10 years, viability and activity significantly decreased. We noted a positive association between higher pre-cryopreservation %CD34 count and colony activity. Cryopreservation of peripheral blood progenitor cells for up to 10 years results in no loss of clonogenic capacity, as determined by culture activity, although longer durations of storage may affect activity. Until validated methods are developed, cryopreserved grafts should be evaluated based on pre-freeze CD34(+) cell counts as assayed by flow cytometry, and post-thaw sample evaluation should be reserved for patients identified as poor mobilizers.

Suitable Concentrations of Uric Acid Can Reduce Cell Death in Models of OGD and Cerebral Ischemia-Reperfusion Injury.

PubMed

Zhang, Bin; Yang, Ning; Lin, Shao-Peng; Zhang, Feng

2017-07-01

Cerebral infarction (CI) is a common clinical cerebrovascular disease, and to explore the pathophysiological mechanisms and seek effective treatment means are the hotspot and difficult point in medical research nowadays. Numerous studies have confirmed that uric acid plays an important role in CI, but the mechanism has not yet been clarified. When treating HT22 and BV-2 cells with different concentrations of uric acid, uric acid below 450 μM does not have significant effect on cell viability, but uric acid more than 500 μM can significantly inhibit cell viability. After establishing models of OGD (oxygen-glucose deprivation) with HT22 and BV-2 cells, uric acid at a low concentration (50 μM) cannot improve cell viability and apoptosis, and Reactive oxygen species (ROS) levels during OGD/reoxygenation; a suitable concentration (300 μM) of uric acid can significantly improve cell viability and apoptosis, and reduce ROS production during OGD/reoxygenation; but a high concentration (1000 μM) of uric acid can further reduce cell viability and enhance ROS production. After establishing middle cerebral artery occlusion of male rats with suture method, damage and increase of ROS production in brain tissue could be seen, and after adding suitable concentration of uric acid, the degree of brain damage and ROS production was reduced. Therefore, different concentrations of uric acid should have different effect, and suitable concentrations of uric acid have neuroprotective effect, and this finding may provide guidance for study on the clinical curative effect of uric acid.

In vitro effects of preserved and unpreserved anti-allergic drugs on human corneal epithelial cells.

PubMed

Guzman-Aranguez, Ana; Calvo, Patricia; Ropero, Inés; Pintor, Jesús

2014-11-01

Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Cells were treated with unpreserved ketotifen solution, benzalkonium chloride (BAC)-containing anti-allergic drugs (ketotifen, olopatadine, levocabastine) as well as BAC alone. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine cell viability. Effects of compounds on barrier function were analyzed measuring transepithelial electrical resistance (TEER) to determine paracellular permeability and rose bengal assays to evaluate transcellular barrier formation. The BAC-preserved anti-allergic formulations and BAC alone significantly reduced cell viability, monolayer cultures being more sensitive to damage by these solutions. Unpreserved ketotifen induced the least diminution in cell viability. The extent of decrease of cell viability was clearly dependent of BAC presence, but it was also affected by the different types of drugs when the concentration of BAC was low and the short time of exposure. Treatment with BAC-containing anti-allergic drugs and BAC alone resulted in increased paracellular permeability and loss of transcellular barrier function as indicated by TEER measurement and rose bengal assays. The presence of the preservative BAC in anti-allergic eye drop formulations contributes importantly to the cytotoxic effects induced by these compounds. Stratified cell cultures seem to be a more relevant model for toxicity evaluation induced on the ocular surface epithelia than monolayer cultures.

Strong attachment of circadian pacemaker neurons on modified ultrananocrystalline diamond surfaces.

PubMed

Voss, Alexandra; Wei, HongYing; Zhang, Yi; Turner, Stuart; Ceccone, Giacomo; Reithmaier, Johann Peter; Stengl, Monika; Popov, Cyril

2016-07-01

Diamond is a promising material for a number of bio-applications, including the fabrication of platforms for attachment and investigation of neurons and of neuroprostheses, such as retinal implants. In the current work ultrananocrystalline diamond (UNCD) films were deposited by microwave plasma chemical vapor deposition, modified by UV/O3 treatment or NH3 plasma, and comprehensively characterized with respect to their bulk and surface properties, such as crystallinity, topography, composition and chemical bonding nature. The interactions of insect circadian pacemaker neurons with UNCD surfaces with H-, O- and NH2-terminations were investigated with respect to cell density and viability. The fast and strong attachment achieved without application of adhesion proteins allowed for advantageous modification of dispersion protocols for the preparation of primary cell cultures. Centrifugation steps, which are employed for pelletizing dispersed cells to separate them from dispersing enzymes, easily damage neurons. Now centrifugation can be avoided since dispersed neurons quickly and strongly attach to the UNCD surfaces. Enzyme solutions can be easily washed off without losing many of the dispersed cells. No adverse effects on the cell viability and physiological responses were observed as revealed by calcium imaging. Furthermore, the enhanced attachment of the neurons, especially on the modified UNCD surfaces, was especially advantageous for the immunocytochemical procedures with the cell cultures. The cell losses during washing steps were significantly reduced by one order of magnitude in comparison to controls. In addition, the integration of a titanium grid structure under the UNCD films allowed for individual assignment of physiologically characterized neurons to immunocytochemically stained cells. Thus, employing UNCD surfaces free of foreign proteins improves cell culture protocols and immunocytochemistry with cultured cells. The fast and strong attachment of neurons was attributed to a favorable combination of topography, surface chemistry and wettability. Copyright © 2016 Elsevier B.V. All rights reserved.

Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells.

PubMed

Valenzuela, Manuel; Bastias, Lorena; Montenegro, Iván; Werner, Enrique; Madrid, Alejandro; Godoy, Patricio; Párraga, Mario; Villena, Joan

2018-01-01

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type. Conclusions . These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells.

Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells

PubMed Central

Valenzuela, Manuel; Bastias, Lorena; Montenegro, Iván; Werner, Enrique; Madrid, Alejandro; Godoy, Patricio

2018-01-01

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type. Conclusions. These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells. PMID:29552079

Yeast viability and concentration analysis using lens-free computational microscopy and machine learning

NASA Astrophysics Data System (ADS)

Feizi, Alborz; Zhang, Yibo; Greenbaum, Alon; Guziak, Alex; Luong, Michelle; Chan, Raymond Yan Lok; Berg, Brandon; Ozkan, Haydar; Luo, Wei; Wu, Michael; Wu, Yichen; Ozcan, Aydogan

2017-03-01

Research laboratories and the industry rely on yeast viability and concentration measurements to adjust fermentation parameters such as pH, temperature, and pressure. Beer-brewing processes as well as biofuel production can especially utilize a cost-effective and portable way of obtaining data on cell viability and concentration. However, current methods of analysis are relatively costly and tedious. Here, we demonstrate a rapid, portable, and cost-effective platform for imaging and measuring viability and concentration of yeast cells. Our platform features a lens-free microscope that weighs 70 g and has dimensions of 12 × 4 × 4 cm. A partially-coherent illumination source (a light-emitting-diode), a band-pass optical filter, and a multimode optical fiber are used to illuminate the sample. The yeast sample is directly placed on a complementary metal-oxide semiconductor (CMOS) image sensor chip, which captures an in-line hologram of the sample over a large field-of-view of >20 mm2. The hologram is transferred to a touch-screen interface, where a trained Support Vector Machine model classifies yeast cells stained with methylene blue as live or dead and measures cell viability as well as concentration. We tested the accuracy of our platform against manual counting of live and dead cells using fluorescent exclusion staining and a bench-top fluorescence microscope. Our regression analysis showed no significant difference between the two methods within a concentration range of 1.4 × 105 to 1.4 × 106 cells/mL. This compact and cost-effective yeast analysis platform will enable automatic quantification of yeast viability and concentration in field settings and resource-limited environments.

Fetal brain extracellular matrix boosts neuronal network formation in 3D bioengineered model of cortical brain tissue.

PubMed

Sood, Disha; Chwalek, Karolina; Stuntz, Emily; Pouli, Dimitra; Du, Chuang; Tang-Schomer, Min; Georgakoudi, Irene; Black, Lauren D; Kaplan, David L

2016-01-01

The extracellular matrix (ECM) constituting up to 20% of the organ volume is a significant component of the brain due to its instructive role in the compartmentalization of functional microdomains in every brain structure. The composition, quantity and structure of ECM changes dramatically during the development of an organism greatly contributing to the remarkably sophisticated architecture and function of the brain. Since fetal brain is highly plastic, we hypothesize that the fetal brain ECM may contain cues promoting neural growth and differentiation, highly desired in regenerative medicine. Thus, we studied the effect of brain-derived fetal and adult ECM complemented with matricellular proteins on cortical neurons using in vitro 3D bioengineered model of cortical brain tissue. The tested parameters included neuronal network density, cell viability, calcium signaling and electrophysiology. Both, adult and fetal brain ECM as well as matricellular proteins significantly improved neural network formation as compared to single component, collagen I matrix. Additionally, the brain ECM improved cell viability and lowered glutamate release. The fetal brain ECM induced superior neural network formation, calcium signaling and spontaneous spiking activity over adult brain ECM. This study highlights the difference in the neuroinductive properties of fetal and adult brain ECM and suggests that delineating the basis for this divergence may have implications for regenerative medicine.

Factors affecting ultraviolet-A photon emission from β-irradiated human keratinocyte cells.

PubMed

Le, M; Mothersill, C E; Seymour, C B; Ahmad, S B; Armstrong, A; Rainbow, A J; McNeill, F E

2015-08-21

The luminescence intensity of 340±5 nm photons emitted from HaCaT (human keratinocyte) cells was investigated using a single-photon-counting system during cellular exposure to (90)Y β-particles. Multiple factors were assessed to determine their influence upon the quantity and pattern of photon emission from β-irradiated cells. Exposure of 1 x 10(4) cells/5 mL to 703 μCi resulted in maximum UVA photoemission at 44.8 x 10(3)±2.5 x 10(3) counts per second (cps) from live HaCaT cells (background: 1-5 cps); a 16-fold increase above cell-free controls. Significant biophoton emission was achieved only upon stimulation and was also dependent upon presence of cells. UVA luminescence was measured for (90)Y activities 14 to 703 μCi where a positive relationship between photoemission and (90)Y activity was observed. Irradiation of live HaCaT cells plated at various densities produced a distinct pattern of emission whereby luminescence increased up to a maximum at 1 x 10(4) cells/5 mL and thereafter decreased. However, this result was not observed in the dead cell population. Both live and dead HaCaT cells were irradiated and were found to demonstrate different rates of photon emission at low β activities (⩽400 μCi). Dead cells exhibited greater photon emission rates than live cells which may be attributable to metabolic processes taking place to modulate the photoemissive effect. The results indicate that photon emission from HaCaT cells is perturbed by external stimulation, is dependent upon the activity of radiation delivered, the density of irradiated cells, and cell viability. It is postulated that biophoton emission may be modulated by a biological or metabolic process.

On-chip gradient generation in 256 microfluidic cell cultures: simulation and experimental validation.

PubMed

Somaweera, Himali; Haputhanthri, Shehan O; Ibraguimov, Akif; Pappas, Dimitri

2015-08-07

A microfluidic diffusion diluter was used to create a stable concentration gradient for dose response studies. The microfluidic diffusion diluter used in this study consisted of 128 culture chambers on each side of the main fluidic channel. A calibration method was used to find unknown concentrations with 12% error. Flow rate dependent studies showed that changing the flow rates generated different gradient patterns. Mathematical simulations using COMSOL Multi-physics were performed to validate the experimental data. The experimental data obtained for the flow rate studies agreed with the simulation results. Cells could be loaded into culture chambers using vacuum actuation and cultured for long times under low shear stress. Decreasing the size of the culture chambers resulted in faster gradient formation (20 min). Mass transport into the side channels of the microfluidic diffusion diluter used in this study is an important factor in creating the gradient using diffusional mixing as a function of the distance. To demonstrate the device's utility, an H2O2 gradient was generated while culturing Ramos cells. Cell viability was assayed in the 256 culture chambers, each at a discrete H2O2 concentration. As expected, the cell viability for the high concentration side channels increased (by injecting H2O2) whereas the cell viability in the low concentration side channels decreased along the chip due to diffusional mixing as a function of distance. COMSOL simulations were used to identify the effective concentration of H2O2 for cell viability in each side chamber at 45 min. The gradient effects were confirmed using traditional H2O2 culture experiments. Viability of cells in the microfluidic device under gradient conditions showed a linear relationship with the viability of the traditional culture experiment. Development of the microfluidic device used in this study could be used to study hundreds of concentrations of a compound in a single experiment.

Active targeting of cancer cells using folic acid-conjugated platinum nanoparticles.

PubMed

Teow, Yiwei; Valiyaveettil, Suresh

2010-12-01

Interaction of nanoparticles with human cells is an interesting topic for understanding toxicity and developing potential drug candidates. Water soluble platinum nanoparticles were synthesized via reduction of hexachloroplatinic acid using sodium borohydride in the presence of capping agents. The bioactivity of folic acid and poly(vinyl pyrrolidone) capped platinum nanoparticles (Pt-nps) has been investigated using commercially available cell lines. In the cell viability experiments, PVP-capped nanoparticles were found to be less toxic (>80% viability), whereas, folic acid-capped platinum nanoparticles showed a reduced viability down to 24% after 72 h of exposure at a concentration of 100 μg ml(-1) for MCF7 breast cancer cells. Such toxicity, combined with the possibility to incorporate functional organic molecules as capping agents, can be used for developing new drug candidates.

Polyphenolic extracts of edible flowers incorporated onto atelocollagen matrices and their effect on cell viability.

PubMed

López-García, Jorge; Kuceková, Zdenka; Humpolíček, Petr; Mlček, Jiři; Sáha, Petr

2013-10-30

The phenolic extract of chives flowers (Allium schoenoprasum, Liliaceae), introduced Sage (Salvia pratensis, Lamiaceae), European elderberry (Sambucus nigra, Caprifoliaceae) and common dandelion (Taraxacum officinale, Asteraceae) were characterised by High Performance Liquid Chromatography and incorporated in different concentrations onto atelocollagen thin films. In order to assess the biological impact of these phenolic compounds on cell viability, human immortalised non-tumorigenic keratinocyte cell line was seeded on the thin films and cell proliferation was determined by using an MTT assay. In addition, their antimicrobial activity was estimated by using an agar diffusion test. Data indicated the concomitance between cell viability and concentration of polyphenols. These findings suggest that these phenolic-endowed atelocollagen films might be suitable for tissue engineering applications, on account of the combined activity of polyphenols and collagen.

Increased efficiency of mammalian somatic cell hybrid production under microgravity conditions during ballistic rocket flight

NASA Technical Reports Server (NTRS)

Schnettler, R.; Gessner, P.; Zimmermann, U.; Neil, G. A.; Urnovitz, H. B.

1989-01-01

The electrofusion of hybridoma cell lines under short-duration microgravity during a flight of the TEXUS 18 Black Brand ballistic sounding rocket at Kiruna, Sweden is reported. The fusion partners, growth medium, cell fusion medium, cell fusion, cell viability in the fusion medium, and postfusion cell culture are described, and the rocket, cell fusion chamber, apparatus, and module are examined. The experimental timeline, the effects of fusion medium and incubation time on cell viability and hybrid yields, and the effect of microgravity on hybrid yields are considered.

LncRNA-LET inhibits cell viability, migration and EMT while induces apoptosis by up-regulation of TIMP2 in human granulosa-like tumor cell line KGN.

