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.

Essential oils--their antimicrobial activity against Escherichia coli and effect on intestinal cell viability.

PubMed

Fabian, Dusan; Dusan, Fabian; Sabol, Marián; Marián, Sabol; Domaracká, Katarína; Katarína, Domaracká; Bujnáková, Dobroslava; Dobroslava, Bujnáková

2006-12-01

Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria. The main objective of this study was to evaluate possible harmful effects of four commonly used essential oils and their major components on intestinal cells. Antimicrobial activity of selected plant extracts against enteroinvasive Escherichia coli was dose dependent. However, doses of essential oils with the ability to completely inhibit bacterial growth (0.05%) showed also relatively high cytotoxicity to intestinal-like cells cultured in vitro. Lower doses of essential oils (0.01%) had only partial antimicrobial activity and their damaging effect on Caco-2 cells was only modest. Cell death assessment based on morphological and viability staining followed by fluorescence microscopy showed that essential oils of cinnamon and clove and their major component eugenol had almost no cytotoxic effect at lower doses. Although essential oil of oregano and its component carvacrol slightly increased the incidence of apoptotic cell death, they showed extensive antimicrobial activity even at lower concentrations. Relatively high cytotoxicity was demonstrated by thyme oil, which increased both apoptotic and necrotic cell death incidence. In contrast, its component thymol showed no cytotoxic effect as well as greatly-reduced ability to inhibit visible growth of the chosen pathogen in the doses used. On the other hand, the addition of all essential oils and their components at lower doses, with the exception of thyme oil, to bacterial suspension significantly reduced the cytotoxic effect of E. coli on Caco-2 cells after 1h culture. In conclusion, it is possible to find appropriate doses of essential oils showing both antimicrobial activity and very low detrimental effect on intestinal cells.

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

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.

Cell viability test after laser guidance

NASA Astrophysics Data System (ADS)

Rosenbalm, Tabitha N.; Owens, Sarah; Bakken, Daniel; Gao, Bruce Z.

2006-02-01

To precisely control the position of multiple types of cells in a coculture for the study of cell-cell interactions, we have developed a laser micropatterning technique. The technique employs the optical forces generated by a weakly focused laser beam. In the beam's focal region, the optical force draws microparticles, such as cells, into the center of the beam, propels them along the beam axis, and guides them onto a target surface. Specific patterns are created through computercontrolled micromanipulation of the substrate relative to the laser beam. Preliminary data have demonstrated cell viability after laser guidance. This project was designed to systematically vary the controllable laser parameters, namely, intensity and exposure time of the laser on single cells, and thus determine the laser parameters that allow negligible cell damage with functional cellular position control. To accomplish this goal, embryonic day 7 (E7) chick forebrain neurons were cultured in 35 mm petri dishes. Control and test cells were selected one hour after cell placement to allow cell attachment. Test cells were subjected to the laser at the focal region. The experimental parameters were chosen as: wavelength - 800 nm, intensities - 100 mW, 200 mW, and 300 mW, and exposure times - 10 s and 60 s. Results were analyzed based on neurite outgrowth and the Live/Dead assay (Viability/Cytoxicity kit from Molecular Probes). No statistical difference (p >> 0.1, student t-test) in viability or function was found between the control neurons and those exposed to the laser. This confirms that laser guidance seems to be a promising method for cellular manipulation.

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.

Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

PubMed

Hansen, Gunda; Johansen, Claus Lindvald; Marten, Gunvor; Wilmes, Jacqueline; Jespersen, Lene; Arneborg, Nils

2016-07-01

In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points.

Redefining the effect of salt on thermophilic starter cell viability, culturability and metabolic activity in cheese.

PubMed

Hickey, C D; Fallico, V; Wilkinson, M G; Sheehan, J J

2018-02-01

This study investigated the differential effect of salt concentration in the outside and inside layers of brine salted cheeses on viability, culturability and enzyme activity of starter bacteria. The high-salt environment of the outside layer caused a sharp decrease in L. helveticus viability as measured by traditional plate counts. Remarkably, this was associated with lower release of intracellular enzymes (LDH), reduced levels of proteolysis and larger membrane integrity as measured by flow cytometry (FC) following classical Live/Dead staining. FC analysis of light scattering properties highlighted a significant reduction in size and granularity of the microbiota located in the cheese surface, suggestive of cell shrinkage and condensation of internal macromolecules probably due to hyperosmotic stress. The microbiota of the cheese surface were found to experience greater oxidative stress, as measured by FC analysis of the total levels of reactive oxygen species, compared to that of the interior layer. These results lead us to postulate that the physiology and health status of the microbiota were significantly different in the outer and inner layers of the cheese. The hyperosmotic environment of the outer layer resulted in reduced cell lysis, as measurable by assays based upon membrane integrity, but rather triggered cell death via mechanisms involving cell shrinkage and ROS-mediated damage of vital intracellular components. This study challenges the current thinking on how salt controls microbial activity in ripening cheese, especially in cheeses which are brine salted as local variations in biochemical ripening indices can differ significantly from the outside to the inside of a ripening cheese. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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.

Cell function and viability in glucose polymer peritoneal dialysis fluids.

PubMed

Liberek, T; Topley, N; Mistry, C D; Coles, G A; Morgan, T; Quirk, R A; Williams, J D

1993-01-01

To investigate the biocompatibility profile of a new peritoneal dialysis fluid containing glucose polymer (GPF). Viability and function of peripheral neutrophils (PMN) from healthy donors and cultured human peritoneal mesothelial cells were assessed in vitro after exposure to dialysis fluids. Phagocytosis, leukotriene B4 synthesis, and respiratory burst activation were measured following stimulation with serum-treated zymosan (STZ) or opsonized Staphylococcus epidermidis (S. epidermidis). Bacterial growth in the fluids was also investigated. In vivo pH equilibration of GPF and subsequent respiratory burst activation following incubation in spent dialysate were studied. For all the host defense parameters measured, commercial dialysis fluids (Dianeal; 1.36% and 3.86% glucose) and GPF (pH 5.2) were significantly more inhibitory than the control buffer (pH 7.3). Mesothelial cell viability was reduced by all the fluids tested irrespective of pH. Glucose polymer fluid was significantly more inhibitory than Dianeal 1.36% for STZ phagocytosis and respiratory burst activation. In contrast, it was less suppressive than Dianeal 3.86% for LTB4 synthesis. For all parameters tested, except LTB4 generation, there was a marked effect of pH, with GPF being significantly more inhibitory at pH 5.2 than at pH 7.3. None of the fluids tested supported the growth of S. epidermidis, although the viable counts in GFP were significantly higher than in Dianeal. Fluid inhibition of PMN respiratory burst activation and cytotoxicity were reduced in a time-dependent manner following increasing dwell time in vivo. GPF does not appear to be significantly different from Dianeal as far as host defense parameters are concerned. However, the cell viability and bacterial survival data suggest some possibly negative aspects of this fluid formation.

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

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

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

Hydroxycinnamic acid decarboxylase activity of Brettanomyces bruxellensis involved in volatile phenol production: relationship with cell viability.

PubMed

Laforgue, R; Lonvaud-Funel, A

2012-12-01

Brettanomyces bruxellensis populations have been correlated with an increase in phenolic off-flavors in wine. The volatile phenols causing the olfactory defect result from the successive decarboxylation and reduction of hydroxycinnamic acids that are normal components of red wines. The growth of B. bruxellensis is preventable by adding sulfur dioxide (SO(2)), with variable effectiveness. Moreover, it was hypothesized that SO(2) was responsible for the entry of B. bruxellensis into a viable but non-culturable (VBNC) state. The aim of this project was to investigate the effects of SO(2) on the remaining enzyme activities of B. bruxellensis populations according to their viability and cultivability, focusing on the hydroxycinnamate decarboxylase enzyme, the first enzyme needed, rather than the metabolites produced. Enzyme activity was determined both in cell-free extracts and resting cells after various SO(2) treatments in synthetic media. After slight sulfiting (around 50 mg/L total SO(2)), the yeasts had lost part of their enzyme activity but not their cultivability. At higher doses (at least 75 mg/L total SO(2)) the majority of yeasts had lost their cultivability but still retained part of their enzyme activity. These results suggested that non culturable cells retained some enzyme activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test--human cell line activation test (h-CLAT).