PubMed

Han, Qingfang; Zhang, Wenke; Meng, Jinlai; Ma, Li; Li, Aihua

2018-04-01

Polycystic ovary syndrome (PCOS) is a common endocrine disease characterized by hyperandrogenism, irregular menses, and polycystic ovaries. Several long non-coding RNAs (lncRNAs) are aberrantly expressed in PCOS patients; however, little is known about the effects of the lncRNA-low expression in tumor (lncRNA-LET) on PCOS. We aimed to explore the effects of lncRNA-LET on human granulosa-like tumor cell line, KGN. Expression of lncRNA-LET in normal IOSE80 cells and granulosa cells was determined by qRT-PCR. KGN cell viability, apoptosis and migration were measured by trypan blue exclusion method, flow cytometry assay and wound healing assay, respectively. TGF-β1 was used to induce epithelial-mesenchymal transition (EMT) process. LncRNA-LET expression and mRNA expressions of TIMP2 and EMT-related proteins were measured by qRT-PCR. Western blot analysis was used to measure the protein expression of apoptosis-related proteins, EMT-related proteins, TIMP2, and the proteins in the Wnt/β-catenin and Notch signaling pathways. lncRNA-LET was down-regulated in KGN cells, and its overexpression inhibited cell viability and migration, and promoted apoptosis in KGN cells. Overexpression of lncRNA-LET increased the expression of E-cadherin and decreased the expressions of N-cadherin and vimentin in KGN cells. These effects of lncRNA-LET on KGN cells were reversed by TIMP2 suppression. Overexpression of TIMP2 inhibited cell viability, migration and EMT process, and increased apoptosis by activating the Wnt/β-catenin and Notch pathways. Overexpression of lncRNA-LET inhibits cell viability, migration and EMT process, and increases apoptosis in KGN cells by up-regulating the expression of TIMP2 and activating the Wnt/β-catenin and notch signaling pathways. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Improvement of Storage Medium for Cultured Human Retinal Pigment Epithelial Cells Using Factorial Design.

PubMed

Pasovic, L; Utheim, T P; Reppe, S; Khan, A Z; Jackson, C J; Thiede, B; Berg, J P; Messelt, E B; Eidet, J R

2018-04-09

Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 different additives. Individual effects of each additive on cell viability were assessed using epifluorescence microscopy. Factorial design identified promising additive combinations by extrapolating their individual effects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifluorescence microscopy. Flow cytometry validated the findings. Proteomics identified 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.

Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.

PubMed

Baar, Marjolein P; Brandt, Renata M C; Putavet, Diana A; Klein, Julian D D; Derks, Kasper W J; Bourgeois, Benjamin R M; Stryeck, Sarah; Rijksen, Yvonne; van Willigenburg, Hester; Feijtel, Danny A; van der Pluijm, Ingrid; Essers, Jeroen; van Cappellen, Wiggert A; van IJcken, Wilfred F; Houtsmuller, Adriaan B; Pothof, Joris; de Bruin, Ron W F; Madl, Tobias; Hoeijmakers, Jan H J; Campisi, Judith; de Keizer, Peter L J

2017-03-23

The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging Xpd TTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored. Copyright © 2017 Elsevier Inc. All rights reserved.

Apple derived cellulose scaffolds for 3D mammalian cell culture.

PubMed

Modulevsky, Daniel J; Lefebvre, Cory; Haase, Kristina; Al-Rekabi, Zeinab; Pelling, Andrew E

2014-01-01

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture

PubMed Central

Modulevsky, Daniel J.; Lefebvre, Cory; Haase, Kristina; Al-Rekabi, Zeinab; Pelling, Andrew E.

2014-01-01

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment. PMID:24842603

Cooperation of HIF- and NCAM-mediated mechanisms in cell viability of hippocampal cultures after oxygen-glucose deprivation.

PubMed

Lushnikova, Iryna; Nikandrova, Yelyzaveta; Skibo, Galyna

2017-10-01

Neurodegenerative diseases of different genesis are the result of cellular damages including those caused by oxygen and glucose deficit. Neuronal survival or death in brain pathologies depends on a variety of interrelated molecular mechanisms. A key role in modulation of neuron viability belongs to HIF (hypoxia-inducible factor) and NCAM (neural cell adhesion molecules) signaling pathways. In this work, we used organotypic and dissociated hippocampal cultures to analyze cell viability and HIF-1α immunopositive (HIF-1α + ) signal after 30 min oxygen-glucose deprivation (OGD) followed by 24 h of reoxygenation in the presence of FGL (synthetic NCAM-derived mimetic peptide). According to LDH- and MTS-assay of cell viability, FGL showed a neuroprotective effect, which was attributed to the association with FGFR. We showed that these effects correlated with changes of the HIF-1α + level suggesting the communications of HIF and NCAM signaling pathways. These data extend our knowledge of neurodegeneration mechanisms and open additional potential for the development of neuroprotection strategies. © 2017 International Federation for Cell Biology.

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

PubMed Central

Bourgognon, Maxime; Klippstein, Rebecca; Al-Jamal, Khuloud T.

2015-01-01

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsrød et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (>95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient. PMID:26327223

Canine adipose-derived stromal cell viability following exposure to synovial fluid from osteoarthritic joints.

PubMed

Kiefer, Kristina M; O'Brien, Timothy D; Pluhar, Elizabeth G; Conzemius, Michael

2015-01-01

Stem cell therapy used in clinical application of osteoarthritis in veterinary medicine typically involves intra-articular injection of the cells, however the effect of an osteoarthritic environment on the fate of the cells has not been investigated. Assess the viability of adipose derived stromal cells following exposure to osteoarthritic joint fluid. Adipose derived stromal cells (ASCs) were derived from falciform adipose tissue of five adult dogs, and osteoarthritic synovial fluid (SF) was obtained from ten patients undergoing surgical intervention on orthopedic diseases with secondary osteoarthritis. Normal synovial fluid was obtained from seven adult dogs from an unrelated study. ASCs were exposed to the following treatment conditions: culture medium, normal SF, osteoarthritic SF, or serial dilutions of 1:1 to 1:10 of osteoarthritic SF with media. Cells were then harvested and assessed for viability using trypan blue dye exclusion. There was no significant difference in the viability of cells in culture medium or normal SF. Significant differences were found between cells exposed to any concentration of osteoarthritic SF and normal SF and between cells exposed to undiluted osteoarthritic SF and all serial dilutions. Subsequent dilutions reduced cytotoxicity. Osteoarthritic synovial fluid in this ex vivo experiment is cytotoxic to ASCs, when compared with normal synovial fluid. Current practice of direct injection of ASCs into osteoarthritic joints should be re-evaluated to determine if alternative means of administration may be more effective.

Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma.

PubMed

Petrachi, Tiziana; Romagnani, Alessandra; Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia

2017-01-24

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.

Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma

PubMed Central

Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia

2017-01-01

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma. PMID:28036292

Effect of photobiomodulation on viability and proliferation of stem cells from exfoliated deciduous teeth under different nutritional conditions

NASA Astrophysics Data System (ADS)

Morato de Souza, Letícia; Guilherme Roque Rinco, Ugo; Aparecida Tavares Aguiar, Daniela; Aparecido de Almeida Junior, Luciano; Cosme-Silva, Leopoldo; Marchini Oliveira, Thais; Teixeira Marques, Nádia Carolina; Thiemy Sakai, Vivien

2018-02-01

This study aimed to evaluate the effect of different doses of low-level laser irradiation on the viability and proliferation of stem cells from exfoliated deciduous teeth (SHED) cultured under nutritional deficit (cellular stress) or regular nutritional conditions. SHED underwent irradiation by a red laser between 1.2 and 6.2 J cm-2. Prior to the irradiation, all groups received culture medium (MEMα, Eagle’s minimum essential medium alpha modification) supplemented with 1% of fetal bovine serum (FBS) for 1 h. After the irradiation, cells received MEMα supplemented with 10% of FBS (regular nutrition) or 1% of FBS (nutritional deficit). Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays 6 and 24 h after irradiation (P  <  0.05). At 24 h, SHED under nutritional deficit showed lower viability and proliferation after 1.2 J cm-2 irradiation. All of the irradiated groups revealed significantly higher viability and proliferation in SHED maintained under nutritional deficit than in regular nutritional conditions, except in the 3.7 and 6.2 J cm-2 groups by MTT assay. In the crystal violet assay, SHED irradiated with 1.2 J cm-2 showed no difference between the different nutritional conditions. Decrease of FBS concentration in the culture medium seems to enhance the sensitivity of SHED to the effects of photobiomodulation therapy. Nutritional stress conditions improved cell viability and proliferation of SHED after laser irradiation, except for 1.2 J cm-2.

Human neural progenitor cells retain viability, phenotype, proliferation, and lineage differentiation when labeled with a novel iron oxide nanoparticle, Molday ION Rhodamine B

PubMed Central

Shen, Wei-Bin; Plachez, Celine; Chan, Amanda; Yarnell, Deborah; Puche, Adam C; Fishman, Paul S; Yarowsky, Paul

2013-01-01

Ultrasmall superparamagnetic iron-oxide particles (USPIOs) loaded into stem cells have been suggested as a way to track stem cell transplantation with magnetic resonance imaging, but the labeling, and post-labeling proliferation, viability, differentiation, and retention of USPIOs within the stem cells have yet to be determined for each type of stem cell and for each type of USPIO. Molday ION Rhodamine B™ (BioPAL, Worcester, MA, USA) (MIRB) has been shown to be a USPIO labeling agent for mesenchymal stem cells, glial progenitor cells, and stem cell lines. In this study, we have evaluated MIRB labeling in human neuroprogenitor cells and found that human neuroprogenitor cells are effectively labeled with MIRB without use of transfection reagents. Viability, proliferation, and differentiation properties are unchanged between MIRB-labeled neuroprogenitors cells and unlabeled cells. Moreover, MIRB-labeled human neuroprogenitor cells can be frozen, thawed, and replated without loss of MIRB or even without loss of their intrinsic biology. Overall, those results show that MIRB has advantageous properties that can be used for cell-based therapy. PMID:24348036

Intercellular cytosolic transfer correlates with mesenchymal stromal cell rescue of umbilical cord blood cell viability during ex vivo expansion

PubMed Central

Chu, Pat P. Y.; Bari, Sudipto; Fan, Xiubo; Gay, Florence P. H.; Ang, Justina M. L.; Chiu, Gigi N. C.; Lim, Sai K.; Hwang, William Y. K.

2012-01-01

Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P<0.01). This was associated with significant enhancement of mitochondrial membrane potential (P<0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P< 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture. PMID:22775077

Rapamycin toxicity in MIN6 cells and rat and human islets is mediated by the inhibition of mTOR complex 2 (mTORC2).

PubMed

Barlow, A D; Xie, J; Moore, C E; Campbell, S C; Shaw, J A M; Nicholson, M L; Herbert, T P

2012-05-01

Rapamycin (sirolimus) is one of the primary immunosuppressants for islet transplantation. Yet there is evidence that the long-term treatment of islet-transplant patients with rapamycin may be responsible for subsequent loss of islet graft function and viability. Therefore, the primary objective of this study was to elucidate the molecular mechanism of rapamycin toxicity in beta cells. Experiments were performed on isolated rat and human islets of Langerhans and MIN6 cells. The effects of rapamycin and the roles of mammalian target of rapamycin complex 2 (mTORC2)/protein kinase B (PKB) on beta cell signalling, function and viability were investigated using cell viability assays, insulin ELISA assays, kinase assays, western blotting, pharmacological inhibitors, small interfering (si)RNA and through the overproduction of a constitutively active mutant of PKB. Rapamycin treatment of MIN6 cells and islets of Langerhans resulted in a loss of cell function and viability. Although rapamycin acutely inhibited mTOR complex 1 (mTORC1), the toxic effects of rapamycin were more closely correlated to the dissociation and inactivation of mTORC2 and the inhibition of PKB. Indeed, the overproduction of constitutively active PKB protected islets from rapamycin toxicity whereas the inhibition of PKB led to a loss of cell viability. Moreover, the selective inactivation of mTORC2 using siRNA directed towards rapamycin-insensitive companion of target of rapamycin (RICTOR), mimicked the toxic effects of chronic rapamycin treatment. This report provides evidence that rapamycin toxicity is mediated by the inactivation of mTORC2 and the inhibition of PKB and thus reveals the molecular basis of rapamycin toxicity and the essential role of mTORC2 in maintaining beta cell function and survival.

Effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell function and viability.

PubMed

McDermott, Catherine; Chess-Williams, Russ; Grant, Gary D; Perkins, Anthony V; McFarland, Amelia J; Davey, Andrew K; Anoopkumar-Dukie, Shailendra

2012-03-01

We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro. RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles. The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment. Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Treatment of prostate cancer cell lines and primary cells using low temperature plasma

NASA Astrophysics Data System (ADS)

O'Connell, Deborah; Hirst, Adam; Frame, Fiona F.; Maitland, Norman J.

2014-10-01

The mechanisms of cell death after plasma treatment of both benign and cancerous prostate epithelial cells are investigated. Prostate cancer tissue was obtained with patient consent from targeted needle core biopsies following radical prostatectomy. Primary cells were cultured from cancer tissue and plated onto a chamber slide at a density of 10,000 cells per well in 200 microliter of stem cell media (SCM). The treated sample was previously identified as Gleason grade 7 cancer through tissue histo-pathology. A dielectric barrier discharge (DBD) jet configuration, with helium as a carrier gas, and 0.3% O2 admixture was used for treating the cells. Reactive oxygen and nitrogen species (RONS) produced by the plasma are believed to be the main mediators of the plasma-cell interaction and response. We found the concentration of reactive oxygen species (ROS) induced inside the cells increased with plasma exposure. Exposure to the plasma for >3 minutes showed high levels of DNA damage compared to untreated and hydrogen peroxide controls. Cell viability and cellular recovery are also investigated and will be presented. All findings were common to both cell lines, suggesting the potential of LTP therapy for both benign and malignant disease.

The effect of five artificial sweeteners on Caco-2, HT-29 and HEK-293 cells.

PubMed

van Eyk, Armorel Diane

2015-01-01

Artificial sweeteners (AS) have been associated with tumor development (including colon cancer) in both animals and humans although evidence has been conflicting. Additional research was thus conducted by studying the effects of 5 AS on the morphology, cell proliferation and DNA in cells by utilizing Caco-2, HT-29 (colon) and HEK-293 (kidney) cell lines. Cells were exposed to sodium cyclamate, sodium saccharin, sucralose and acesulfame-K (0-50 mM) and aspartame (0-35 mM) over 24, 48 and 72 hours. Morphological changes were presented photographically and % cell viability was determined by using the MTT cell viability assay. Possible DNA damage (comet assay) induced by the AS (0.1, 1 and 10 mM, treated for 24, 48 and 72 hours) was studied. The appearance of "comets" was scored from no damage to severe damage (0-4). Cells became flatter and less well defined at higher AS concentrations (>10 mM). At concentrations >10 mM, decreased cell viability was noted with both increasing concentration and increasing incubation time for all cell lines tested. In general, HEK-293 cells seemed to be less affected then the colon cancer cells. Sucralose and sodium saccharin seemed to elicit the greatest degree of DNA fragmentation of all the sweeteners tested in all the cell lines used. Morphological cell alterations, cell viability and DNA fragmentation seemed to be more in the colon cancer cells. Further studies have to be performed to clarify mechanisms involved causing these alterations in mammalian cells.

Towards gene banking amphibian maternal germ lines: short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.