PubMed

Sakaguchi, Hitoshi; Ashikaga, Takao; Miyazawa, Masaaki; Kosaka, Nanae; Ito, Yuichi; Yoneyama, Katsurako; Sono, Sakiko; Itagaki, Hiroshi; Toyoda, Hidekazu; Suzuki, Hiroyuki

2009-04-01

Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20-95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65-90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of "CD86 > or = 150 or CD54 > or = 200" resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.

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.

Cell viability of Candida albicans against the antifungal activity of thymol.

PubMed

de Vasconcelos, Laís César; Sampaio, Fabio Correia; Albuquerque, Allan de Jesus dos Reis; Vasconcelos, Laurylene César de Souza

2014-01-01

Candida albicans is a commensal fungus, but circumstantially it may cause superficial infections of the mucous membranes, such as denture stomatitis, when a biofilm is formed on the surface of dental prostheses. This study evaluated the cell viability of C. albicans biofilms against the antifungal activity of thymol when compared with miconazole, by the fluorescence imaging using SYTO 9 and propidium iodide dyes, and counting of colony forming units. C. albicans standard strains (ATCC 11006) were used. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of drugs were determined by broth microdilution tests and the inoculum was standardized to match 0.5 on the McFarland scale (106 cfu/mL). Biofilms were grown on the surface of acrylic resin disks in parallel flow chambers from Sabouraud broth supplemented with 10% dextrose. For counting of colony forming units, the fungal solution was sequentially diluted and plated in Sabouraud dextrose agar. Data were analyzed using two-way ANOVA and Tukey's test (a=5%). Biofilms treated with thymol and miconazole presented low numbers of viable cells at the evaluated exposure times. There was statistically significant difference (p<0.05) when compared with control, and the mean value of the exposure times between miconazole and thymol did not differ significantly (p>0.05). In conclusion, both drugs have similar efficiency as antifungal agents against biofilms of C. albicans formed on acrylic surfaces.

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.

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.

Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability.

PubMed

Bukhari, Maurish; Burm, Hayley; Samways, Damien S K

2016-10-01

Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.

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.

New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma

PubMed Central

Maugg, Doris; Rothenaigner, Ina; Schorpp, Kenji; Potukuchi, Harish Kumar; Korsching, Eberhard; Baumhoer, Daniel; Hadian, Kamyar

2015-01-01

Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS. PMID:26039064

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.

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.

Innovative Microcapsules for Pancreatic β-Cells Harvested from Mature Double-Transgenic Mice: Cell Imaging, Viability, Induced Glucose-Stimulated Insulin Measurements and Proinflammatory Cytokines Analysis.

PubMed

Mooranian, Armin; Tackechi, Ryu; Jamieson, Emma; Morahan, Grant; Al-Salami, Hani

2017-06-01

Recently we demonstrated that microencapsulation of a murine pancreatic β-cell line using an alginate-ursodeoxycholic acid (UDCA) matrix produced microcapsules with good stability and cell viability. In this study, we investigated if translation of this formulation to microencapsulation of primary β-cells harvested from mature double-transgenic healthy mice would also generate stable microcapsules with good cell viability. Islets of Langerhans were isolated from Ngn3-GFP/RIP-DsRED mice by intraductal collagenase P digestion and density gradient centrifugation, dissociated into single cells and the β-cell population purified by Fluorescence Activated Cell Sorting. β-cells were microencapsulated using either alginate-poly-l-ornithine (F1; control) or alginate-poly-l-ornithine-UDCA (F2; test) formulations. Microcapsules were microscopically examined and microencapsulated cells were analyzed for viability, insulin and cytokine release, 2 days post-microencapsulation. Microcapsules showed good uniformity and morphological characteristics and even cell distribution within microcapsules with or without UDCA. Two days post microencapsulation cell viability, mitochondrial ATP and insulin production were shown to be optimized in the presence of UDCA whilst production of the proinflammatory cytokine IL-1β was reduced. Contradictory to our previous studies, UDCA did not reduce production of any other pro-inflammatory biomarkers. These results suggest that UDCA incorporation improves microcapsules' physical and morphological characteristics and improves the viability and function of encapsulated mature primary pancreatic β-cells.

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.

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.

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.

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.

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.

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

Effect of low-level laser irradiation on proliferation and viability of human dental pulp stem cells.

PubMed

Zaccara, Ivana Maria; Ginani, Fernanda; Mota-Filho, Haroldo Gurgel; Henriques, Águida Cristina Gomes; Barboza, Carlos Augusto Galvão

2015-12-01

A positive effect of low-level laser irradiation (LLLI) on the proliferation of some cell types has been observed, but little is known about its effect on dental pulp stem cells (DPSCs). The aim of this study was to identify the lowest energy density able to promote the proliferation of DPSCs and to maintain cell viability. Human DPSCs were isolated from two healthy third molars. In the third passage, the cells were irradiated or not (control) with an InGaAlP diode laser at 0 and 48 h using two different energy densities (0.5 and 1.0 J/cm²). Cell proliferation and viability and mitochondrial activity were evaluated at intervals of 24, 48, 72, and 96 h after the first laser application. Apoptosis- and cell cycle-related events were analyzed by flow cytometry. The group irradiated with an energy density of 1.0 J/cm² exhibited an increase of cell proliferation, with a statistically significant difference (p < 0.05) compared to the control group at 72 and 96 h. No significant changes in cell viability were observed throughout the experiment. The distribution of cells in the cell cycle phases was consistent with proliferating cells in all three groups. We concluded that LLLI, particularly a dose of 1.0 J/cm², contributed to the growth of DPSCs and maintenance of its viability. This fact indicates this therapy to be an important future tool for tissue engineering and regenerative medicine involving stem cells.

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.

Leptin-induced ER-α-positive breast cancer cell viability and migration is mediated by suppressing CCN5-signaling via activating JAK/AKT/STAT-pathway.

PubMed

Haque, Inamul; Ghosh, Arnab; Acup, Seth; Banerjee, Snigdha; Dhar, Kakali; Ray, Amitabha; Sarkar, Sandipto; Kambhampati, Suman; Banerjee, Sushanta K

2018-01-25

In menopausal women, one of the critical risk factors for breast cancer is obesity/adiposity. It is evident from various studies that leptin, a 16 kDa protein hormone overproduced in obese people, plays the critical role in neovascularization and tumorigenesis in breast and other organs. However, the mechanisms by which obesity influences the breast carcinogenesis remained unclear. In this study, by analyzing different estrogen receptor-α (ER-α)-positive and ER-α-negative BC cell lines, we defined the role of CCN5 in the leptin-mediated regulation of growth and invasive capacity. We analyzed the effect of leptin on cell viability of ER-α-positive MCF-7 and ZR-75-1 cell lines and ER-α-negative MDA-MB-231 cell line. Additionally, we also determined the effect of leptin on the epithelial-mesenchymal transition (EMT) bio-markers, in vitro invasion and sphere-formation of MCF-7 and ZR-75-1 cell lines. To understand the mechanism, we determined the impact of leptin on CCN5 expression and the functional role of CCN5 in these cells by the treatment of human recombinant CCN5 protein(hrCCN5). Moreover, we also determined the role of JAK-STAT and AKT in the regulation of leptin-induced suppression of CCN5 in BC cells. Present studies demonstrate that leptin can induce cell viability, EMT, sphere-forming ability and migration of MCF-7 and ZR-75-1 cell lines. Furthermore, these studies found that leptin suppresses the expression of CCN5 at the transcriptional level. Although the CCN5 suppression has no impact on the constitutive proliferation of MCF-7 and ZR-75-1 cells, it is critical for leptin-induced viability and necessary for EMT, induction of in vitro migration and sphere formation, as the hrCCN5 treatment significantly inhibits the leptin-induced viability, EMT, migration and sphere-forming ability of these cells. Mechanistically, CCN5-suppression by leptin is mediated via activating JAK/AKT/STAT-signaling pathways. These studies suggest that CCN5 serves as a

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.

Activin receptor-like kinase 5 inhibition reverses impairment of endothelial cell viability by endogenous islet mesenchymal stromal cells.

PubMed

Clarkin, Claire E; King, Aileen J; Dhadda, Paramjeet; Chagastelles, Pedro; Nardi, Nance; Wheeler-Jones, Caroline P; Jones, Peter M

2013-03-01

Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-β signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-β signaling by these cells influences islet EC viability. Cultured islets exhibited reduced expression of EC markers (VEGFR2, VE-cadherin and CD31), which was associated with diminished but sustained expression of endoglin a marker of both ECs and MSCs. Double fluorescent labeling of islets in situ with the EC marker CD31 disclosed a population of CD31-negative cells which were positive for endoglin. In vitro coculture of microvascular ECs with endoglin-positive, CD31-negative islet MSCs reduced VEGFR2 protein expression, disrupted EC angiogenic behavior, and increased EC detachment. Medium conditioned by islet MSCs significantly decreased EC viability and increased EC caspase 3/7 activity. EC:MSC cocultures showed enhanced Smad2 phosphorylation consistent with altered ALK5 signaling. Pharmacological inhibition of ALK5 activity with SB431542 (SB) improved EC survival upon contact with MSCs, and SB-treated cultured islets retained EC marker expression and sensitivity to exogenous VEGF164 . Thus, endoglin-expressing islet MSCs influence EC ALK5 signaling in vitro, which decreases EC viability, and changes in ALK5 activity in whole cultured islets contribute to islet EC loss. Modifying TGF-β signaling may enable maintenance of islet ECs during islet isolation and thus improve islet graft revascularization post-transplantation. Copyright © 2013 AlphaMed Press.

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.

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

PubMed Central

Johnson, M. Brittany; Criss, Alison K.

2013-01-01

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells. PMID:24056524

Chrysin Attenuates Cell Viability of Human Colorectal Cancer Cells through Autophagy Induction Unlike 5-Fluorouracil/Oxaliplatin.

PubMed

Lin, Yueh-Ming; Chen, Chih-I; Hsiang, Yi-Ping; Hsu, Yung-Chia; Cheng, Kung-Chuan; Chien, Pei-Hsuan; Pan, Hsiao-Lin; Lu, Chien-Chang; Chen, Yun-Ju

2018-06-14

Chemotherapeutic 5-fluorouracil (5-FU) combined with oxaliplatin is often used as the standard treatment for colorectal cancer (CRC). The disturbing side effects and drug resistance commonly observed in chemotherapy motivate us to develop alternative optimal therapeutic options for CRC treatment. Chrysin, a natural and biologically active flavonoid abundant in propolis, is reported to have antitumor effects on a few CRCs. However, whether and how chrysin achieves similar effectiveness to the 5-FU combination is not clear. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), western blotting, fluorescence microscopy, and reactive oxygen species (ROS) production were assayed. We found that chrysin exhibited similar inhibition of cell viability as the 5-FU combination in a panel of human CRC cells. Furthermore, the results showed that chrysin significantly increased the levels of LC3-II, an autophagy-related marker, in CRC cells, which was not observed with the 5-FU combination. More importantly, blockage of autophagy induction restored chrysin-attenuated CRC cell viability. Further mechanistic analysis revealed that chrysin, not the 5-FU combination, induced ROS generation, and in turn, inhibited the phosphorylation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Collectively, these results imply that chrysin may be a potential replacement for the 5-FU and oxaliplatin combination to achieve antitumor activity through autophagy for CRC treatment in the future.

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.

Monitoring of microbial cell viability using nanostructured electrodes modified with Graphene/Alumina nanocomposite.

PubMed

Hassan, Rabeay Y A; Mekawy, Moataz M; Ramnani, Pankaj; Mulchandani, Ashok

2017-05-15

Microbial infections are rapidly increasing; however most of the existing microbiological and molecular detection methods are time consuming and/or cannot differentiate between the viable and dead cells which may overestimate the risk of infections. Therefore, a bioelectrochemical sensing platform with a high potential to the microbial-electrode interactions was designed based on decorated graphene oxide (GO) sheet with alumina (Al 2 O 3 ) nanocrystals. GO-Al 2 O 3 nanocomposite was synthesized using self-assembly of GO and Al 2 O 3 and characterized using the scanning electron microscopy (SEM), transmission electron microscopy (TEM), x-ray diffraction (XRD), Raman-spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Enhancement of electrocatalytic activity of the composite-modified electrode was demonstrated. Thus, using the GO-Al 2 O 3 nanocomposite modified electrode, the cell viability was determined by monitoring the bioelectrochemical response of the living microbial cells (bacteria and yeast) upon stimulation with carbon source. The bioelectrochemical assay was optimized to obtain high sensitivity and the method was applied to monitor cell viability and screen susceptibility of metabolically active cells (E. coli, B. subtilis, Enterococcus, P. aeruginosa and Salmonella typhi) to antibiotics such as ampicillin and kanamycin. Therefore, the developed assay is suitable for cell proliferation and cytotoxicity testing. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Development, glycolytic activity, and viability of preimplantation mouse embryos subjected to different periods of glucose starvation.

PubMed

Leppens-Luisier, G; Sakkas, D

1997-03-01

After compaction, the preimplantation mouse embryo switches to a glucose-based metabolism, whereas for the 2- to 4-cell stage embryo, glucose can be inhibitory. In this study, we investigated the adaptability of preimplantation embryos to different periods of glucose starvation by culturing in vitro fertilized (IVF) and in vivo-fertilized 1-cell OF1 mouse embryos. Blastocysts obtained from exposure to glucose starvation for different periods of time were examined for the number of cells in the trophectoderm and inner cell mass, and for glycolytic activity and viability. A high percentage of blastocysts was obtained when 1-cell embryos fertilized in vitro or in vivo were cultured in M16 until the 2-cell stage, were transferred to M16 without glucose (M16-G) until the 4- or 8-cell stage, and then were transferred to fresh M16-G. When in vivo-fertilized 1-cell embryos were cultured to the 2-cell stage and then left in M16, less than 5% formed blastocysts compared to 26% of those transferred into M16-G. Blastocysts obtained when in vivo-fertilized 1-cell embryos were left in M16-G after the 2-cell stage, however, showed a significantly elevated glycolytic activity compared to those transferred to fresh M16 or M16-G medium at the 4- or 8-cell stage. Interestingly, even though these embryos displayed elevated glycolytic activity, they did not exhibit differences in the numbers of inner cell mass and trophectoderm cells or in viability compared to embryos cultured according to other protocols. Blastocysts from all cultured protocols had a significantly lower total cell number and a lower trophectoderm, but not inner cell mass, cell number compared to blastocysts developed in vivo. This study documents the metabolic adaptability of the preimplantation embryo by highlighting its ability to proceed with development and retain viability when challenged with glucose starvation at different periods.

Fluorescein Diacetate Microplate Assay in Cell Viability Detection.