PubMed

Lawson, Bianca; Clulow, Simon; Mahony, Michael J; Clulow, John

2013-01-01

Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40-60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca(2+)-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both-indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for cryopreserving amphibian maternal germ lines.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burch, S.W.; Goven, A.J.; Fitzpatrick, L.C.

An in vitro assay has been developed for rapid (48 h) evaluation of cytotoxic effects of exposure (24 h) of earthworm coelomocytes. The assay, inhibition of phagocytosis (24 h) of stained yeast cells and cell viability, links a traditional soil bioassay organism (Lumbricus terrestris) with a laboratory protocol for use in evaluating physical/chemical fractions resulting from terrestrial TIE manipulations. The assay was developed using copper sulfate as a reference toxicant. Copper exposures as low as 2--4 pg/ml. resulted in 20--60% inhibition of phagocytosis without significant decrease in cell viability. Exposures above 10 pg/ml resulted in reduced cell viability and inhibitionmore » of phagocytosis. The assay was successfully applied to terrestrial TIE fractions derived from extractions of soil from a PCP contaminated wood treatment site.« less

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

PubMed Central

Pipparelli, Aurélien; Arsenijevic, Yvan; Thuret, Gilles; Gain, Philippe

2013-01-01

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy. PMID:23626771

Kimchi methanol extract and the kimchi active compound, 3'-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid, downregulate CD36 in THP-1 macrophages stimulated by oxLDL.

PubMed

Yun, Ye-Rang; Kim, Hyun-Ju; Song, Yeong-Ok

2014-08-01

Macrophage foam cell formation by oxidized low-density lipoprotein (oxLDL) is a key step in the progression of atherosclerosis, which is involved in cholesterol influx and efflux in macrophages mediated by related proteins such as peroxisome proliferator-activated receptor γ (PPARγ), CD36, PPARα, liver-X receptor α (LXRα), and ATP-binding cassette transporter A1 (ABCA1). The aim of this study was to investigate the beneficial effects of kimchi methanol extract (KME) and a kimchi active compound, 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA) on cholesterol flux in THP-1-derived macrophages treated with oxLDL. The effects of KME and HDMPPA on cell viability and lipid peroxidation were determined. Furthermore, the protein expression of PPARγ, CD36, PPARα, LXRα, and ABCA1 was examined. OxLDL strongly induced cell death and lipid peroxidation in THP-1-derived macrophages. However, KME and HDMPPA significantly improved cell viability and inhibited lipid peroxidation induced by oxLDL in THP-1-derived macrophages (P<.05). Moreover, KME and HDMPPA suppressed CD36 and PPARγ expressions, both of which participate in cholesterol influx. In contrast, KME and HDMPPA augmented LXRα, PPARα, and ABCA1 expression, which are associated with cholesterol efflux. Consequently, KME and HDMPPA suppressed lipid accumulation. These results indicate that KME and HDMPPA may inhibit lipid accumulation, in part, by regulating cholesterol influx- and efflux-related proteins. These findings will thus be useful for future prevention strategies against atherosclerosis.

Insights in spatio-temporal characterization of human fetal neural stem cells.

PubMed

Martín-Ibáñez, Raquel; Guardia, Inés; Pardo, Mónica; Herranz, Cristina; Zietlow, Rike; Vinh, Ngoc-Nga; Rosser, Anne; Canals, Josep M

2017-05-01

Primary human fetal cells have been used in clinical trials of cell replacement therapy for the treatment of neurodegenerative disorders such as Huntington's disease (HD). However, human fetal primary cells are scarce and difficult to work with and so a renewable source of cells is sought. Human fetal neural stem cells (hfNSCs) can be generated from human fetal tissue, but little is known about the differences between hfNSCs obtained from different developmental stages and brain areas. In the present work we characterized hfNSCs, grown as neurospheres, obtained from three developmental stages: 4-5, 6-7 and 8-9weeks post conception (wpc) and four brain areas: forebrain, cortex, whole ganglionic eminence (WGE) and cerebellum. We observed that, as fetal brain development proceeds, the number of neural precursors is diminished and post-mitotic cells are increased. In turn, primary cells obtained from older embryos are more sensitive to the dissociation process, their viability is diminished and they present lower proliferation ratios compared to younger embryos. However, independently of the developmental stage of derivation proliferation ratios were very low in all cases. Improvements in the expansion rates were achieved by mechanical, instead of enzymatic, dissociation of neurospheres but not by changes in the seeding densities. Regardless of the developmental stage, neurosphere cultures presented large variability in the viability and proliferation rates during the initial 3-4 passages, but stabilized achieving significant expansion rates at passage 5 to 6. This was true also for all brain regions except cerebellar derived cultures that did not expand. Interestingly, the brain region of hfNSC derivation influences the expansion potential, being forebrain, cortex and WGE derived cells the most expandable compared to cerebellar. Short term expansion partially compromised the regional identity of cortical but not WGE cultures. Nevertheless, both expanded cultures were multipotent and kept the ability to differentiate to region specific mature neuronal phenotypes. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of laser treatment on the attachment and viability of mesenchymal stem cell responses on shape memory NiTi alloy.

PubMed

Chan, C W; Hussain, I; Waugh, D G; Lawrence, J; Man, H C

2014-09-01

The objectives of this study were to investigate the effect of laser-induced surface features on the morphology, attachment and viability of mesenchymal stem cells (MSCs) at different periods of time, and to evaluate the biocompatibility of different zones: laser-melted zone (MZ), heat-affected zone (HAZ) and base metal (BM) in laser-treated NiTi alloy. The surface morphology and composition were studied by scanning electron microscope (SEM) and X-ray photoemission spectroscopy (XPS), respectively. The cell morphology was examined by SEM while the cell counting and viability measurements were done by hemocytometer and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The results indicated that the laser-induced surface features, such as surface roughening, presence of anisotropic dendritic pattern and complete surface Ni oxidation were beneficial to improve the biocompatibility of NiTi as evidenced by the highest cell attachment (4 days of culture) and viability (7 days of culture) found in the MZ. The biocompatibility of the MZ was the best, followed by the BM with the HAZ being the worst. The defective and porous oxide layer as well as the coarse grained structure might attribute to the inferior cell attachment (4 days of culture) and viability (7 days of culture) on the HAZ compared with the BM which has similar surface morphology. Copyright © 2014 Elsevier B.V. All rights reserved.

Viability of a human melanoma cell after single and combined treatment with fotemustine, dacarbazine, and proton irradiation.

PubMed

Petrović, Ivan M; Korićanac, Lela B; Todorović, Danijela V; Ristić-Fira, Aleksandra M; Valastro, Lucia M; Privitera, Giuseppe; Cuttone, Giacomo

2007-01-01

Viability of human HTB140 melanoma cells after being exposed to fotemustine (FM) and dacarbazine (DTIC) as well as to proton irradiation was studied. Effects of 100 and 250 microM drugs were assessed after incubation of 6, 24, 48, 72, and 96 h. Irradiations were performed with 62 MeV therapeutic protons, delivering to the cell monolayer single doses of 2, 4, 8, 12, and 16 Gy. Viability was evaluated 7 days after irradiation. Inactivation level was estimated using microtetrasolium (MTT) and sulforhodamine B (SRB) assays. Combined effects of each drug and protons, were carried out using the same drug concentrations. Proton doses applied were those used in therapy, that is, 12 and 16 Gy. With the increase of drug concentration or irradiation dose, level of cell inactivation reached approximately 60%, 48 h after drug treatment or 7 days after irradiation at 16 Gy. Considering the rate of drug concentrations used, as well as the level of doses applied, it appears that HTB140 cells are more resistant to proton irradiation than to alkylating agents tested. The combined treatment with FM or DTIC and protons did not show significant changes of cell viability as compared to the effects of single agents. Since the time point for measuring cumulative effects of drug and irradiation was 48 h post irradiation, it seems that the obtained level of viability could be attributed primarily to the effects of drugs.

Methylene blue mediated photobiomodulation on human osteoblast cells.

PubMed

Ateş, Gamze Bölükbaşı; Ak, Ayşe; Garipcan, Bora; Gülsoy, Murat

2017-11-01

Photobiomodulation (PBM) and photodynamic therapy (PDT) are two major methods, which use light in medicine and dentistry. PBM uses low-level laser light to induce cell proliferation and activity. In contrast, PDT use laser light combined with a photosensitizer (PS) to cause cell death. Due to similar, not fully understood mechanisms and biphasic response of light, unexpected and complex outcomes may be observed. In the present study, the effect of 635 nm laser light, with power density 50 mW/cm 2 , at three different energy densities (0.5, 1, and 2 J/cm 2 which last 10, 20, and 40 s, respectively) mediated by methylene blue (MB) on the human osteoblast cell line (ATCC-CRL-11372, Rockville, MD, USA) was investigated. Cell viability (MTT assay and acridine orange/propidium iodide staining) and proliferation (Alamar Blue assay) were assessed at 24, 48, and 72 h post irradiation. Alkaline phosphatase (ALP) activity, mineralization (Alizarin Red staining) and gene expressions (RT-PCR analysis) were analyzed at 7th and 14th days after treatment. Five groups were formed as the control group (no MB, no irradiation), MB (only 0.05 μM MB), MB + 0.5 J/cm 2 , MB + 1 J/cm 2 , and MB + 2 J/cm 2 . Cell viability was decreased at 72 h (ANOVA; p < 0.05) for MB + 0.5 J/cm 2 , MB + 1 J/cm 2 , and MB + 2 J/cm 2 groups. Although proliferation does not seem to be effected by MB-mediated laser application, osteo-anabolic activity is altered. ALP activity was significantly increased at day 7 (ANOVA; p < 0.05) for MB-combined laser groups; on the other hand, mineralization was significantly decreased (ANOVA; p < 0.05) in all treatment groups. Alkaline phosphatase and collagen-I expressions were upregulated in MB + 2 J/cm 2 group at 7th and 14th days, respectively. These results may contribute to the low-dose PDT researches and understanding PBM effects on osteoblast behavior but further studies are needed since inappropriate conditions may lead to undesirable results for both therapies.

Residual HEMA and TEGDMA Release and Cytotoxicity Evaluation of Resin-Modified Glass Ionomer Cement and Compomers Cured with Different Light Sources

PubMed Central

Botsali, Murat Selim; Kuşgöz, Adem; Altintaş, Subutay Han; Ülker, Hayriye Esra; Kiliç, Serdar; Başak, Feridun; Ülker, Mustafa

2014-01-01

The purpose of this study was first to evaluate the elution of 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) monomers from resin-modified glass ionomer cement (RMGIC) and compomers cured with halogen and light-emitting diode (LED) light-curing units (LCUs). The effect of cured materials on the viability of L929 fibroblast cells was also evaluated. One RMGIC (Ketac N100) and two compomers (Dyract Extra and Twinkystar) were tested. Materials were prepared in teflon disks and light-cured with LED or halogen LCUs. The residual monomers of resin materials in solution were identified using high-performance liquid chromatography. The fibroblast cells' viability was analyzed using MTT assay. The type of LCU did not have a significant effect on the elution of HEMA and TEGDMA. A greater amount of HEMA than TEGMDA was eluted. The amount of TEGDMA eluted from Twinkystar was greater than Dyract Extra (P < 0.05) when cured with a halogen LCU. All material-LCU combinations decreased the fibroblast cells' viability more than the control group (P < 0.01), except for Dyract Extra cured with a halogen LCU (P > 0.05). Curing with the LED LCU decreased the cells' viability more than curing with the halogen LCU for compomers. For Ketac N100, the halogen LCU decreased the cells' viability more than the LED LCU. PMID:24592149

The influence of size and charge of chitosan/polyglutamic acid hollow spheres on cellular internalization, viability and blood compatibility.

PubMed

Dash, Biraja C; Réthoré, Gildas; Monaghan, Michael; Fitzgerald, Kathleen; Gallagher, William; Pandit, Abhay

2010-11-01

Polymeric hollow spheres can be tailored as efficient carriers of various therapeutic molecules due to their tunable properties. However, the entry of these synthetic vehicles into cells, their cell viability and blood compatibility depend on their physical and chemical properties e.g. size, surface charge. Herein, we report the effect of size and surface charge on cell viability and cellular internalization behaviour and their effect on various blood components using chitosan/polyglutamic acid hollow spheres as a model system. Negatively charged chitosan/polyglutamic acid hollow spheres of various sizes 100, 300, 500 and 1000 nm were fabricated using a template based method and covalently surface modified using linear polyethylene glycol and methoxyethanol amine to create a gradient of surface charge from negative to neutrally charged spheres respectively. The results here suggest that both size and surface charge have a significant influence on the sphere's behaviour, most prominently on haemolysis, platelet activation, plasma recalcification time, cell viability and internalization over time. Additionally, cellular internalization behaviour and viability was found to vary with different cell types. These results are in agreement with those of inorganic spheres and liposomes, and can serve as guidelines for tailoring polymeric solid spheres for specific desired applications in biological and pharmaceutical fields, including the design of nanometer to submicron-sized delivery vehicles. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

An In vitro Comparison of Coconut Water, Milk, and Saline in Maintaining Periodontal Ligament Cell Viability

PubMed Central

D’Costa, Vivian Flourish; Bangera, Madhu Keshava; Kini, Shravan; Kutty, Shakkira Moosa; Ragher, Mallikarjuna

2017-01-01

Background and Objectives: Two of the most critical factors affecting the prognosis of an avulsed tooth after replantation are extraoral dry time and the storage media in which the tooth is placed before treatment is rendered. The present study is undertaken to evaluate the periodontal ligament (PDL) cell viability after storage of teeth in different storage media, namely, coconut water, milk, and saline. Materials and Methods: Forty sound human premolars undergoing extraction for orthodontic purpose were selected. The teeth were allowed to lie dry on sand/mud for 30 min followed by which they were randomly divided and stored in three different media, i.e., coconut water, milk, and saline. After 45-min storage in their respective media, the root surface was then scraped for PDL tissue. Results: The ANOVA and Newman–Keuls post hoc procedure for statistical analysis of viable cell count under a light microscope using hemocytometer demonstrated that coconut water preserved significantly more PDL cells viable (P < 0.05) compared with milk and saline. Conclusion: Storage media help in preserving the viability of PDL cells when immediate replantation is not possible. This study evaluated the posttraumatic PDL cells’ viability following storage in three different storage media. Within the parameters of this study, it was found that coconut water is the most effective media for maintaining the viability of PDL. PMID:29284947

Effect of storage time on the viability of cryopreserved bovine spermatozoa

USDA-ARS?s Scientific Manuscript database

Long term cryopreserved semen viability can impact the National Animal Germplasm Program’s (NAGP) sampling strategy and ability to reconstitute livestock populations. Therefore, the purpose of this project was to determine if prolonged storage of cryopreserved sperm impacts cell viability. Cryoprese...

Hyaluronic acid increases tendon derived cell viability and collagen type I expression in vitro: Comparative study of four different Hyaluronic acid preparations by molecular weight.

PubMed

Osti, Leonardo; Berardocco, Martina; di Giacomo, Viviana; Di Bernardo, Graziella; Oliva, Francesco; Berardi, Anna C

2015-10-06

Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate in human rotator cuff tendon derived cells the effects of four different HA on cell viability, proliferation, apoptosis and the expression of collagen type I and collagen type III. An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of four different HA preparations (Ps) (sodium hyaluronate MW: 500-730 KDa - Hyalgan®, 1000 kDa Artrosulfur HA®, 1600 KDa Hyalubrix® and 2200 KDa Synolis-VA®) at various concentrations. Tendon derived cells morphology were evaluated after 0, 7 and 14 d of culture. Viability, proliferation, apoptosis were evaluated after 0, 24 and 48 h of culture. The expression and deposition of collagen type I and collagen type III were evaluated after 1, 7 and 14 d of culture. All HAPs tested increased viability and proliferation, in dose dependent manner. HAPs already reduce apoptosis at 24 h compared to control cells (without HAPs). Furthermore, HAPs stimulated the synthesis of collagen type I in a dose dependent fashion over 14 d, without increase in collagen type III; moreover, in the presence of Synolis-VA® the expression and deposition of collagen type I was significantly higher as compare with the other HAPs. HAPs enhanced viability, proliferation and expression of collagen type I in tendon derived cells.

[Cytotoxicity induced by gasoline engine exhausts associated with oxidative stress].

PubMed

Che, Wangjun; Zhang, Zunzhen; Wu, Mei; Wang, Ling

2008-09-01

To evaluate the relationship between cytotoxic effects of the extracts of condensate, particulates and semivolatile organic compounds from gasoline engine exhausts (EGE) and oxidative stress. After A549 cells were treated with various concentrations of EGE for 2h, and cell viabilities were detected induced by EGE were examined by MTT assay. Meanwhile, the reactive oxygen species (ROS) in A549 cells induced by EGE were examined, 2',7'-dichlorodihy-drofluorescin diacetate (DCFH-DA) was used to catch ROS and its level measured by value of pixel fluorescence intensity. Furthermore, A549 cells pretreated with different concentrations of glutathione (GSH) were exposed to various concentrations of EGE for 2h, and then cell viabilities were examined. Viabilities of A549 cells significantly decreased in comparison to the solvent group when the concentrations of EGE were more than 3.9 ml/ml (P < 0.05). There were a dose-response relationships between the viabilities and the concentration of EGE (r = -0.81, P < 0.01). At the concentrations of 31.3 ml/ml and 62.5 ml/ml, the values of pixel fluorescence intensity were (125.0 +/- 19.2) and (168.9 +/- 16.9), which were significantly higher than those of control (8.5 +/- 1.4). In addition, the viabilities of cells pretreated with GSH gradually increased with the increases of the concentrations of GSH. There were also a significant difference between the pretreated and non-pretreated group at the concentrations of 0.5 mmol/L and 1.0 mmol/L. Oxidative stress could be one of the mechanisms of cytotoxic effects of EGE.

Nur77 inhibits oxLDL induced apoptosis of macrophages via the p38 MAPK signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Qin; Han, Fei; Peng, Shi

2016-03-18

The interaction between macrophages and oxLDL plays a crucial role in the initiation and progression of atherosclerosis. As a key initiator in a number of plaque promoting processes, oxLDL induces variable effects such as cell apoptosis or proliferation. Orphan nuclear receptor Nur77 is potently induced in macrophages by diverse stimuli, suggesting that it is of importance in vascular inflammation resulting in atherosclerosis, but whether Nur77 induction is detrimental or protective is unclear. In our study, we explore the role of Nur77 in the regulation of oxLDL-induced macrophage apoptosis and the signaling pathways that are involved. We found that oxLDL inducedmore » Nur77 expression in a dose and time dependent fashion, and cell viability was decreased in parallel. To determine whether Nur77 induction contributes to the loss of cell viability or is a protective mechanism, the effect of Nur77 overexpression was examined. Importantly, Nur77 overexpression inhibited the oxLDL-induced decrease of cell viability, inhibited the production of apoptotic bodies and restored DNA synthesis following oxLDL exposure. Furthermore, we found that Nur77 induction is mediated through the p38 MAPK signaling pathway. After pretreatment with SB203580, cell viability was decreased, the expression of CyclinA2 and PCNA was attenuated and the percentage of cell apoptosis was enhanced. Likewise, Nur77 overexpression increased the expression of the cell cycle genes PCNA and p21, and attenuated the increase in caspase-3. On the other hand, knockdown of Nur77 expression by specific siRNA resulted in the increased expression of caspase 3. The results demonstrate that Nur77 is induced by oxLDL via the p38 MAPK signaling pathway, which is involved in the regulation of cell survival. Nur77 enhanced cell survival via suppressing apoptosis, without affecting cell proliferation of activated macrophages, which may be beneficial in patients with atherosclerosis. - Highlights: • oxLDL could induce Nur77 expression. • Nur77 overexpression inhibited oxLDL-induced cell viability, production of apoptotic bodies and restored DNA synthesis. • Cell viability, CyclinA2 and PCNA expression and cell apoptosis were mediated through the p38 MAPK signaling pathway. • Nur77 overexpression mediated the expression of genes PCNA, p21, and caspase-3.« less

Diminished origin licensing capacity specifically sensitises tumour cells to replication stress

PubMed Central

Zimmerman, Kristin M.; Jones, Rebecca M.; Petermann, Eva; Jeggo, Penelope A.

2013-01-01

Previous studies have shown that dormant licensed replication origins can be exploited to enhance recovery from replication stress. Since tumour cells express high levels of origin licensing proteins, we examined whether depletion of such factors might specifically sensitise tumour versus non-tumour cells. Consistent with previous findings, we observed that three tumour-derived cell lines overexpress ORC1, a licensing component, compared to four non-tumour cell lines and that a greater level of ORC1 was required to maintain viability in the tumour cells. We determined siRNA-mediated knockdown conditions for each line that maximally reduced ORC1 but did not impact upon viability, which we considered would optimally deplete dormant origins. ORC1 depletion hypersensitised the tumour-derived cells to hydroxyurea (HU) and H202 but did not affect the sensitivity of the non-tumour lines. Similar results were observed following depletion of ORC6 or CDC6. Further, co-depletion of p53 and ORC1 modestly impaired viability of 1BR3hTERT non-tumour fibroblasts and more dramatically caused hypersensitivity to HU. Finally, overexpression of the c-Myc oncogene combined with ORC1 depletion in non-tumour BJhTERT cells diminished viability. Collectively, these findings suggest that tumour cells may have a reliance on origin licensing capacity, suggesting that licensing factors could represent a target for drug-based cancer therapy. PMID:23364533

Untying a nanoscale knotted polymer structure to linear chains for efficient gene delivery in vitro and to the brain

NASA Astrophysics Data System (ADS)

Newland, B.; Aied, A.; Pinoncely, A. V.; Zheng, Y.; Zhao, T.; Zhang, H.; Niemeier, R.; Dowd, E.; Pandit, A.; Wang, W.

2014-06-01

The purpose of this study was to develop a platform transfection technology, for applications in the brain, which could transfect astrocytes without requiring cell specific functionalization and without the common cause of toxicity through high charge density. Here we show that a simple and scalable preparation technique can be used to produce a ``knot'' structured cationic polymer, where single growing chains can crosslink together via disulphide intramolecular crosslinks (internal cyclizations). This well-defined knot structure can thus ``untie'' under reducing conditions, showing a more favorable transfection profile for astrocytes compared to 25 kDa-PEI (48-fold), SuperFect® (39-fold) and Lipofectamine®2000 (18-fold) whilst maintaining neural cell viability at over 80% after four days of culture. The high transfection/lack of toxicity of this knot structured polymer in vitro, combined with its ability to mediate luciferase transgene expression in the adult rat brain, demonstrates its use as a platform transfection technology which should be investigated further for neurodegenerative disease therapies.The purpose of this study was to develop a platform transfection technology, for applications in the brain, which could transfect astrocytes without requiring cell specific functionalization and without the common cause of toxicity through high charge density. Here we show that a simple and scalable preparation technique can be used to produce a ``knot'' structured cationic polymer, where single growing chains can crosslink together via disulphide intramolecular crosslinks (internal cyclizations). This well-defined knot structure can thus ``untie'' under reducing conditions, showing a more favorable transfection profile for astrocytes compared to 25 kDa-PEI (48-fold), SuperFect® (39-fold) and Lipofectamine®2000 (18-fold) whilst maintaining neural cell viability at over 80% after four days of culture. The high transfection/lack of toxicity of this knot structured polymer in vitro, combined with its ability to mediate luciferase transgene expression in the adult rat brain, demonstrates its use as a platform transfection technology which should be investigated further for neurodegenerative disease therapies. Electronic supplementary information (ESI) available: 1H NMR spectroscopy data and gel permeation chromatography data. See DOI: 10.1039/c3nr06737h

The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability

NASA Astrophysics Data System (ADS)

Yi, Zong-Chun; Li, Xiao-Fei; Wang, Yan; Wang, Jie; Sun, Yan; Zhuang, Feng-Yuan

2011-06-01

The budding yeast Saccharomyces cerevisiae has been proposed as an ideal model organism for clarifying the biological effects caused by spaceflight conditions. The postmitotic S. cerevisiae cells onboard Practice eight recoverable satellite were subjected to spaceflight for 15 days. After recovery, the viability, the glycogen content, the activities of carbohydrate metabolism enzymes, the DNA content and the lipid peroxidation level in yeast cells were analyzed. The viability of the postmitotic yeast cells after spaceflight showed a three-fold increase as compared with that of the ground control cells. Compared to the ground control cells, the lipid peroxidation level in the spaceflight yeast cells markedly decreased. The spaceflight yeast cells also showed an increase in G2/M cell population and a decrease in Sub-G1 cell population. The glycogen content and the activities of hexokinase and succinate dehydrogenase significantly decreased in the yeast cells after spaceflight. In contrast, the activity of malate dehydrogenase showed an obvious increase after spaceflight. These results suggested that microgravity or spaceflight could promote the survival of postmitotic S. cerevisiae cells through regulating carbohydrate metabolism, ROS level and cell cycle progression.

On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves.

PubMed

Ding, Xiaoyun; Lin, Sz-Chin Steven; Kiraly, Brian; Yue, Hongjun; Li, Sixing; Chiang, I-Kao; Shi, Jinjie; Benkovic, Stephen J; Huang, Tony Jun

2012-07-10

Techniques that can dexterously manipulate single particles, cells, and organisms are invaluable for many applications in biology, chemistry, engineering, and physics. Here, we demonstrate standing surface acoustic wave based "acoustic tweezers" that can trap and manipulate single microparticles, cells, and entire organisms (i.e., Caenorhabditis elegans) in a single-layer microfluidic chip. Our acoustic tweezers utilize the wide resonance band of chirped interdigital transducers to achieve real-time control of a standing surface acoustic wave field, which enables flexible manipulation of most known microparticles. The power density required by our acoustic device is significantly lower than its optical counterparts (10,000,000 times less than optical tweezers and 100 times less than optoelectronic tweezers), which renders the technique more biocompatible and amenable to miniaturization. Cell-viability tests were conducted to verify the tweezers' compatibility with biological objects. With its advantages in biocompatibility, miniaturization, and versatility, the acoustic tweezers presented here will become a powerful tool for many disciplines of science and engineering.

BMI-1 suppression increases the radiosensitivity of oesophageal carcinoma via the PI3K/Akt signaling pathway.

PubMed

Yang, Xing-Xiao; Ma, Ming; Sang, Mei-Xiang; Zhang, Xue-Yuan; Liu, Zhi-Kun; Song, Heng; Zhu, Shu-Chai

2018-02-01

B-cell‑specific Moloney murine leukaemia virus integration site-1 (BMI-1) contributes to the growth of tumour cells post-irradiation (IR). The aim of the present study was to characterize the effects of BMI-1 on cell viability, radiosensitivity and its mechanisms of action in oesophageal squamous cell cancer (ESCC). Western blotting and immunohistochemistry were employed to evaluate the protein expression of BMI-1 in ESCC cells and specimens, respectively. Additionally, the protein expression levels of BMI-1, H2AK119ub and γH2AX in ESCC cells were detected following different doses of IR and at different times after IR. The protein expression levels of MDC1 and 53BP1 were also measured. Flow cytometry and MTT assays were used to determine cell cycle progression, apoptosis and cell viability. The phosphatidylinositol 3-kinase inhibitor LY294002 and the agonist IGF-1 were employed to suppress or induce the phosphorylation of Akt to determine whether BMI-1 induces radioresistance in ESCC cells via activation of the PI3K/Akt pathway. The expression of BMI-1 was higher in ESCC tissues and cells compared with that in normal oesophageal tissues and cells. In addition, BMI-1 was positively related to tumour size and lymph node metastases and negatively to the overall survival of ESCC patients. IR induced the expression of BMI-1, H2AK119ub and γH2AX in a dose- and time-dependent manner. BMI-1 knockdown lowered the expression of γH2AX, MDC1 and 53BP1, suppressed cell viability and increased radiosensitivity. G2/M phase arrest was eliminated; this was followed by an increased proportion of cells entering the G0/G1 phase after IR and BMI-1 knockdown via the upregulation of P16 and downregulation of cyclin D2 and cyclin-dependent kinase-4. Moreover, BMI-1 knockdown increased cell apoptosis, downregulated MCL-1 and p-Akt and upregulated Bax. Additionally, the inhibitory effect of the downregulation of p-Akt by LY294002 on tumour cell viability was identical to that of BMI-1 knockdown, while the kinase agonist IGF-1 reversed the effects of BMI-1 knockdown on cell viability and radiosensitivity. Taken together, BMI-1 knockdown induces radiosensitivity in ESCC and significantly inhibits cell viability, which may contribute to an increased proportion of cells in the G0/G1 phase and cell apoptosis via suppression of the PI3K/Akt signalling pathway.

Three-dimensional culture of dental pulp stem cells in direct contact to tricalcium silicate cements.

PubMed

Widbiller, M; Lindner, S R; Buchalla, W; Eidt, A; Hiller, K-A; Schmalz, G; Galler, K M

2016-03-01

Calcium silicate cements are biocompatible dental materials applicable in contact with vital tissue. The novel tricalcium silicate cement Biodentine™ offers properties superior to commonly used mineral trioxide aggregate (MTA). Objective of this study was to evaluate its cytocompatibility and ability to induce differentiation and mineralization in three-dimensional cultures of dental pulp stem cells after direct contact with the material. Test materials included a new tricalcium silicate (Biodentine™, Septodont, Saint-Maur-des-Fossés, France), MTA (ProRoot® MTA, DENSPLY Tulsa Dental Specialities, Johnson City, TN, USA), glass ionomer (Ketac™ Molar Aplicap™, 3M ESPE, Seefeld, Germany), human dentin disks and polystyrene. Magnetic activated cell sorting for to the surface antigen STRO-1 was performed to gain a fraction enriched with mesenchymal stem cells. Samples were allowed to set and dental pulp stem cells in collagen carriers were placed on top. Scanning electron microscopy of tricalcium silicate cement surfaces with and without cells was conducted. Cell viability was measured for 14 days by MTT assay. Alkaline phosphatase activity was evaluated (days 3, 7, and 14) and expression of mineralization-associated genes (COL1A1, ALP, DSPP, and RUNX2) was quantified by real-time quantitative PCR. Nonparametric statistical analysis for cell viability and alkaline phosphatase data was performed to compare different materials as well as time points (Mann-Whitney U test, α = 0.05). Cell viability was highest on tricalcium silicate cement, followed by MTA. Viability on glass ionomer cement and dentin disks was significantly lower. Alkaline phosphatase activity was lower in cells on new tricalcium silicate cement compared to MTA, whereas expression patterns of marker genes were alike. Increased cell viability and similar levels of mineralization-associated gene expression in three-dimensional cell cultures on the novel tricalcium silicate cement and mineral trioxide aggregate indicate that the material is cytocompatible and bioactive. The tested new tricalcium silicate cement confirms its suitability as an alternative to MTA in vital pulp therapy.