PubMed

Chen, Xi; Yang, Xiu-Ying; Fang, Lian-Hua; DU, Guan-Hua

2016-12-20

Objective To investigate the application of the fluorescein diacetate (FDA) microplate assay in cell viability detection. Methods Cells were seeded in a 96-well culture plate until detection. After incubated with FDA,the plate was detected by fluorescence microplate analyzer. The effects of FDA incubation duration,concentration,and other factors on the assay's accuracy and stability were assessed. We also compared the results of FDA with methyl thiazolyl(MTT) in terms of cell numbers and H 2 O 2 injury. Results Within 0-30 minutes,the fluorescence-cell number coefficient of FDA assay increased with duration and reached 0.99 in 27-30 minutes. The optimum concentration of final FDA in this study was 10-30 μg/ml. On cell viability detection,the result of FDA method was equivalent to MTT method. As to H 2 O 2 injury assay,the sensitivity of FDA method was superior to MTT on the higher concentration H 2 O 2 treatment due to a relative shorter duration for detection. Conclusion As a stable and reliable method,FDA is feasible for cell variability detection under varied conditions.

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.

Dendritic Cells Promote Pancreatic Viability in Mice with Acute Pancreatitis

PubMed Central

Bedrosian, Andrea S.; Nguyen, Andrew H.; Hackman, Michael; Connolly, Michael K.; Malhotra, Ashim; Ibrahim, Junaid; Cieza-Rubio, Napoleon E.; Henning, Justin R.; Barilla, Rocky; Rehman, Adeel; Pachter, H. Leon; Medina-Zea, Marco V.; Cohen, Steven M.; Frey, Alan B.; Acehan, Devrim; Miller, George

2011-01-01

Background & Aims Acute pancreatitis increases morbidity and mortality from organ necrosis by mechanisms that are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis. Methods Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier analysis. Results Numbers of MHC II+CD11c+DC increased 100-fold in pancreas of mice with acute pancreatitis, to account for nearly 15% of intra-pancreatic leukocytes. Intra-pancreatic DC acquired an immune phenotype in mice with acute pancreatitis; they expressed higher levels of MHC II and CD86 and increased production of interleukin-6, membrane cofactor protein (MCP)-1, and tumor necrosis factor (TNF)-α. However, rather than inducing an organ-destructive inflammatory process, DC were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DC and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DC died from acinar cell death within 4 days. Depletion of DC from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DC did not require infiltrating neutrophils, activation of NF-κB, or signaling by mitogen-activated protein kinase or TNF-α. Conclusions DC are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress. PMID:21801698

Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells.

PubMed

Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung; Yoo, Eun-Sun; Chan Ra, Jeong; Ryu, Kyung-Ha

2012-06-22

Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-β in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-α, G-CSF, and TGF-β. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function. Copyright © 2012 Elsevier Inc. All rights reserved.

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

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.

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.

Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

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

Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah

Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cellsmore » by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.« less

Effect of Irrigation Time of Antiseptic Solutions on Bone Cell Viability and Growth Factor Release.

PubMed

Sawada, Kosaku; Nakahara, Ken; Haga-Tsujimura, Maiko; Fujioka-Kobayashi, Masako; Iizuka, Tateyuki; Miron, Richard J

2018-03-01

Antiseptic solutions are commonly utilized to treat local infection in the oral and maxillofacial region. However, surrounding vital bone is also exposed to antiseptic agents during irrigation and may have a potential negative impact on bone survival. The aim of the present study was therefore to investigate the effect of rinsing time with various antiseptic solutions on bone cell viability, as well as their subsequent release of growth factors important for bone regeneration. The bone samples collected from porcine mandible were rinsed in the following commonly utilized antiseptic solutions; povidone-iodine (0.5%), chlorhexidine digluconate (CHX, 0.2%), hydrogen peroxide (1%), and sodium hypochlorite (0.25%) for 1, 5, 10, 20, 30, or 60 minutes and assessed for cell viability and release of growth factors including vascular endothelial growth factor, transforming growth factor beta 1, bone morphogenetic protein 2, receptor activator of nuclear factor kappa-B ligand, and interleukin-1 beta by enzyme-linked immunosorbent assay. It was found in all the tested groups that the long exposure of any of the tested antiseptic solutions drastically promoted higher cell death. Sodium hypochlorite demonstrated the significantly highest cell death and at all time points. Interestingly, bone cell viability was highest in the CHX group post short-term rinsing of 1, 5, or 10 minutes when compared with the other 4 tested groups. A similar trend was also observed in subsequent growth factor release. The present study demonstrated that of the 4 tested antiseptic solutions, short-term CHX rinsing (ideally within 1 minute) favored bone cell viability and growth factor release. Clinical protocols should be adapted accordingly.

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.

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.

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.

Diesel Exhaust Particulate Extracts Inhibit Transcription of Nuclear Respiratory Factor-1 and Cell Viability in Human Umbilical Vein Endothelial Cells

PubMed Central

Mattingly, Kathleen A.; Klinge, Carolyn M.

2011-01-01

Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1 regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription and this suppression was not ablated by concomitant treatment with E2, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E2 increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. PMID:22105178

Antimony trichloride induces a loss of cell viability via reactive oxygen species-dependent autophagy in A549 cells.

PubMed

Zhao, Xinyuan; Xing, Fengjun; Cong, Yewen; Zhuang, Yin; Han, Muxi; Wu, Zhiqiang; Yu, Shali; Wei, Haiyan; Wang, Xiaoke; Chen, Gang

2017-12-01

Antimony (Sb) is one of the most prevalent heavy metals and frequently leads to biological toxicity. Although autophagy is believed to be involved in metal-associated cytotoxicity, there is no evidence of its involvement following exposure. Moreover, the underlying mechanism of autophagy remains unclear. In this study, treatment with antimony trichloride caused autophagy in a dose- and time-dependent manner in A549 cells but did not affect the level of Atg5 or Atg7 mRNA expression. Furthermore, Sb enhanced autophagic flux while upregulating p62 gene and protein levels. The classic mechanistic target of rapamycin (mTOR) pathway is not involved in Sb-induced autophagy. However, Sb-induced autophagy and the upregulation of p62 were inhibited by treatment with the antioxidant N-acetylcysteine (NAC). Subsequent analyses demonstrated that the inhibition of autophagy protected A549 cells from a loss of cell viability, while the activation of autophagy by rapamycin had the opposite effect. These data suggest that reactive oxygen species-dependent autophagy mediates Sb-stimulated cell viability loss in A549 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro Cell Viability by CellProfiler® Software as Equivalent to MTT Assay.

PubMed

Gasparini, Luciana S; Macedo, Nayana D; Pimentel, Elisângela F; Fronza, Marcio; Junior, Valdemar L; Borges, Warley S; Cole, Eduardo R; Andrade, Tadeu U; Endringer, Denise C; Lenz, Dominik

2017-07-01

This study evaluated in vitro cell viability by the colorimetric MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay compared to image analysis by CellProfiler ® software. Hepatoma (Hepa-1c1c7) and fibroblast (L929) cells were exposed to isolated substances, camptothecin, lycorine, tazettine, albomaculine, 3-epimacronine, trispheridine, galanthine and Padina gymnospora , Sargassum sp. methanolic extract, and Habranthus itaobinus Ravenna ethyl acetate in different concentrations. After MTT assay, cells were stained with Panotic dye kit. Cell images were obtained with an inverted microscope equipped with a digital camera. The images were analyzed by CellProfiler ® . No cytotoxicity at the highest concentration analyzed for 3-epimacronine, albomaculine, galanthine, trispheridine, P. gymnospora extract and Sargassum sp. extract where detected. Tazettine offered cytotoxicity only against the Hepa1c1c7 cell line. Lycorine, camptothecin, and H. itaobinus extract exhibited cytotoxic effects in both cell lines. The viability methods tested were correlated demonstrated by Bland-Atman test with normal distribution with mean difference between the two methods close to zero, bias value 3.0263. The error was within the limits of the confidence intervals and these values had a narrow difference. The correlation between the two methods was demonstrated by the linear regression plotted as R 2 . CellProfiler ® image analysis presented similar results to the MTT assay in the identification of viable cells, and image analysis may assist part of biological analysis procedures. The presented methodology is inexpensive and reproducible. In vitro cell viability assessment with MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay may be replaced by image analysis by CellProfiler ® . The viability methods

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

Diosmin reduces cell viability of A431 skin cancer cells through apoptotic induction.

PubMed

Buddhan, Rajamanickam; Manoharan, Shanmugam

2017-01-01

Aim of the present study was to evaluate the in vitro cytotoxic potential of the diosmin in A431 skin cancer cells. The cytotoxic (anti-cell proliferative) potential of diosmin in A431 cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (cell viability), dual staining (apoptotic induction), dichloro-dihydro-fluorescein diacetate assay (reactive oxygen species [ROS] generation), DNA fragmentation study, Western blotting analysis (apoptotic markers expression) and flow cytometry (cell cycle arrest). Diosmin reduced the cell viability of A431 cells in a dose-dependent fashion and the inhibitory concentration 50% value was attained at 45 μg/ml using MTT assay. Diosmin at a concentration of 45 μg/ml generated excessive ROS in A431 cells, as compared to untreated cells. Diosmin treated A431 cells also revealed multiple DNA fragments than the untreated cells. Diosmin upregulated the expression of p53, caspases 3 and 9 and downregulated the expression of Bcl-2, matrix metalloproteinases-2 and 9 in A431 cells. The cytotoxic or anti-cell proliferative potential of diosmin is due to its ROS-mediated apoptotic induction potential, as well as due to its role in the inhibition of invasion in the A431 cells.

Ibrutinib (ImbruvicaTM) potently inhibits ErbB receptor phosphorylation and cell viability of ErbB2-positive breast cancer cells.

PubMed

Grabinski, Nicole; Ewald, Florian

2014-12-01

Ibrutinib (formerly PCI-32765) is a specific, irreversible, and potent inhibitor of Burton's tyrosine kinase (BTK) developed for the treatment of several forms of blood cancer. It is now an FDA-approved drug marketed under the name Imbruvica(TM) (Pharmacyclics, Inc.) and successfully used as an orally administered second-line drug in the treatment of mantle cell lymphoma. Since BTK is predominantly expressed in hematopoietic cells, the sensitivity of solid tumor cells to Ibrutinib has not been analyzed. In this study, we determined the effect of Ibrutinib on breast cancer cells. We demonstrate that Ibrutinib efficiently reduces the phosphorylation of the receptor tyrosine kinases ErbB1, ErbB2 and ErbB3, thereby suppressing AKT and MAPK signaling in ErbB2-positive (ErbB2+) breast cancer cell lines. Treatment with Ibrutinib significantly reduced the viability of ErbB2+ cell lines with IC50 values at nanomolar concentrations, suggesting therapeutic potential of Ibrutinib in breast cancer. Combined treatment with Ibrutinib and the dual PI3K/mTOR inhibitor BEZ235 synergistically reduces cell viability of ErbB2+ breast cancer cells. Combination indices below 0.25 at 50% inhibition of cell viability were determined by the Chou-Talalay method. Therefore, the combination of Ibrutinib and canonical PI3K pathway inhibitors could be a new and effective approach in the treatment of breast cancer with activated ErbB receptors. Ibrutinib could thus become a valuable component of targeted therapy in aggressive ErbB2+ breast cancer.

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.

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.

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.

Human periodontal ligament cell viability in milk and milk substitutes.

PubMed

Pearson, Robert M; Liewehr, Frederick R; West, Leslie A; Patton, William R; McPherson, James C; Runner, Royce R

2003-03-01

The purpose of this study was to determine the efficacy of several milk substitutes compared to whole milk in maintaining the viability of human periodontal ligament (PDL) cells on avulsed teeth. PDL cells were obtained from freshly extracted, healthy third molars and cultured in Eagle's minimal essential media (EMEM). The cells were plated onto 24-well culture plates and allowed to attach for 24 h. EMEM was replaced with refrigerated whole milk (positive control), reconstituted powdered milk, evaporated milk, or one of two baby formulas (Similac or Enfamil). Tap water served as the negative control. Tissue culture plates were incubated with the experimental media at 37 degrees C for 1, 2, 4, or 8 h. Cell viability was determined by a cell proliferation assay (CellTiter 96 AQ Assay), with absorbance read at 450 nM. A two-way ANOVA (p < 0.001) indicated that at 1 h there was no difference in the effect on PDL cell viability between any of the materials and whole milk. At 2 h, Enfamil and Similac performed significantly better than whole milk, whereas evaporated milk performed worse. At 4 h, Enfamil performed better than whole milk, whereas all other milk substitutes performed worse. At 8 h, all substitutes performed worse than whole milk. These results suggest that Enfamil, which is supplied in powder form that does not require special storage and has a shelf life of 18 months, is a more effective storage medium for avulsed teeth than pasteurized milk for at least 4 h.

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

In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis.

PubMed

Helm, Katharina; Beyreis, Marlena; Mayr, Christian; Ritter, Markus; Jakab, Martin; Kiesslich, Tobias; Plaetzer, Kristjan

2017-01-01

For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis. A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H2O2 and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity. Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays. Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments. © 2017 The Author(s)Published by S. Karger AG, Basel.

FGF1-gold nanoparticle conjugates targeting FGFR efficiently decrease cell viability upon NIR irradiation

PubMed Central

Szlachcic, Anna; Pala, Katarzyna; Zakrzewska, Malgorzata; Jakimowicz, Piotr; Wiedlocha, Antoni; Otlewski, Jacek

2012-01-01

Fibroblast growth factor receptors (FGFRs) are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we designed, constructed, and characterized FGFR-targeted gold nanoconjugates suitable for infrared-induced thermal ablation (localized heating leading to cancer cell death) based on gold nanoparticles (AuNPs). We showed that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs. To assure thermal stability, protease resistance, and prolonged half-life of the targeting protein, we employed highly stable FGF1 variant that retains the biological activities of the wild type FGF1. Novel FGF1 variant, AuNP conjugates are specifically internalized only by the cells expressing FGFRs, and they significantly reduce their viability after irradiation with near-infrared light (down to 40% of control cell viability), whereas the proliferation potential of cells lacking FGFRs is not affected. These results demonstrate the feasibility of FGF1-coated AuNPs for targeted cancer therapy. PMID:23226697

Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives.

PubMed

Stockert, Juan C; Horobin, Richard W; Colombo, Lucas L; Blázquez-Castro, Alfonso

2018-04-01

For many years various tetrazolium salts and their formazan products have been employed in histochemistry and for assessing cell viability. For the latter application, the most widely used are 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC) for viability assays of eukaryotic cells and bacteria, respectively. In these cases, the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to strongly colored and lipophilic formazan products, which are then quantified by absorbance (MTT) or fluorescence (CTC). More recently, certain sulfonated tetrazolium, which give rise to water-soluble formazans, have also proved useful for cytotoxicity assays. We describe several aspects of the application of tetrazolium salts and formazans in biomedical cell biology research, mainly regarding formazan-based colorimetric assays, cellular reduction of MTT, and localization and fluorescence of the MTT formazan in lipidic cell structures. In addition, some pharmacological and labeling perspectives of these compounds are also described. Copyright © 2018 Elsevier GmbH. All rights reserved.

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.

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.

Prokaryotic RNA Associated to Bacterial Viability Induces Polymorphonuclear Neutrophil Activation.