Autophagic effects of Hibiscus sabdariffa leaf polyphenols and epicatechin gallate (ECG) against oxidized LDL-induced injury of human endothelial cells.

PubMed

Chen, Jing-Hsien; Lee, Ming-Shih; Wang, Chi-Ping; Hsu, Cheng-Chin; Lin, Hui-Hsuan

2017-08-01

Oxidized low-density lipoprotein (ox-LDL) contributes to the pathogenesis of atherosclerosis by promoting vascular endothelial cell injury. Hibiscus sabdariffa leaf polyphenols (HLP), rich in flavonoids, have been shown to possess antioxidant and antiatherosclerotic activities. In this study, we examined the protective role of HLP and its main compound (-)-epicatechin gallate (ECG) in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL in vitro. In a model of ox-LDL-impaired HUVECs, assessments of cell viability, cytotoxicity, cell proliferation, apoptosis, and autophagy were detected. To highlight the mechanisms of the antiapoptotic effects of HLP and ECG, the expressions of molecular proteins were measured by Western blotting, real-time PCR, and so on. HLP or ECG improved the survival of HUVECs from ox-LDL-induced viability loss. In addition, HLP or ECG showed potential in reducing ox-LDL-dependent apoptosis. Next, the ox-LDL-induced formation of acidic vesicular organelles and upregulation of the autophagy-related genes were increased by HLP or ECG. The HLP-triggered autophagic flux was further confirmed by increasing the LC3-II level under the pretreatment of an autophagy inhibitor chloroquine. Molecular data indicated the autophagic effect of HLP or ECG might be mediated via class III PI3K/Beclin-1 and PTEN/class I PI3K/Akt cascade signaling, as demonstrated by the usage of a class III PI3K inhibitor 3-methyladenine (3-MA) and a PTEN inhibitor SF1670. Our data imply that ECG-enriched HLP upregulates the autophagic pathway, which in turn led to reduce ox-LDL-induced HUVECs injury and apoptosis and provide a new mechanism for its antiatherosclerotic activity.

Effect of chemical composition on corneal cellular response to photopolymerized materials comprising 2-hydroxyethyl methacrylate and acrylic acid.

PubMed

Lai, Jui-Yang

2013-10-01

Characterization of corneal cellular response to hydrogel materials is an important issue in ophthalmic applications. In this study, we aimed to investigate the relationship between the feed composition of 2-hydroxyethyl methacrylate (HEMA)/acrylic acid (AAc) and material compatibility towards corneal stromal and endothelial cells. The monomer solutions of HEMA and AAc were mixed at varying volume ratios of 92:0, 87:5, 82:10, 77:15, and 72:20, and were subjected to UV irradiation. Results of electrokinetic measurements showed that an increase in absolute zeta potential of photopolymerized membranes is observed with increasing the volume ratios of AAc/HEMA. Following 4 days of incubation with various hydrogels, the primary rabbit corneal stromal and endothelial cell cultures were examined for viability, proliferation, and pro-inflammatory gene expression. The samples prepared from the solution mixture containing 0-10 vol.% AAc displayed good cytocompatibility. However, with increasing volume ratio of AAc and HEMA from 15:77 to 20:72, the decreased viability, inhibited proliferation, and stimulated inflammation were noted in both cell types, probably due to the stronger charge-charge interactions. On the other hand, the ionic pump function of corneal endothelial cells exposed to photopolymerized membranes was examined by analyzing the Na(+),K(+)-ATPase alpha 1 subunit (ATP1A1) expression level. The presence of material samples having higher anionic charge density (i.e., zeta potential of -38 to -56 mV) may lead to abnormal transmembrane transport. It is concluded that the chemical composition of HEMA/AAc has an important influence on the corneal stromal and endothelial cell responses to polymeric biomaterials. Copyright © 2013 Elsevier B.V. All rights reserved.

Investigation into the distinct subcellular effects of docosahexaenoic acid loaded low-density lipoprotein nanoparticles in normal and malignant murine liver cells

PubMed Central

Moss, Lacy R.; Mulik, Rohit S.; Van Treuren, Tim; Kim, Soo Young; Corbin, Ian R.

2016-01-01

Background Recent studies have shown that low density lipoproteins reconstituted with the natural omega 3 fatty acid docosahexaenoic acid (LDL-DHA) is selectively cytotoxic to liver cancer cells over normal hepatocytes. To date, little is known about the subcellular events which transpire following LDL-DHA treatment. Methods Herein, murine noncancer and cancer liver cells, TIB-73 and TIB-75 respectively, were investigated utilizing confocal microscopy, flow cytometry and viability assays to demonstrate differential actions of LDL-DHA nanoparticles in normal versus malignant cells. Results Our studies first showed that basal levels of oxidative stress are significantly higher in the malignant TIB-75 cells compared to the normal TIB-73 cells. As such, upon entry of LDL-DHA into the malignant TIB-75 cells, DHA is rapidly oxidized precipitating global and lysosomal lipid peroxidation along with increased lysosomal permeability. This leakage of lysosomal contents and lipid peroxidation products trigger subsequent mitochondrial dysfunction and nuclear injury. The cascade of LDL-DHA mediated lipid peroxidation and organelle damage was partially reversed by the administration of the antioxidant, N-acetylcysteine, or the iron-chelator, deferoxamine. LDL-DHA treatment in the normal TIB-73 cells was well tolerated and did not elicit any cell or organelle injury. Conclusion These studies have shown that LDL-DHA is selectively cytotoxic to liver cancer cells and that increased levels of ROS and iron catalyzed reactions promote the peroxidation of DHA which lead to organelle dysfunction and ultimately the demise of the cancer cell. General significance LDL-DHA selectively disrupts lysosomal, mitochondrial and nuclear function in cancer cells as a novel pathway for eliminating cancer cells. PMID:27418237

A perfusion culture system using a stirred ceramic membrane reactor for hyperproduction of IgG2a monoclonal antibody by hybridoma cells.

PubMed

Dong, Haodi; Tang, Ya-Jie; Ohashi, Ryo; Hamel, Jean-François P

2005-01-01

A novel perfusion culture system for efficient production of IgG2a monoclonal antibody (mAb) by hybridoma cells was developed. A ceramic membrane module was constructed and used as a cell retention device installed in a conventional stirred-tank reactor during the perfusion culture. Furthermore, the significance of the control strategy of perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was investigated. With the highest increasing rate (deltaD, vvd per day, vvdd) of perfusion rate, the maximal viable cell density of 3.5 x 10(7) cells/mL was obtained within 6 days without any limitation and the cell viability was maintained above 95%. At lower deltaD's, the cell growth became limited. Under nutrient-limited condition, the specific cell growth rate (mu) was regulated by deltaD. During the nonlimited growth phase, the specific mAb production rate (qmAb) remained constant at 0.26 +/- 0.02 pg/cell x h in all runs. During the cell growth-limited phase, qmAb was regulated by deltaD within the range of 0.25-0.65 vvdd. Under optimal conditions, qmAb of 0.80 and 2.15 pg/cell x h was obtained during the growth-limited phase and stationary phase, respectively. The overall productivity and yield were 690 mg/L x day and 340 mg/L x medium, respectively. This study demonstrated that this novel perfusion culture system for suspension mammalian cells can support high cell density and efficient mAb production and that deltaD is an important control parameter to regulate and achieve high mAb production.

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

PubMed Central

Wu, Qing; Lin, Wei-Dong; Liao, Guan-Qun; Zhang, Li-Guo; Wen, Shun-Qian; Lin, Jia-Ying

2015-01-01

AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity. PMID:25624718

Impact of lithium alone or in combination with haloperidol on oxidative stress parameters and cell viability in SH-SY5Y cell culture.

PubMed

Gawlik-Kotelnicka, Oliwia; Mielicki, Wojciech; Rabe-Jabłońska, Jolanta; Lazarek, Jerry; Strzelecki, Dominik

2016-02-01

It has been reported that lithium may inhibit lipid peroxidation and protein oxidation. Lithium salts also appear to stimulate cell proliferation, increase neurogenesis, and delay cell death. Oxidative stress and neurodegeneration may play an important role in the pathophysiology of bipolar disorder and the disease course thereof. The aim of this research is to estimate the influence of lithium (alone and in combination with haloperidol) on the parameters of oxidative stress and viability of SH-SY5Y cell lines in neutral and pro-oxidative conditions. The evaluated oxidative stress parameter was lipid peroxidation. The viability of the cell lines was measured utilising the MTT test. In neutral conditions, higher levels of thiobarbituric acid reactive substances were observed in those samples which contained both haloperidol and lithium than in other samples. However, these differences were not statistically significant. Cell viability was significantly higher in therapeutic lithium samples than in the controls; samples of haloperidol alone as well as those of haloperidol with lithium did not differ from controls. The results of our study may indicate that lithium possess neuroprotective properties that may be partly due to antioxidative effects. The combination of lithium and haloperidol may generate increased oxidative stress.

HEMOXCell, a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate-Supplemented Media.

PubMed

Le Pape, Fiona; Cosnuau-Kemmat, Lucie; Richard, Gaëlle; Dubrana, Frédéric; Férec, Claude; Zal, Franck; Leize, Elisabeth; Delépine, Pascal

2017-04-01

Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

In vitro time- and dose-effect response of JP-8 and S-8 jet fuel on alveolar type II epithelial cells of rats.

PubMed

Robb, Tiffany M; Rogers, Michael J; Woodward, Suann S; Wong, Simon S; Witten, Mark L

2010-07-01

This study was designed to characterize and compare the effects of jet propellant-8 (JP-8) fuel and synthetic-8 (S-8) on cell viability and nitric oxide synthesis in cultured alveolar type II epithelial cells of rats. Exposure times varied from 0.25, 0.5, 1, and 6 hours at the following concentrations of jet fuel: 0.0, 0.1, 0.4, and 2.0 microg/mL. Data indicate that JP-8 presents a gradual decline in cell viability and steady elevation in nitric oxide release as exposure concentrations increase. At a 2.0 microg/mL concentration of JP-8, nearly all of the cells are not viable. Moreover, S-8 exposure to rat type II lung cells demonstrated an abrupt fall in percentage cell viability and increases in nitric oxide measurement, particularly after the 2.0 microg/mL was reached at 1 and 6 hours. At 0.0, 0.2, and 0.4 microg/mL concentrations of S-8, percentage viability was sustained at steady concentrations. The results suggest different epithelial toxicity and mechanistic effects of S-8 and JP-8, providing further insight concerning the impairment imposed at specific levels of lung function and pathology induced by the different fuels.

Comparative studies of cellular viability levels on 2D and 3D in vitro culture matrices.

PubMed

Gargotti, M; Lopez-Gonzalez, U; Byrne, H J; Casey, A

2018-02-01

In this study, the cellular viability and function of immortalized human cervical and dermal cells are monitored and compared in conventional 2D and two commercial 3D membranes, Collagen and Geltrex, of varying working concentration and volume. Viability was monitored with the aid of the Alamar Blue assay, cellular morphology was monitored with confocal microscopy, and cell cycle studies and cell death mechanism studies were performed with flow cytometry. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to 3D environment causing alterations to effective resazurin concentration, uptake and conversion rates, which was dependent on exposure time, but also due to the effect of the membrane itself on cellular function. These effects were verified by flow cytometry, in which no significant differences in viable cell numbers between 2D and 3D systems were observed after 24 h culture. The results showed the observed effect was different after shorter exposure periods, was also dependent on working concentration of the 3D system and could be mediated by altering the culture vessel size. Cell cycle analysis revealed cellular function could be altered by growth on the 3D substrates and the alterations were noted to be dependent on 3D membrane concentration. The use of 3D culture matrices has been widely interpreted to result in "improved viability levels" or "reduced" toxicity or cellular "resistance" compared to cells cultured on traditional 2D systems. The results of this study show that cellular health and viability levels are not altered by culture in 3D environments, but their normal cycle can be altered as indicated in the cell cycle studies performed and such variations must be accounted for in studies employing 3D membranes for in vitro cellular screening.

Molecular size and origin do not influence the harmful side effects of hydroxyethyl starch on human proximal tubule cells (HK-2) in vitro.

PubMed

Bruno, Raphael R; Neuhaus, Winfried; Roewer, Norbert; Wunder, Christian; Schick, Martin A

2014-09-01

Recently, clinical trials revealed renal impairment induced by hydroxyethyl starch (HES) in septic patients. In prior studies, we managed to demonstrate that HES accumulated in renal proximal tubule cells (PTCs). The related pathomechanism has not yet been discovered. To validate our hypothesis that the HES molecule itself is harmful, regardless of its molecule size or origin, we conducted a comprehensive study to elucidate the influences of different HES preparations on PTC viability in vitro. Cell viability of human PTC was measured with a cytotoxicity assay, quantifying the reduction of tetrazolium salt to colored formazan. Experiments were performed by assessing the influence of different carrier solutions of HES (balanced, nonbalanced, culture medium), different average molecular weights (70, 130, 200 kDa), different origins (potato or corn derived), and various durations of incubation (2-21 hours). Furthermore, HES 130/0.4 was fractionated by ultrafiltration, and the impact on cell viability of average single-size fractions with <3, 3 to 10, 10 to 30, 30 to 50, 50 to 100, and >100 kDa was investigated. We also tested the possible synergistic effects of inflammation induced by tumor necrosis factor-α. All tested HES solutions, regardless of origin or carrier matrix, decreased cell viability in an equivalent, dose-dependent manner. Coincubation with tumor necrosis factor-α did not reduce HES-induced reduction of cell viability. Minor differences were detected comparing 70, 130, and 200 kDa preparations. Analysis of fractionated HES revealed that each fraction decreased cell viability. Even small HES molecules (10-30 kDa) were significantly deleterious. For the first time, we were able to show that only the total mass of HES molecules applied is responsible for the harmful impact on renal PTC in vitro. Neither molecular size nor their origin showed any relevance.

Co-regulation of Primary Mouse Hepatocyte Viability and Function by Oxygen and Matrix

PubMed Central

Buck, Lorenna D.; Inman, S. Walker; Rusyn, Ivan; Griffith, Linda G.

2014-01-01

Although oxygen and extracellular matrix cues both influence differentiation state and metabolic function of primary rat and human hepatocytes, relatively little is known about how these factors together regulate behaviors of primary mouse hepatocytes in culture. To determine the effects of pericellular oxygen tension on hepatocellular function, we employed 2 methods of altering oxygen concentration in the local cellular microenvironment of cells cultured in the presence or absence of an extracellular matrix (Matrigel) supplement. By systematically altering medium depth and gas phase oxygen tension, we created multiple oxygen regimes (hypoxic, normoxic, and hyperoxic) and measured the local oxygen concentrations in the pericellular environment using custom-designed oxygen microprobes. From these measurements of oxygen concentrations, we derived values of oxygen consumption rates under a spectrum of environmental contexts, thus providing the first reported estimates of these values for primary mouse hepatocytes. Oxygen tension and matrix microenvironment were found to synergistically regulate hepatocellular survival and function as assessed using quantitative image analysis for cells stained with vital dyes, and assessment of secretion of albumin. Hepatocellular viability was affected only at strongly hypoxic conditions. Surprisingly, albumin secretion rates were greatest at a moderately supra-physiological oxygen concentration, and this effect was mitigated at still greater supra-physiological concentrations. Matrigel enhanced the effects of oxygen on retention of function. This study underscores the importance of carefully controlling cell density, medium depth and gas phase oxygen, as the effects of these parameters on local pericellular oxygen tension and subsequent hepatocellular function are profound. PMID:24222008

Low temperature co-fired ceramic packaging of CMOS capacitive sensor chip towards cell viability monitoring.