PubMed

Rodriguez-Rodrigues, Nahuel; Castillo, Luis A; Landoni, Verónica I; Martire-Greco, Daiana; Milillo, M Ayelén; Barrionuevo, Paula; Fernández, Gabriela C

2017-01-01

Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live Escherichia coli (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria

Prokaryotic RNA Associated to Bacterial Viability Induces Polymorphonuclear Neutrophil Activation

PubMed Central

Rodriguez-Rodrigues, Nahuel; Castillo, Luis A.; Landoni, Verónica I.; Martire-Greco, Daiana; Milillo, M. Ayelén; Barrionuevo, Paula; Fernández, Gabriela C.

2017-01-01

Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live Escherichia coli (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria

In Vitro Anti/Pro-oxidant Activities of R. ferruginea Extract and Its Effect on Glioma Cell Viability: Correlation with Phenolic Compound Content and Effects on Membrane Dynamics.

PubMed

Dos Santos, Desirée Magalhães; Rocha, Camila Valesca Jardim; da Silveira, Elita Ferreira; Marinho, Marcelo Augusto Germani; Rodrigues, Marisa Raquel; Silva, Nichole Osti; da Silva Ferreira, Ailton; de Moura, Neusa Fernandes; Darelli, Gabriel Jorge Sagrera; Braganhol, Elizandra; Horn, Ana Paula; de Lima, Vânia Rodrigues

2018-04-01

Rapanea ferruginea antioxidant and antitumoral properties were not explored before in literature. This study aimed to investigate these biological activities for the R. ferruginea leaf extract and correlate them with its phenolic content and influence in biological membrane dynamics. Thus, in this study, anti/pro-oxidative properties of R. ferruginea leaf extract by in vitro DPPH and TBARS assays, with respect to the free radical reducing potential and to its activity regarding membrane free radical-induced peroxidation, respectively. Furthermore, preliminary tests related to the extract effect on in vitro glioma cell viability were also performed. In parallel, the phenolic content was detected by HPLC-DAD and included syringic and trans-cinnamic acids, quercetrin, catechin, quercetin, and gallic acid. In an attempt to correlate the biological activity of R. ferruginea extract and its effect on membrane dynamics, the molecular interaction between the extract and a liposomal model with natural-sourced phospholipids was investigated. Location and changes in vibrational, rotational, and translational lipid motions, as well as in the phase state of liposomes, induced by R. ferruginea extract, were monitored by Fourier-transform infrared spectroscopy, nuclear magnetic resonance, differential scanning calorimetry, and UV-visible spectroscopy. In its free form, the extract showed promising in vitro antioxidant properties. Free-form extract (at 1000µ g/mL) exposure reduced glioma cell in vitro viability in 40%, as evidenced by MTT tests. Pro-oxidant behavior was observed when the extract was loaded into liposomes. A 70.8% cell viability reduction was achieved with 500 µg/mL of liposome-loaded extract. The compounds of R. ferruginea extract ordered liposome interface and disorder edits a polar region. Phenolic content, as well as membrane interaction and modulation may have an important role in the oxidative and antitumoral activities of the R. ferruginea leaf extract.

Influence of Cu-Ti thin film surface properties on antimicrobial activity and viability of living cells.

PubMed

Wojcieszak, Damian; Kaczmarek, Danuta; Antosiak, Aleksandra; Mazur, Michal; Rybak, Zbigniew; Rusak, Agnieszka; Osekowska, Malgorzata; Poniedzialek, Agata; Gamian, Andrzej; Szponar, Bogumila

2015-11-01

The paper describes properties of thin-film coatings based on copper and titanium. Thin films were prepared by co-sputtering of Cu and Ti targets in argon plasma. Deposited coatings consist of 90at.% of Cu and 10at.% of Ti. Characterization of the film was made on the basis of investigations of microstructure and physicochemical properties of the surface. Methods such as scanning electron microscopy, x-ray microanalysis, x-ray diffraction, x-ray photoelectron spectroscopy, atomic force microscopy, optical profilometry and wettability measurements were used to assess the properties of deposited thin films. An impact of Cu-Ti coating on the growth of selected bacteria and viability of the living cells (line L929, NCTC clone 929) was described in relation to the structure, surface state and wettability of the film. It was found that as-deposited films were amorphous. However, in such surroundings the nanocrystalline grains of 10-15nm and 25-35nm size were present. High surface active area with a roughness of 8.9nm, had an effect on receiving relatively high water contact angle value (74.1°). Such wettability may promote cell adhesion and result in an increase of the probability of copper ion transfer from the film surface into the cell. Thin films revealed bactericidal and fungicidal effects even in short term-contact. High activity of prepared films was directly related to high amount (ca. 51 %) of copper ions at 1+ state as x-ray photoelectron spectroscopy results have shown. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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.

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.

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.

Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

PubMed

Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

2017-01-01

Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

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.

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.

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.

Fluorescein diacetate for determination of cell viability in 3D fibroblast-collagen-GAG constructs.

PubMed

Powell, Heather M; Armour, Alexis D; Boyce, Steven T

2011-01-01

Quantification of cell viability and distribution within engineered tissues currently relies on representative histology, phenotypic assays, and destructive assays of viability. To evaluate uniformity of cell density throughout 3D collagen scaffolds prior to in vivo use, a nondestructive, field assessment of cell viability is advantageous. Here, we describe a field measure of cell viability in lyophilized collagen-glycosaminoglycan (C-GAG) scaffolds in vitro using fluorescein diacetate (FdA). Fibroblast-C-GAG constructs are stained 1 day after cellular inoculation using 0.04 mg/ml FdA followed by exposure to 366 nm UV light. Construct fluorescence quantified using Metamorph image analysis is correlated with inoculation density, MTT values, and histology of corresponding biopsies. Construct fluorescence correlates significantly with inoculation density (p  <  0.001) and MTT values (p  <  0.001) of biopsies collected immediately after FdA staining. No toxicity is detected in the constructs, as measured by MTT assay before and after the FdA assay at different time points; normal in vitro histology is demonstrated for the FdA-exposed constructs. In conclusion, measurement of intracellular fluorescence with FdA allows for the early, comprehensive measurement of cellular distributions and viability in engineered tissue.

The preservative effect of Thai propolis extract on the viability of human periodontal ligament cells.

PubMed

Prueksakorn, Attaporn; Puasiri, Subin; Ruangsri, Supanigar; Makeudom, Anupong; Sastraruji, Thanapat; Krisanaprakornkit, Suttichai; Chailertvanitkul, Pattama

2016-12-01

Tooth avulsion causes an injury to the periodontal ligament (PDL). The success of tooth replantation depends on the quantity and quality of PDL cells. The aim of this study was to examine the preservative and proliferative effects of Thai propolis extract, previously shown to exert anti-inflammatory and antioxidant activities, on human PDL cells. Ninety-six premolars were left to air dry for 30 min and stored in Hank's balanced salt solution (HBSS), milk, or various concentrations of propolis extract from 0.25 to 10 mg ml -1 for 3 h. PDL cells were isolated by collagenase and trypsin digestion, and their viability was determined by a trypan blue dye exclusion assay. PDL tissues were also scraped off the root surface and cultured to determine cell growth and morphology. The alamarBlue ® and BrdU assays were performed to determine the cytotoxic and proliferative effects of the extract on cultured PDL cells, respectively. A non-toxic dose of 2.5 mg ml -1 of propolis extract yielded the greatest percentage of cell viability (78.84 ± 3.34%), which was significantly higher than those of the other concentrations (P < 0.001). Nevertheless, this percentage was not significantly different from that of HBSS (80.14 ± 2.44%; P = 1.00), but was significantly higher than that of milk (71.27 ± 2.79%; P < 0.001). The cells grown from PDL explants looked like fibroblasts. However, 2.5 mg ml -1 of the extract did not induce PDL cell proliferation. Thai propolis extract at 2.5 mg ml -1 appears to be the most effective dose for preserving the viability of PDL cells, and this was comparable to HBSS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

PubMed Central

Perdomo-Celis, Federico; Salgado, Doris M.; Castañeda, Diana M.