PubMed

Halonen, Niina; Kilpijärvi, Joni; Sobocinski, Maciej; Datta-Chaudhuri, Timir; Hassinen, Antti; Prakash, Someshekar B; Möller, Peter; Abshire, Pamela; Kellokumpu, Sakari; Lloyd Spetz, Anita

2016-01-01

Cell viability monitoring is an important part of biosafety evaluation for the detection of toxic effects on cells caused by nanomaterials, preferably by label-free, noninvasive, fast, and cost effective methods. These requirements can be met by monitoring cell viability with a capacitance-sensing integrated circuit (IC) microchip. The capacitance provides a measurement of the surface attachment of adherent cells as an indication of their health status. However, the moist, warm, and corrosive biological environment requires reliable packaging of the sensor chip. In this work, a second generation of low temperature co-fired ceramic (LTCC) technology was combined with flip-chip bonding to provide a durable package compatible with cell culture. The LTCC-packaged sensor chip was integrated with a printed circuit board, data acquisition device, and measurement-controlling software. The packaged sensor chip functioned well in the presence of cell medium and cells, with output voltages depending on the medium above the capacitors. Moreover, the manufacturing of microfluidic channels in the LTCC package was demonstrated.

Novel type 1 photosensitizers: viability of leukemia cells exposed to reactive intermediates generated in situ by in vitro photofragmentation

NASA Astrophysics Data System (ADS)

Rajagopalan, Raghavan; Karwa, Amol; Lusiak, Przemyslaw M.; Srivastava, Kripa; Poreddy, Amruta R.; Pandurangi, Raghootama S.; Galen, Karen P.; Neumann, William L.; Cantrell, Gary E.; Dorshow, Richard B.

2009-06-01

Photodynamic therapy of tumors involving Type 2 photosenstizers has been conspicuously successful, but the Type 1 process, in contrast, has not received much attention despite its considerable potential. Accordingly, several classes of molecules containing fragile bonds such as azido (-N=N=N), azo (-N=N-), sulfenato (-S-O-) and oxaza (-N-O-) functional groups that produce reactive intermediates such as radicals and nitrenes upon photoexcitation were prepared and tested for cell viability using U397 leukemia cell line. The azido photosensitizer was conjugated to leukemia cell binding peptide, SFFWRLS, for targeted cell viability study. The cells were incubated with the photosensitizer at various concentrations, and were illuminated for 5, 10, and 20 minutes. The results show that all the photosensitizers caused cell death compared to the controls when exposed to both the photosensitizers and light. Most importantly, selective cell death was observed with the azido peptide conjugate 6, which clearly demonstrates that these Type 1 sensitizers are useful for phototherapeutic applications.

Electroinduced Delivery of Hydrogel Nanoparticles in Colon 26 Cells, Visualized by Confocal Fluorescence System.

PubMed

Atanasova, Severina; Nikolova, Biliana; Murayama, Shuhei; Stoyanova, Elena; Tsoneva, Iana; Zhelev, Zhivko; Aoki, Ichio; Bakalova, Rumiana

2016-09-01

Nano-scale drug delivery systems (nano-DDS) are under intense investigation. Nano-platforms are developed for specific administration of small molecules, drugs, genes, contrast agents [quantum dots (QDs)] both in vivo and in vitro. Electroporation is a biophysical phenomenon which consists of the application of external electrical pulses across the cell membrane. The aim of this study was to research electro-assisted Colon 26 cell line internalization of QDs and QD-loaded nano-hydrogels (polymersomes) visualized by confocal microscopy and their influence on cell viability. The experiments were performed on the Colon 26 cancer cell line, using a confocal fluorescent imaging system and cell viability test. Electroporation facilitated the delivery of nanoparticles in vivo. We demonstrated increased voltage-dependent delivery of nanoparticles into cells after electrotreatment, without significant cell viability reduction. The delivery and retention of the polymersomes in vitro is a promising tool for future cancer treatment strategies and nanomedcine. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

In Vitro Effects of Preserved and Unpreserved Anti-Allergic Drugs on Human Corneal Epithelial Cells

PubMed Central

Calvo, Patricia; Ropero, Inés; Pintor, Jesús

2014-01-01

Abstract Purpose: Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Methods: Cells were treated with unpreserved ketotifen solution, benzalkonium chloride (BAC)-containing anti-allergic drugs (ketotifen, olopatadine, levocabastine) as well as BAC alone. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine cell viability. Effects of compounds on barrier function were analyzed measuring transepithelial electrical resistance (TEER) to determine paracellular permeability and rose bengal assays to evaluate transcellular barrier formation. Results: The BAC-preserved anti-allergic formulations and BAC alone significantly reduced cell viability, monolayer cultures being more sensitive to damage by these solutions. Unpreserved ketotifen induced the least diminution in cell viability. The extent of decrease of cell viability was clearly dependent of BAC presence, but it was also affected by the different types of drugs when the concentration of BAC was low and the short time of exposure. Treatment with BAC-containing anti-allergic drugs and BAC alone resulted in increased paracellular permeability and loss of transcellular barrier function as indicated by TEER measurement and rose bengal assays. Conclusions: The presence of the preservative BAC in anti-allergic eye drop formulations contributes importantly to the cytotoxic effects induced by these compounds. Stratified cell cultures seem to be a more relevant model for toxicity evaluation induced on the ocular surface epithelia than monolayer cultures. PMID:25100331

A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYS

EPA Science Inventory

AbstractTITLE: A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYSABSTRACT BODY: Microelectrode array (MEA) recordings are increasingly being used as an in vitro method to detect and characte...

Upright and Inverted Single-Junction GaAs Solar Cells Grown by Hydride Vapor Phase Epitaxy

DOE PAGES

Simon, John; Schulte, Kevin L.; Jain, Nikhil; ...

2016-10-19

Hydride vapor phase epitaxy (HVPE) is a low-cost alternative to conventional metal-organic vapor phase epitaxy (MOVPE) growth of III-V solar cells. In this work, we show continued improvement of the performance of HVPE-grown single-junction GaAs solar cells. We show over an order of magnitude improvement in the interface recombination velocity between GaAs and GaInP layers through the elimination of growth interrupts, leading to increased short-circuit current density and open-circuit voltage compared with cells with interrupts. One-sun conversion efficiencies as high as 20.6% were achieved with this improved growth process. Solar cells grown in an inverted configuration that were removed frommore » the substrate showed nearly identical performance to on-wafer cells, demonstrating the viability of HVPE to be used together with conventional wafer reuse techniques for further cost reduction. As a result, these devices utilized multiple heterointerfaces, showing the potential of HVPE for the growth of complex and high-quality III-V devices.« less

Quasi-perpetual discharge behaviour in p-type Ge-air batteries.

PubMed

Ocon, Joey D; Kim, Jin Won; Abrenica, Graniel Harne A; Lee, Jae Kwang; Lee, Jaeyoung

2014-11-07

Metal-air batteries continue to become attractive energy storage and conversion systems due to their high energy and power densities, safer chemistries, and economic viability. Semiconductor-air batteries - a term we first define here as metal-air batteries that use semiconductor anodes such as silicon (Si) and germanium (Ge) - have been introduced in recent years as new high-energy battery chemistries. In this paper, we describe the excellent doping-dependent discharge kinetics of p-type Ge anodes in a semiconductor-air cell employing a gelled KOH electrolyte. Owing to its Fermi level, n-type Ge is expected to have lower redox potential and better electronic conductivity, which could potentially lead to a higher operating voltage and better discharge kinetics. Nonetheless, discharge measurements demonstrated that this prediction is only valid at the low current regime and breaks down at the high current density region. The p-type Ge behaves extremely better at elevated currents, evident from the higher voltage, more power available, and larger practical energy density from a very long discharge time, possibly arising from the high overpotential for surface passivation. A primary semiconductor-air battery, powered by a flat p-type Ge as a multi-electron anode, exhibited an unprecedented full discharge capacity of 1302.5 mA h gGe(-1) (88% anode utilization efficiency), the highest among semiconductor-air cells, notably better than new metal-air cells with three-dimensional and nanostructured anodes, and at least two folds higher than commercial Zn-air and Al-air cells. We therefore suggest that this study be extended to doped-Si anodes, in order to pave the way for a deeper understanding on the discharge phenomena in alkaline metal-air conversion cells with semiconductor anodes for specific niche applications in the future.

Osteoarticular cells tolerate short-term exposure to nitisinone-implications in alkaptonuria.

PubMed

Mistry, J B; Jackson, D J; Bukhari, M; Taylor, A M

2016-02-01

Alkaptonuria (AKU) is a rare genetic disease resulting in severe, rapidly progressing, early onset multi-joint osteoarthropathy. A potential therapy, nitisinone, is being trialled that reduces the causative agent; homogentisic acid (HGA) and in a murine model has shown to prevent ochronosis. Little is currently known about the effect nitisinone has on osteoarticular cells; these cells suffer most from the presence of HGA and its polymeric derivatives. This led us to investigate nitisinone's effect on chondrocytes and osteoblast-like cells in an in vitro model. Human C20/A4 immortalized chondrocytes, and osteosarcoma cells MG63 cultured in DMEM, as previously described. Confluent cells were then plated into 24-well plates at 4 × 10(4) cells per well in varying concentrations of nitisinone. Cells were cultured for 7 days with medium changes every third day. Trypan blue assay was used to determine viability and the effect of nitisinone concentration on cells. Statistical analysis was performed using analysis of variance, and differences between groups were determined by Newman-Keuls post-test. Analysis of C20/A4 chondrocyte and MG63 osteoblast-like cell viability when cultured in different concentrations of nitisinone demonstrates that there is no statistically significant difference in cell viability compared to control cultures. There is currently no literature surrounding the use of nitisinone in human in vitro models, or its effect on chondrocytes or osteoblast like cells. Our results show that nitisinone does not appear detrimental to cell viability of chondrocytes or osteoblast-like cells, which adds to the evidence that this therapy could be useful in treating AKU.

The effect of uranium on bacterial viability and cell surface morphology using atomic force microscopy in the presence of bicarbonate ions.

PubMed

Sepulveda-Medina, Paola; Katsenovich, Yelena; Musaramthota, Vishal; Lee, Michelle; Lee, Brady; Dua, Rupak; Lagos, Leonel

2015-06-01

Past disposal practices at nuclear production facilities have led to the release of liquid waste into the environment creating multiple radionuclide plumes. Microorganisms are known for the ability to interact with radionuclides and impact their mobility in soils and sediments. Gram-positive Arthrobacter sp. are one of the most common bacterial groups in soils and are found in large numbers in subsurface environments contaminated with radionuclides. This study experimentally analyzed changes on the bacteria surface at the nanoscale level after uranium exposure and evaluated the effect of aqueous bicarbonate ions on U(VI) toxicity of a low uranium-tolerant Arthrobacter oxydans strain G968 by investigating changes in adhesion forces and cell dimensions via atomic force microscopy (AFM). Experiments were extended to assess cell viability by the Live/Dead BacLight Bacterial Viability Kit (Molecular Probes) and quantitatively illustrate the effect of uranium exposure in the presence of varying concentrations of bicarbonate ions. AFM and viability studies showed that samples containing bicarbonate were able to withstand uranium toxicity and remained viable. Samples containing no bicarbonate exhibited deformed surfaces and a low height profile, which, in conjunction with viability studies, indicated that the cells were not viable. Copyright © 2015 Institut Pasteur. All rights reserved.

Fibroblast Viability after Storage at 20 °C in Milk, Hank's Balanced Salt Solution and Coconut Water.

PubMed

Souza, Beatriz Dulcineia Mendes de; Alves, Ana Maria Hecke; Santos, Luciane Geanini Pena Dos; Simões, Claudia Maria de Oliveira; Felippe, Wilson Tadeu; Felippe, Mara Cristina Santos

2016-01-01

The objective of this study was to evaluate the effectiveness of various storage media at 20 °C in maintaining the viability of human periodontal ligament fibroblasts (HPLF) over time. HPLF were maintained at 20 °C in skim milk (SM), whole milk (WM), freshly prepared Hank's balanced salt solution (HBSS), Save-A-Tooth(r), natural coconut water (NCW), coconut water industrialized (ICW) and tap water (negative control) for 3, 6, 24, 48, 72, 96 and 120 h. Cells maintained in Minimal Essential Medium (MEM-37) at 37 °C served as a positive control. Cell viability was determined by MTT assay. Statistical analysis was performed by Kruskal-Wallis test and Scheffe test (α = 5%). From 24 h, NCW was significantly better in maintaining cell viability than all other tested storage media (p<0.05). SM and WM were significantly better than HBSS for up to 72 h. Save-A-Tooth(r) and ICW were the worst conservation storage media. In conclusion, the effectiveness of the tested storage media to maintain the viability of the periodontal ligament cells was as follows, in a descending order: NCW > MEM-37> SM and IM> HBSS> ICW > Save-A-Tooth(r)> tap water.

Propofol protects hippocampal neurons from apoptosis in ischemic brain injury by increasing GLT-1 expression and inhibiting the activation of NMDAR via the JNK/Akt signaling pathway.

PubMed

Gong, Hong-Yan; Zheng, Fang; Zhang, Chao; Chen, Xi-Yan; Liu, Jing-Jing; Yue, Xiu-Qin

2016-09-01

Ischemic brain injury (IBI) can cause nerve injury and is a leading cause of morbidity and mortality worldwide. The neuroprotective effects of propofol against IBI have been previously demonstrated. However, the neuroprotective effects of propofol on hippocampal neurons are not yet entirely clear. In the present study, models of IBI were established in hypoxia-exposed hippocampal neuronal cells. Cell viability assay and apoptosis assay were performed to examine the neuroprotective effects of propofol on hippocampal neurons in IBI. A significant decrease in cell viability and a significant increase in cell apoptosis were observed in the IBI group compared with the control group, accompanied by a decrease in glial glutamate transporter-1 (GLT‑1) expression as determined by RT-qPCR and western blot analysis. The effects of IBI were reversed by propofol treatment. The siRNA-mediated knockdown of GLT‑1 in the hypoxia-exposed hippocampal neuronal cells led to an increase in cell apoptosis, Jun N-terminal kinase (JNK) activation and N-methyl-D‑aspartate (NMDA) receptor (NR1 and NR2B) activation, as well as to a decrease in cell viability and a decrease in Akt activation. The effects of RNA interference-mediated GLT‑1 gene silencing on cell viability, JNK activation, NMDAR activation, cell apoptosis and Akt activation in the hippocampal neuronal cells were slightly reversed by propofol treatment. The JNK agonist, anisomycin, and the Akt inhibitor, LY294002, both significantly blocked the effects of propofol on hippocampal neuronal cell viability and apoptosis in IBI. The decrease in JNK activation and the increase in Akt activation caused by GLT‑1 overexpression were reversed by NMDA. Collectively, our findings suggest that propofol treatment protects hippocampal neurons against IBI by enhancing GLT‑1 expression and inhibiting the activation of NMDAR via the JNK/Akt signaling pathway.

Morphology, cell viability, karyotype, expression of surface markers and plasticity of three human primary cell line cultures before and after the cryostorage in LN2 and GN2.