2016-01-01

Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3+ CD8+ amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3+ cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = −0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs. PMID:26961858

Gallic acid reduces cell viability, proliferation, invasion and angiogenesis in human cervical cancer cells

PubMed Central

ZHAO, BING; HU, MENGCAI

2013-01-01

Gallic acid is a trihydroxybenzoic acid, also known as 3,4,5-trihydroxybenzoic acid, which is present in plants worldwide, including Chinese medicinal herbs. Gallic acid has been shown to have cytotoxic effects in certain cancer cells, without damaging normal cells. The objective of the present study was to determine whether gallic acid is able to inhibit human cervical cancer cell viability, proliferation and invasion and suppress cervical cancer cell-mediated angiogenesis. Treatment of HeLa and HTB-35 human cancer cells with gallic acid decreased cell viability in a dose-dependent manner. BrdU proliferation and tube formation assays indicated that gallic acid significantly decreased human cervical cancer cell proliferation and tube formation in human umbilical vein endothelial cells, respectively. Additionally, gallic acid decreased HeLa and HTB-35 cell invasion in vitro. Western blot analysis demonstrated that the expression of ADAM17, EGFR, p-Akt and p-Erk was suppressed by gallic acid in the HeLa and HTB-35 cell lines. These data indicate that the suppression of ADAM17 and the downregulation of the EGFR, Akt/p-Akt and Erk/p-Erk signaling pathways may contribute to the suppression of cancer progression by Gallic acid. Gallic acid may be a valuable candidate for the treatment of cervical cancer. PMID:24843386

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.

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.

Comparisons of human amniotic mesenchymal stem cell viability in FDA-approved collagen-based scaffolds: Implications for engineered diaphragmatic replacement.

PubMed

Shieh, Hester F; Graham, Christopher D; Brazzo, Joseph A; Zurakowski, David; Fauza, Dario O

2017-06-01

We sought to examine amniotic fluid mesenchymal stem cell (afMSC) viability within two FDA-approved collagen-based scaffolds, as a prerequisite to clinical translation of afMSC-based engineered diaphragmatic repair. Human afMSCs were seeded in a human-derived collagen hydrogel and in a bovine-derived collagen sheet at 3 matching densities. Cell viability was analyzed at 1, 3, and 5days using an ATP-based 3D bioluminescence assay. Statistical comparisons were by ANOVA (P<0.05). There was a highly significant 3-way interaction between scaffold type, seeding density, and time in 3D culture as determinants of cell viability, clearly favoring the human hydrogel (P<0.001). In both scaffolds, cell viability was highest at the highest seeding density of 150,000 cells/mL. Time in 3D culture impacted cell viability at the optimal seeding density in the human hydrogel, with the highest levels on days 1 (P<0.001) and 5 (P=0.05) with no significant effect in the bovine sheet (P=0.39-0.96). Among clinically-approved cell delivery vehicles, mesenchymal stem cell viability is significantly enhanced in a collagen hydrogel when compared with a collagen sheet. Cell viability can be further optimized by seeding density and time in 3D culture. These data further support the regulatory viability of clinical trials of engineered diaphragmatic repair. N/A (animal and laboratory study). Copyright © 2017 Elsevier Inc. All rights reserved.

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

PubMed

Genz, Berit; Thomas, Maria; Pützer, Brigitte M; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

2014-11-01

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. Copyright © 2014 Elsevier Inc. All rights reserved.

Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory †

PubMed Central

Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K.; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V. McNeil; Segarra, Verónica A.

2017-01-01

Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented—one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research. PMID:28861134

Effects of drinking desalinated seawater on cell viability and proliferation.

PubMed

Macarrão, Camila Longhi; Bachi, André Luis Lacerda; Mariano, Mario; Abel, Lucia Jamli

2017-06-01

Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.

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

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

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.

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.

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

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.

Red Maca (Lepidium meyenii) did not affect cell viability despite increased androgen receptor and prostate-specific antigen gene expression in the human prostate cancer cell line LNCaP.

PubMed

Díaz, P; Cardenas, H; Orihuela, P A

2016-10-01

We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80 μg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48 hr after treatment. Furthermore, LNCaP cells were treated with 80 μg/ml of red Maca plus Taxol or 2ME 5 μM and viability was assessed 48 hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80 μg/ml, and 12 hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40 μg/ml, but not at 80 μg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells. © 2016 Blackwell Verlag GmbH.

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

The in vitro impact of toothpaste extracts on cell viability.

PubMed

Cvikl, Barbara; Lussi, Adrian; Gruber, Reinhard

2015-06-01

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity. © 2015 Eur J Oral Sci.

The effects of vibration loading on adipose stem cell number, viability and differentiation towards bone-forming cells

PubMed Central

Tirkkonen, Laura; Halonen, Heidi; Hyttinen, Jari; Kuokkanen, Hannu; Sievänen, Harri; Koivisto, Anna-Maija; Mannerström, Bettina; Sándor, George K. B.; Suuronen, Riitta; Miettinen, Susanna; Haimi, Suvi

2011-01-01

Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d–1 for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells. PMID:21613288

Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

NASA Astrophysics Data System (ADS)

Shen, Yajing; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Kojima, Masaru; Fukuda, Toshio

2011-11-01

Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

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.

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

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.

Osteochondral Tissue Cell Viability Is Affected by Total Impulse during Impaction Grafting

PubMed Central

Balash, Paul; Kang, Richard W.; Schwenke, Thorsten; Cole, Brian J.; Wimmer, Markus A.

2010-01-01

Objective: Osteochondral graft transplantation has garnered significant attention because of its ability to replace the lesion with true hyaline cartilage. However, surgical impaction of the graft to anchor it into the defect site can be traumatic and lead to cell death and cartilage degeneration. This study aimed to test the hypothesis that increasing impulse magnitude during impaction of osteochondral plugs has a direct effect on loss of cell viability. Design: In this controlled laboratory study, the impaction force was kept constant while the impulse was varied. Ninety-six osteochondral plugs were extracted from the trochlea of bovine stifle joints and were randomly assigned into 3 experimental and 1 (nonimpacted) control group. The transferred impulse of the experimental groups reflected the median and the lower and upper quartiles of preceding clinical measurements. Data were obtained at day 0, day 4, and day 8; at each point, cell viability was assessed using the Live/Dead staining kit and histological assessments were performed to visualize matrix structural changes. Results: After impaction, cartilage samples stayed intact and did not show any histological signs of matrix disruption. As expected, higher impulse magnitudes introduced more cell death; however, this relationship was lost at day 8 after impaction. Conclusion: Impulse magnitude has a direct effect on cell viability of the graft. Because impulse magnitude is mostly governed by the press-fit characteristics of the recipient site, this study aids in the definition of optimal insertion conditions for osteochondral grafts. PMID:26069558

Lactate calcium salt affects the viability of colorectal cancer cells via betaine homeostasis.

PubMed

Jang, Yeong-Su; Jo, Young-Kwon; Sim, Jae Jun; Ji, Eunhee; Jeong, Keun-Yeong; Kim, Hwan Mook

2016-02-15

Betaine plays an important role in cellular homeostasis. However, the physiological roles of betaine-γ-aminobutyric acid (GABA) transporter (BGT-1) are still being disputed in cancer. In this study, we tried to find the possibility of the antitumor effect on colorectal cancer (CRC) cell via lactate calcium salt (CaLa)-induced BGT-1 downregulation. The CRC cell viability and clonogenic assay was performed using different doses of BGT-1 inhibitor. The expression level of BGT-1 was measured following the treatment of 2.5mM CaLa. Betaine was treated to confirm the resistance of the antitumor activity by CaLa. Tumor growth was also measured using a xenograft animal model. Long-term exposure of 2.5mM CaLa clearly decreased the expression of BGT-1 in the CRC cells. As a result of the downregulation of BGT-1 expression, the clonogenic ability of CRC cells was also decreased in the 2.5mM CaLa-treated group. Reversely, the number of colonies and cell viability was increased by combination treatment with betaine and 2.5mM CaLa, as compared with a single treatment of 2.5mM CaLa. Tumor growth was significantly inhibited in the xenograft model depending on BGT-1 downregulation by 2.5mM CaLa treatment. These results support the idea that long-lasting calcium supplementation via CaLa contributes to disruption of betaine homeostasis in the CRC cells and is hypothesized to reduce the risk of CRC. In addition, it indicates the possibility of CaLa being a potential incorporating agent with existing therapeutics against CRC. Copyright © 2016 Elsevier Inc. All rights reserved.