PubMed

Del Pino, Alberto; Ligero, Gertrudis; López, María B; Navarro, Héctor; Carrillo, Jose A; Pantoll, Siobhan C; Díaz de la Guardia, Rafael

2015-02-01

Primary cell line cultures from human skin biopsies, adipose tissue and tumor tissue are valuable samples for research and therapy. In this regard, their derivation, culture, storage, transport and thawing are important steps to be studied. Towards this end, we wanted to establish the derivation, and identify the culture characteristics and the loss of viability of three human primary cell line cultures (human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs), and primary culture of tumor cells from lung adenocarcinoma (PCTCLA)). Compared to fresh hADFs, hMSCs and PCTCLA, thawed cells stored in a cryogenic Dewar tanks with liquid nitrogen (LN2), displayed 98.20% ± 0.99, 95.40% ± 1.41 and 93.31% ± 3.83 of cell viability, respectively. Thawed cells stored in a Dry Vapor Shipper container with gas phase (GN2), for 20 days, in addition displayed 4.61% ± 2.78, 3.70% ± 4.09 and 9.13% ± 3.51 of average loss of cells viability, respectively, showing strong correlation between the loss of viability in hADFs and the number of post-freezing days in the Dry Vapor Shipper. No significant changes in morphological characteristics or in the expression of surface markers (being hADFs, hMSCs and PCTCLA characterized by positive markers CD73+; CD90+; CD105+; and negative markers CD14-; CD20-; CD34-; and CD45-; n=2) were found. Chromosome abnormalities in the karyotype were not found. In addition, under the right conditions hMSCs were differentiated into adipogenic, osteogenic and chondrogenic lineages in vitro. In this paper, we have shown the characteristics of three human primary cell line cultures when they are stored in LN2 and GN2. Copyright © 2014 Elsevier Inc. All rights reserved.

Alternative therapeutic approach to renal-cell carcinoma: induction of apoptosis with combination of vitamin K3 and D-fraction.

PubMed

Degen, Michael; Alexander, Bobby; Choudhury, Muhammad; Eshghi, Majid; Konno, Sensuke

2013-12-01

Because of a dismal prognosis for advanced renal-cell carcinoma (RCC), an alternative therapeutic approach, using vitamin K3 (VK3) and D-fraction (DF) was investigated. VK3 is a synthetic VK derivative and DF is a bioactive mushroom extract, and they have been shown to have antitumor activity. We examined if the combination of VK3 and DF would exhibit the improved anticancer effect on RCC in vitro. Human RCC, ACHN cell line, were treated with varying concentrations of VK3, DF, or a combination of the two. Cell viability was assessed at 72 hours by MTT assay. To explore the possible anticancer mechanism, studies on cell cycle, chromatin modifications, and apoptosis were conducted. VK3 alone led to a ~20% reduction in cell viability at 4 μM, while DF alone induced a 20% to 45% viability reduction at ≥ 500 μg/mL. A combination of VK3 (4 μM) and DF (300 μg/mL) led to a drastic >90% viability reduction, however. Cell cycle analysis indicated that VK3/DF treatment induced a G1 cell cycle arrest, accompanied by the up-regulation of p21(WAF1) and p27(Kip1). Histone deacetylase (HDAC) was also significantly (~60%) inactivated, indicating chromatin modifications. In addition, Western blot analysis revealed that the up-regulation of Bax and activation of poly-(ADP-ribose)-polymerase (PARP) were seen in VK3/DF-treated cells, indicating induction of apoptosis. The combination of VK3 and DF can lead to a profound reduction in ACHN cell viability, through a p21(WAF1)-mediated G1 cell cycle arrest, and ultimately induces apoptosis. Therefore, the combination of VK3/DF may have clinical implications as an alternative, improved therapeutic modality for advanced RCC.

Long Noncoding RNA H19 Inhibits Cell Viability, Migration, and Invasion Via Downregulation of IRS-1 in Thyroid Cancer Cells

PubMed Central

Wang, Peng; Xu, Weimin; Liu, Haixia; Bu, Qingao; Sun, Diwen

2017-01-01

Thyroid cancer is a common endocrine gland malignancy which exhibited rapid increased incidence worldwide in recent decades. This study was aimed to investigate the role of long noncoding RNA H19 in thyroid cancer. Long noncoding RNA H19 was overexpressed or knockdown in thyroid cancer cells SW579 and TPC-1, and the expression of long noncoding RNA H19 was detected by real-time polymerase chain reaction. The cell viability, migration, and invasion were determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay, Transwell assay, and wound healing assay, respectively. Furthermore, cell apoptosis was analyzed by flow cytometry, and expressions of some factors that were related to phosphatidyl inositide 3-kinases/protein kinase B and nuclear factor κB signal pathway were measured by Western blotting. This study revealed that cell viability and migration/invasion of SW579 and TPC-1 were significantly decreased by long noncoding RNA H19 overexpression compared with the control group (P < .05), whereas cell apoptosis was statistically increased (P < .001). Meanwhile, cell viability and migration/invasion were significantly increased after long noncoding RNA H19 knockdown (P < .05). Furthermore, long noncoding RNA H19 negatively regulated the expression of insulin receptor substrate 1 and thus effect on cell proliferation and apoptosis. Insulin receptor substrate 1 regulated the activation of phosphatidyl inositide 3-kinases/AKT and nuclear factor κB signal pathways. In conclusion, long noncoding RNA H19 could suppress cell viability, migration, and invasion via downregulation of insulin receptor substrate 1 in SW579 and TPC-1 cells. These results suggested the important role of long noncoding RNA H19 in thyroid cancer, and long noncoding RNA H19 might be a potential target of thyroid cancer treatment. PMID:29332545

In Vitro Model for Predicting the Protective Effect of Ultraviolet-Blocking Contact Lens in Human Corneal Epithelial Cells.

PubMed

Abengózar-Vela, Antonio; Arroyo, Cristina; Reinoso, Roberto; Enríquez-de-Salamanca, Amalia; Corell, Alfredo; González-García, María Jesús

2015-01-01

To develop an in vitro method to determine the protective effect of UV-blocking contact lenses (CLs) in human corneal epithelial (HCE) cells exposed to UV-B radiation. SV-40-transformed HCE cells were covered with non-UV-blocking CL, UV-blocking CL or not covered, and exposed to UV-B radiation. As control, HCE cells were covered with both types of CLs or not covered, but not exposed to UV-B radiation. Cell viability at 24, 48 and 72 h, after UV-B exposure and removing CLs, was determined by alamarBlue(®) assay. Percentage of live, dead and apoptotic cells was also assessed by flow cytometry after 24 h of UV-B exposure. Intracellular reactive oxygen species (ROS) production after 1 h of exposure was assessed using the dye H(2)DCF-DA. Cell viability significantly decreased, apoptotic cells and intracellular ROS production significantly increased when UVB-exposed cells were covered with non-UV-blocking CL or not covered compared to non-irradiated cells. When cells were covered with UV-blocking CL, cell viability significantly increased and apoptotic cells and intracellular ROS production did not increase compared to exposed cells. UV-B radiation induces cell death by apoptosis, increases ROS production and decreases viable cells. UV-blocking CL is able to avoid these effects increasing cell viability and protecting HCE cells from apoptosis and ROS production induced by UV-B radiation. This in vitro model is an alternative to in vivo methods to determine the protective effect of UV-blocking ophthalmic biomaterials because it is a quicker, cheaper and reliable model that avoids the use of animals.

Suivi in situ de cultures tridimensionnelles en bioreacteur a perfusion grace a la tomographie d'emission par positrons

NASA Astrophysics Data System (ADS)

Chouinard, Julie

The continuous assessment of developing tissue substitutes is crucial to understand their evolution over time. However, this represents quite a challenge when thick samples must be evaluated with standard microscopy techniques. Common characterization methods are time consuming and usually result in the destruction of the culture. Real-time, in situ, non-invasive and non-destructives methods are needed to monitor the growth of large non-transparent constructs in tissue engineering. Medical imaging modalities, which can provide information on the structure and function of internal organs and tissues in living organisms, have the potential of allowing repetitive monitoring of these 3D cultures in vitro. The working hypothesis of this thesis was to establish standard noninvasive and nondestructive real-time bioreactor imaging protocols for in situ monitoring of the viability and metabolism of endothelial cells when grown in perfused 3D fibrin gel scaffolds. To achieve this goal, a culture chamber with hollow fibers was designed and a pulsatile perfusion bioreactor system, able to promote cell survival and proliferation, was constructed and validated. Standard imaging protocols in Positron Emission Tomography (PET) are not adapted to image bioreactor systems. A suitable method had to be devised using the well-known radiotracer 18F-fluorodeoxyglucose ( 18FDG), a marker of glucose metabolism. Optimal uptake conditions were determined using cell monolayers and the best parameters were then applied on perfused 3D cultures to evaluate perfusion, cell viability and emerging cell structures. After only 12 hours of culture, the cell density could be estimated and cell structures were localized within the fibrin gels after 1-2 weeks of culture. PET is a promising tool for tissue engineering with many specific tracers available that might eventually be able to reveal new information on tissue development. Key words: Endothelial cells, Perfusion bioreactor, Positron Emission Tomography (PET), 18F-fluorodeoxyglucose ( 18FDG), Tissue Engineering, 3D cultures, Fibrin.

Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

PubMed Central

Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

2015-01-01

Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

Human Cytomegalovirus-Encoded miR-US25-1 Aggravates the Oxidised Low Density Lipoprotein-Induced Apoptosis of Endothelial Cells

PubMed Central

Fan, Jianmin; Zhang, Wen; Liu, Qiming

2014-01-01

Human cytomegalovirus (HCMV) infection is linked to the development and severity of the cardiovascular disease atherosclerosis; however, there is little known about the promotion of atherosclerosis. miR-US25-1 is one of HCMV-encoded miRNAs and targets cellular genes that are essential for virus growth to control the life cycle of the virus and host cells. The prominent regulation on cell cycle genes of the miR-US25-1 attracts us to explore its role in the atherosclerosis promotion. It was indicated that miR-US25-1 level was upregulated in subjects or in endothelial cells with HCMV infection; and the miR-US25-1 downregulated the expression of BRCC 3 by targeting the 5′ UTR of BRCC 3. And a miR-US25-1 mimics transfection could reduce the EAhy926 cell viability but did not induce apoptosis in EAhy926 cells. And what is more, miR-US25-1 mimicis transfection deteriorated the ox-LDL-induced apoptosis and aggravated the upregulation of apoptosis-associated molecules by oxidised low density lipoprotein (ox-LDL) in EAhy926 cells. And we have also confirmed the deregulation of BRCC 3 expression by miR-US25-1 by targeting the 5′ UTR of it. Given the vital role of BRCC 3 in DNA damage repairing, we speculated that the targeting inhibition of BRCC 3 by miR-US25-1 may contribute to the aggravation of ox-LDL-promoted apoptosis of endothelial EAhy926 cells. PMID:24895586

Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells

PubMed Central

Colafarina, Sabrina; Aruffo, Eleonora; Zarivi, Osvaldo; Bonfigli, Antonella; Di Bucchianico, Sebastiano; Di Carlo, Piero

2017-01-01

Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn’t differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication. PMID:28886142

The expression and role of serotonin receptor 5HTR2A in canine osteoblasts and an osteosarcoma cell line.

PubMed

Bracha, Shay; Viall, Austin; Goodall, Cheri; Stang, Bernadette; Ruaux, Craig; Seguin, Bernard; Chappell, Patrick E

2013-12-12

The significance of the serotonergic system in bone physiology and, more specifically, the importance of the five hydroxytryptamine receptor 2A (5HTR2A) in normal osteoblast proliferation have been previously described; however the role of serotonin in osteosarcoma remains unclear. Particularly, the expression and function of 5HTR2A in canine osteosarcoma has not yet been studied, thus we sought to determine if this indoleamine modulates cellular proliferation in vitro. Using real time quantitative reverse transcription PCR and immunoblot analyses, we explored receptor expression and signaling differences between non-neoplastic canine osteoblasts (CnOb) and an osteosarcoma cell line (COS). To elucidate specific serotonergic signaling pathways triggered by 5HTR2A, we performed immunoblots for ERK and CREB. Finally, we compared cell viability and the induction of apoptosis in the presence 5HTR2A agonists and antagonists. 5HTR2A was overexpressed in the malignant cell line in comparison to normal cells. In CnOb cells, ERK phosphorylation (ERK-P) decreased in response to both serotonin and a specific 5HTR2A antagonist, ritanserin. In contrast, ERK-P abundance increased in COS cells following either treatment. While endogenous CREB was undetectable in CnOb, CREB was observed constitutively in COS, with expression and exhibited increased CREB phosphorylation following escalating concentrations of ritanserin. To determine the influence of 5HTR2A signaling on cell viability we challenged cells with ritanserin and serotonin. Our findings confirmed that serotonin treatment promoted cell viability in malignant cells but not in normal osteoblasts. Conversely, ritanserin reduced cell viability in both the normal and osteosarcoma cells. Further, ritanserin induced apoptosis in COS at the same concentrations associated with decreased cell viability. These findings confirm the existence of a functional 5HTR2A in a canine osteosarcoma cell line. Results indicate that intracellular second messenger signal coupling of 5HTR2A is different between normal and malignant cells, warranting further research to investigate its potential as a novel therapeutic target for canine osteosarcoma.

Viability and biomass of Micrococcus luteus DE2008 at different salinity concentrations determined by specific fluorochromes and CLSM-image analysis.

PubMed

Puyen, Zully M; Villagrasa, Eduard; Maldonado, Juan; Esteve, Isabel; Solé, Antonio

2012-01-01

In previous studies, our group developed a method based on Confocal Laser Scanning Microscopy and Image Analysis (CLSM-IA) to analyze the diversity and biomass of cyanobacteria in microbial mats. However, this method cannot be applied to heterotrophic microorganisms, as these do not have autofluorescence. In this article, we present a method that combines CLSM-IA and Hoechst 33342 and SYTOX Green fluorochromes (FLU-CLSM-IA) to determine the viability and biomass of Micrococcus luteus DE2008, isolated from a saline microbial mat (Ebro Delta, Tarragona, Spain). The method has been applied to assess the effect of salinity on this microorganism. A reduction in viability and biomass (live cells) was observed as the salt concentration increases. The largest effect was at 100‰ NaCl with a cell death of 27.25% and a decrease in total and individual biomass of 39.75 and 0.009 mgC/cm(3), respectively, both with respect to optimal growth (10 ‰ NaCl). On the other hand, another important contribution of this article was that combining the FLU-CLSM-IA results with those achieved by plate counts enabled us to determine, for first time, the viability and the total biomass of the "dormant cells" (66.75% of viability and 40.59 mgC/cm(3) of total biomass at 100‰ NaCl). FLU-CLSM-IA is an efficient, fast, and reliable method for making a total count of cells at pixel level, including the dormant cells, to evaluate the viability and the biomass of a hetetrophic microorganism, M. luteus DE2008.

Novel in vivo flow cytometry platform for early prognosis of metastatic activity of circulating tumor cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

Nolan, Jacqueline; Cai, Chenzhoung; Nedosekin, Dmitry A.; Zharov, Vladimir P.

2017-02-01

Approximately 8 million people lose their lives due to cancer each year. Metastatic disease is responsible for 90% of those cancer-related deaths. Only viable circulating tumor cells (CTCs) that can survive in the blood circulation can create secondary tumors. Thus, real-time enumeration of CTCs and assessment of their viability in vivo has great biological significance. However, little progress has been made in this field. Conventional flow cytometry is the current technique being used for the assessment of cell viability, but there are many limitations to this technique: 1) cell properties may be altered during the extraction and processing method; 2) collection of cells from blood prevents the long-term study of individual cells in their natural biological environment; and 3) there are time-consuming preparation procedures. Whether it be for the assessment of antitumor drugs, where induction of apoptosis or necrosis is the preferred event, or the identification of nanoparticle-induced toxicity during nanotherapeutic treatment, it is clear that new approaches for assessment of the viability circulating blood cells and CTCs are urgently needed. We have developed a novel high speed, multicolor in vivo flow cytometry (FC) platform that integrates photoacoustic (PA) and fluorescence FC (PAFFC) and demonstrate its ability to enumerate rare circulating normal and abnormal (e.g. tumor) cells and assess their viability (e.g. apoptotic and necrotic) in a mouse model.

Effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 osteoblast-like cells.

PubMed

Torshabi, Maryam; Esfahrood, Zeinab Rezaei; Gholamin, Parisan; Karami, Elahe

2016-11-01

Evidence shows that oxidative stress induced by nicotine plays an important role in bone loss. Vitamin E with its antioxidative properties may be able to reverse the effects of nicotine on bone. This study aimed to assess the effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 (osteosarcoma) human osteoblast-like cells. We treated the cells with 5 mM nicotine. The viability and morphology of cells were evaluated respectively using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) and crystal violet assays. The effect of nicotine on osteogenic gene expression in MG-63 cells was assessed by real-time reverse-transcription polymerase chain reaction of osteoblast markers, namely, alkaline phosphatase, osteocalcin and bone sialoprotein. The results revealed that survival and proliferation of MG-63 cells were suppressed following exposure to nicotine, and cytoplasm vacuolization occurred in the cells. Nicotine significantly down-regulated the expression of osteogenic marker genes. Such adverse effects on morphology, viability and osteogenic gene expression of MG-63 cells were reversed by vitamin E therapy. In conclusion, vitamin E supplementation may play a role in proliferation and differentiation of osteoblasts, and vitamin E can be considered as an anabolic agent to treat nicotine-induced bone loss.

In vitro evidence of glucose-induced toxicity in GnRH secreting neurons: high glucose concentrations influence GnRH secretion, impair cell viability, and induce apoptosis in the GT1-1 neuronal cell line.

PubMed

Pal, Lubna; Chu, Hsiao-Pai; Shu, Jun; Topalli, Ilir; Santoro, Nanette; Karkanias, George

2007-10-01

To evaluate for direct toxic effects of high glucose concentrations on cellular physiology in GnRH secreting immortalized GT1-1 neurons. Prospective experimental design. In vitro experimental model using a cell culture system. GT1-1 cells were cultured in replicates in media with two different glucose concentrations (450 mg/dL and 100 mg/dL, respectively) for varying time intervals (24, 48, and 72 hours). Effects of glucose concentrations on GnRH secretion by the GT1-1 neurons were evaluated using a static culture model. Cell viability, cellular apoptosis, and cell cycle events in GT1-1 neurons maintained in two different glucose concentrations were assessed by flow cytometry (fluorescence-activated cell sorter) using Annexin V-PI staining. Adverse influences of high glucose concentrations on GnRH secretion and cell viability were noted in cultures maintained in high glucose concentration (450 mg/dL) culture medium for varying time intervals. A significantly higher percentage of cells maintained in high glucose concentration medium demonstrated evidence of apoptosis by a fluorescence-activated cell sorter. We provide in vitro evidence of glucose-induced cellular toxicity in GnRH secreting GT1-1 neurons. Significant alterations in GnRH secretion, reduced cell viability, and a higher percentage of apoptotic cells were observed in GT1-1 cells maintained in high (450 mg/dL) compared with low (100 mg/dL) glucose concentration culture medium.

Effect of pore architecture on oxygen diffusion in 3D scaffolds for tissue engineering.

PubMed

Ahn, Geunseon; Park, Jeong Hun; Kang, Taeyun; Lee, Jin Woo; Kang, Hyun-Wook; Cho, Dong-Woo

2010-10-01

The aim of this study was to maximize oxygen diffusion within a three-dimensional scaffold in order to improve cell viability and proliferation. To evaluate the effect of pore architecture on oxygen diffusion, we designed a regular channel shape with uniform diameter, referred to as cylinder shaped, and a new channel shape with a channel diameter gradient, referred to as cone shaped. A numerical analysis predicted higher oxygen concentration in the cone-shaped channels than in the cylinder-shaped channels, throughout the scaffold. To confirm these numerical results, we examined cell proliferation and viability in 2D constructs and 3D scaffolds. Cell culture experiments revealed that cell proliferation and viability were superior in the constructs and scaffolds with cone-shaped channels.

Fibrin hydrogels to deliver dental stem cells of the apical papilla for regenerative medicine.

PubMed

Germain, Loïc; De Berdt, Pauline; Vanacker, Julie; Leprince, Julian; Diogenes, Anibal; Jacobs, Damien; Vandermeulen, Gaëlle; Bouzin, Caroline; Préat, Véronique; Dupont-Gillain, Christine; des Rieux, Anne

2015-01-01

Evaluation of survival, proliferation and neurodifferentiation of dental stem cells from the apical papilla (SCAP) in fibrin hydrogels. We hypothesized that fibrin composition will influence cell behavior. Modulus, pore and fiber size were measured. SCAP in vitro viability, proliferation and neural differentiation, as well as in vivo proliferation and angiogenesis were studied. Hydrogel moduli were influenced by fibrin formulation but not hydrogel morphology, SCAP in vitro viability and proliferation. In total 60% of SCAP expressed PanNeurofilament in vitro without induction in Fibrinogen50-Thrombin10. SCAP proliferated when implanted in vivo and stimulated host endothelial cell infiltration. Fibrinogen30-Thrombin10 or Thrombin50 would be more favorable to in vitro SCAP viability and in vivo proliferation, while Fibrinogen 50-Thrombin50 would be more adapted to neurodifferentiation.

The effect of gestational diabetes on proliferation capacity and viability of human umbilical cord-derived stromal cells.

PubMed

Wajid, Nadia; Naseem, Rashida; Anwar, Sanam Saiqa; Awan, Sana Javaid; Ali, Muhammad; Javed, Sara; Ali, Fatima

2015-09-01

Stomal cells derived from Wharton's jelly of human umbilical cord (WJMSCs) are considered as the potential therapeutic agents for regeneration and are getting famous for stem cell banking. Our study aims to evaluate the effects of gestational diabetes on proliferation capacity and viability of WJMSCs. Mesenchymal stromal cells were isolated from Wharton's jelly of human umbilical cords from normal and gestational diabetic (DWJMSCs) mothers. Growth patterns of both types of cells were analyzed through MTT assay and population doubling time. Cell survival, cell death and glucose utilization were estimated through trypan blue exclusion assay, LDH assay and glucose detection assay respectively. Angiogenic ability was evaluated by immunocytochemistry and ELISA for VEGF A. Anti-cancerous potential was analyzed on HeLa cells. DWJMSCs exhibited low proliferative rate, increased population doubling time, reduced cell viability and increased cell death. Interestingly, DWJMSCs were found to have a reduced glucose utilization and anti-cancerous ability while enhanced angiogenic ability. Gestational diabetes induces adverse effects on growth, angiogenic and anti-cancerous potential of WJMSCs.

Electric-field driven assembly of live bacterial cell microarrays for rapid phenotypic assessment and cell viability testing.

PubMed

Goel, Meenal; Verma, Abhishek; Gupta, Shalini

2018-07-15

Microarray technology to isolate living cells using external fields is a facile way to do phenotypic analysis at the cellular level. We have used alternating current dielectrophoresis (AC-DEP) to drive the assembly of live pathogenic Salmonella typhi (S.typhi) and Escherichia coli (E.coli) bacteria into miniaturized single cell microarrays. The effects of voltage and frequency were optimized to identify the conditions for maximum cell capture which gave an entrapment efficiency of 90% in 60 min. The chip was used for calibration-free estimation of cellular loads in binary mixtures and further applied for rapid and enhanced testing of cell viability in the presence of drug via impedance spectroscopy. Our results using a model antimicrobial sushi peptide showed that the cell viability could be tested down to 5 μg/mL drug concentration under an hour, thus establishing the utility of our system for ultrafast and sensitive detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Multimodality molecular imaging and extracellular vesicle release based genetic profiling with porphyrin nanodroplets (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zemp, Roger J.; Paproski, Robert J.

2017-03-01

For emerging tissue-engineering applications, transplants, and cell-based therapies it is important to assess cell viability and function in vivo in deep tissues. Bioluminescence and fluorescence methods are poorly suited to deep monitoring applications with high resolution and require genetically-engineered reporters which are not always feasible. We report on a method for imaging cell viability using deep, high-resolution photoacoustic imaging. We use an exogenous dye, Resazurin, itself weakly fluorescent until it is reduced from blue to a pink color with bright red fluorescence. Upon cell death fluorescence is lost and an absorption shift is observed. The irreversible reaction of resazurin to resorufin is proportional to aerobic respiration. We detect colorimetric absorption shifts using multispectral photoacoustic imaging and quantify the fraction of viable cells. SKOV-3 cells with and without ±80oC heat treatment were imaged after Resazurin treatment. High 575nm:620nm ratiometric absorption and photoacoustic signals in viable cells were observed with a much lower ratio in low-viability populations.

Cell viability viscoelastic measurement in a rheometer used to stress and engineer tissues at low sonic frequencies.

PubMed

Klemuk, Sarah A; Jaiswal, Sanyukta; Titze, Ingo R

2008-10-01

Effects of vibration on human vocal fold extracellular matrix composition and the resultant tissue viscoelastic properties are difficult to study in vivo. Therefore, an in vitro bioreactor, simulating the in vivo physiological environment, was explored. A stress-controlled commercial rheometer was used to administer shear vibrations to living tissues at stresses and frequencies corresponding to male phonation, while simultaneously measuring tissue viscoelastic properties. Tissue environment was evaluated and adjustments made in order to sustain cell life for short term experimentation up to 6 h. Cell nutrient medium evaporation, osmolality, pH, and cell viability of cells cultured in three-dimensional synthetic scaffolds were quantified under comparably challenging environments to the rheometer bioreactor for 4 or 6 h. The functionality of the rheometer bioreactor was demonstrated by applying three vibration regimes to cell-seeded three-dimensional substrates for 2 h. Resulting strain was quantified throughout the test period. Rheologic data and cell viability are reported for each condition, and future improvements are discussed.

Cell viability study of thermo-responsive core-shell superparamagnetic nanoparticles for multimodal cancer therapy

NASA Astrophysics Data System (ADS)

Shah, Saqlain A.; Majeed, A.; Shafique, M. A.; Rashid, K.; Awan, Saif-Ullah

2014-02-01

This is a vital extension of our previously published work. Thermo-responsive copolymer coated superparamagnetic MnFe2O4 nanoparticles are tested for cell viability and affinity on HeLa carcinoma cells under different conditions. Nanoparticles were loaded with anticancer drug doxorubicin. Composite nanoparticles of average diameter 45 nm were of core-shell structure having magnetic core of about 18 nm. Magnetic hyperthermia effects on cell viability and drug delivery were studied by exposing the cell suspension to high frequency magnetic field, and living cells were quantified using MTT method. There was almost absence of drug release at 37 °C. Drug was released at temperatures above lower critical solution temperature (LCST) by magnetic heating. LCST of the thermo-responsive copolymer was observed to be around 39 °C. Below this temperature, copolymer was hydrophilic and swelled. But above LCST, copolymer could become hydrophobic, expel water and drug and shrink in volume. Combination of hyperthermia and drug delivery effectively treated cancer cells.

Involvement of polyubiquitin chains via specific chain linkages in stress response in mammalian cells.

PubMed

Fujimuro, Masahiro; Nishiya, Tadashi; Nomura, Yasuyuki; Yokosawa, Hideyoshi

2005-12-01

Polyubiquitination plays key roles in various proteasome-dependent and independent cellular events. To elucidate roles in stress response of polyubiquitin chains formed via specific chain linkages in mammalian cells, we established NIH3T3 stable cell lines that are capable of conditionally expressing K29R, K48R and K63R ubiquitin mutants, in which the Lys29, Lys48 and Lys63 residues of ubiquitin had been changed to Arg, and we examined the effects of various stresses on their cell viabilities. The expression of K63R ubiquitin mutant decreased viability of the cells post-exposed to ethanol, H(2)O(2) and methyl methanesulfonate (MMS), while that of K48R mutant decreased viability of the cells post-exposed to heat shock as well as ethanol, H(2)O(2) and MMS. Thus, these results suggest that polyubiquitin chains formed via specific chain linkages are involved in the respective stress responses in mammalian cells.

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsurumi, Chizuko; Firat, Elke; Gaedicke, Simone

2009-09-04

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPIImore » expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.« less

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts.

PubMed

Tsurumi, Chizuko; Firat, Elke; Gaedicke, Simone; Huai, Jisen; Mandal, Pankaj Kumar; Niedermann, Gabriele

2009-09-04

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.

Effects of short-chain chlorinated paraffins exposure on the viability and metabolism of human hepatoma HepG2 cells.

PubMed

Geng, Ningbo; Zhang, Haijun; Zhang, Baoqin; Wu, Ping; Wang, Feidi; Yu, Zhengkun; Chen, Jiping

2015-03-03

Short-chain chlorinated paraffins (SCCPs) have attracted considerable attention for their characteristic of persistent organic pollutants. However, very limited information is available for their toxic effects at environmentally relevant doses, limiting the evaluation of their health risks. In this study, cell viability assay and targeted metabolomic approach was used to evaluate the environmental dose (<100 μg/L) effect of SCCPs on HepG2 cells. Cell viability was found to be decreased with increases in exposure dose of SCCPs. Exposure for 48 h to C10-CPs resulted in a significant reduction in cell viability compared with 24 h, even at 1 μg/L. SCCPs exposure altered the intracellular redox status and caused significant metabolic disruptions. As a kind of peroxisome proliferator, SCCPs specifically stimulated the β-oxidation of unsaturated fatty acids and long-chain fatty acids. Meanwhile, SCCPs exposure disturbed glycolysis and amino acid metabolism, and led to the up-regulation of glutamate metabolism and urea cycle. The toxic effects of SCCPs might mainly involve the perturbation of energy production, protein biosynthesis, fatty acid metabolism, and ammonia recycling.

Osthole induces apoptosis, suppresses cell-cycle progression and proliferation of cancer cells.

PubMed

Jarząb, Agata; Grabarska, Aneta; Kiełbus, Michał; Jeleniewicz, Witold; Dmoszyńska-Graniczka, Magdalena; Skalicka-Woźniak, Krystyna; Sieniawska, Elwira; Polberg, Krzysztof; Stepulak, Andrzej

2014-11-01

The aim of the present study was to determine the effects of osthole on cell proliferation and viability, cell-cycle progression and induction of apoptosis in human laryngeal cancer RK33 and human medulloblastoma TE671 cell lines. Cell viability was measured by means of the MTT method and cell proliferation by the 5-bromo-2-deoxyuridine (BrdU) incorporation assay. Cell-cycle progression was determined by flow cytometry, and induction of apoptosis by release of oligonucleosomes to the cytosol. The gene expression was estimated by a quantitative polymerase chain reaction (qPCR) method. High-performance counter-current chromatography (HPCCC) was applied for isolation of osthole from fruits of Mutellina purpurea. Osthole decreased proliferation and cell viability of cancer cells in a dose-dependent manner. The tested compound induced apoptosis, increased the cell numbers in G1 and decreased cell number in S/G2 phases of the cell cycle, differentially regulating CDKN1A and TP53 gene expression depending on cancer cell type. Osthole could be considered as a potential compound for cancer therapy and chemoprevention. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.