Photodithazine photodynamic effect on viability of 9L/lacZ gliosarcoma cell line.

PubMed

Fontana, Leticia C; Pinto, Juliana G; Pereira, André H C; Soares, Cristina P; Raniero, Leandro J; Ferreira-Strixino, Juliana

2017-08-01

Even with the advances of conventional treatment techniques, the nervous system cancer prognosis is still not favorable to the patient which makes alternative therapies needed to be studied. Photodynamic therapy (PDT) is presented as a promising therapy, which employs a photosensitive (PS) agent, light wavelength suitable for the PS agent, and molecular oxygen, producing reactive oxygen species in order to induce cell death. The aim of this study is to observe the PDT action in gliosarcoma cell using a chlorin (Photodithazine, PDZ). The experiments were done with 9L/lacZ lineage cells, grown in a DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution and put in a culture chamber at 37 °C with an atmosphere of 5% CO 2 . The PS agent used was the PDZ to an LED light source device (Biopdi/IRRAD-LED 660) in the 660-nm region. The location of the PS agent was analyzed by fluorescence microscopy, and cell viability was analyzed by MTT assay (mitochondrial activity), exclusion by trypan blue (cell viability), and morphological examination through an optical microscope (Leica MD 2500). In the analysis of the experiments with PDZ, there was 100% cell death at different concentrations and clear morphological differences in groups with and without treatment. Furthermore, it was observed that the photodithazine has been focused on all nuclear and cytoplasmic extension; however, it cannot be said for sure whether the location is in the inside core region or on the plasma membrane. In general, the PDZ showed a promising photosensitive agent in PDT for the use of gliosarcoma.

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.

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

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

Genz, Berit; Thomas, Maria; Pützer, Brigitte M.

2014-11-01

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluatedmore » an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.« less

Drug/Cell-line Browser: interactive canvas visualization of cancer drug/cell-line viability assay datasets.

PubMed

Duan, Qiaonan; Wang, Zichen; Fernandez, Nicolas F; Rouillard, Andrew D; Tan, Christopher M; Benes, Cyril H; Ma'ayan, Avi

2014-11-15

Recently, several high profile studies collected cell viability data from panels of cancer cell lines treated with many drugs applied at different concentrations. Such drug sensitivity data for cancer cell lines provide suggestive treatments for different types and subtypes of cancer. Visualization of these datasets can reveal patterns that may not be obvious by examining the data without such efforts. Here we introduce Drug/Cell-line Browser (DCB), an online interactive HTML5 data visualization tool for interacting with three of the recently published datasets of cancer cell lines/drug-viability studies. DCB uses clustering and canvas visualization of the drugs and the cell lines, as well as a bar graph that summarizes drug effectiveness for the tissue of origin or the cancer subtypes for single or multiple drugs. DCB can help in understanding drug response patterns and prioritizing drug/cancer cell line interactions by tissue of origin or cancer subtype. DCB is an open source Web-based tool that is freely available at: http://www.maayanlab.net/LINCS/DCB CONTACT: avi.maayan@mssm.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

Influence of different air-abrasive powders on cell viability at biologically contaminated titanium dental implants surfaces.

PubMed

Schwarz, Frank; Ferrari, Daniel; Popovski, Kristian; Hartig, Brigitte; Becker, Jürgen

2009-01-01

Studies have indicated that oral biofilm formation at structured titanium surfaces interferes with cell adhesion and proliferation, and its removal by means of conventional treatment procedures may not be sufficient to render these surfaces biologically acceptable. Therefore, the aim of the study was to evaluate the influence of different air-abrasive powders on cell viability at biologically contaminated titanium dental implant surfaces. Intraoral splints were used to collect an in vivo biofilm on sandblasted and acid-etched titanium discs for 48 h. A single (1x) and repeated (2x) use of four different powders (amino acid glycine or sodium bicarbonate particles; range of mean particle size (d(v50)):20-75 microm) was applied at two distances (1 and 2 mm) and angles (30 degrees and 90 degrees) to the surfaces. Specimens (2x) were incubated with SaOs-2 cells for 7 days. Residual biofilm (RB) areas (%), and surface alterations (SEM) (1x and 2x), as well as SaOs-2 cell viability, expressed as mitochondrial cell activity (MA) (counts/second) (2x specimens), were assessed. Comparable mean RB areas were observed within and between groups after both 1x (RB: 0.0% +/- 0.0% to 5.7% +/- 5.7%) and 2x (RB: 0.0% +/- 0.0%) treatments. All surface treatments did not lead to MA (2x) values comparable to the sterile control group. However, sodium bicarbonate particles resulted in significantly higher MA (2x) values than amino acid glycine powders of different sizes. This was associated with pronounced alterations of the surface morphology (2x). Within the limits of the present study, it was concluded that SaOs-2 cell viability at biologically contaminated titanium surfaces was mainly influenced by the particle type of the powder. (c) 2008 Wiley Periodicals, Inc.

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

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.

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.

Bioactive gel-glasses with distinctly different compositions: Bioactivity, viability of stem cells and antibiofilm effect against Streptococcus mutans.

PubMed

Siqueira, Renato L; Maurmann, Natasha; Burguêz, Daniela; Pereira, Daniela P; Rastelli, Alessandra N S; Peitl, Oscar; Pranke, Patricia; Zanotto, Edgar D

2017-07-01

In this study, an evaluation was performed to determine the in vitro bioactivity, viability of stem cells, and antibiofilm effect against Streptococcus mutans of two bioactive gel-glass 60SiO 2 -36CaO-4P 2 O 5 (BG-A) and 80SiO 2 -15CaO-5P 2 O 5 (BG-B) compositions. Both materials were bioactive and undergo the formation of hydroxycarbonate apatite (HCA) on their surfaces when immersed in simulated body fluid (SBF) after 12h, but the BG-A composition showed a more significant formation rate. The pH variation of the samples during the test in SBF indicated that an abrupt change had occurred for the BG-A composition within the first few hours, and the pH was subsequently maintained over time, supporting its stronger antibacterial effects against S. mutans. For the in vitro viability test using mesenchymal stem cells (MSCs), the BG-B showed significantly higher cell viability compared to the BG-A composition at concentrations of 0.125, 1.25 and 12.50mg/mL for 2days. These results indicated that the higher solubility of the BG-A glass favors bioactivity and antibacterial effects. However, as a result of rapid degradation, the increase in the concentration of ions in the cell culture medium was not favorable for cell proliferation. Thus, by varying the composition of glasses, and consequently their dissolution rate, it is possible to favor bioactivity, antimicrobial activity or stem cell proliferation for a particular application of interest. Copyright © 2017 Elsevier B.V. All rights reserved.

Shikonin inhibits the cell viability, adhesion, invasion and migration of the human gastric cancer cell line MGC-803 via the Toll-like receptor 2/nuclear factor-kappa B pathway.

PubMed

Liu, Ji Ping; Liu, Dan; Gu, Jun Fei; Zhu, Mao Mao; Cui, Li

2015-08-01

Shikonin is an active naphthoquinone pigment isolated from the root of Lithospermum erythrorhizon. This study was designed to explore the inhibition of Shikonin on cell viability, adhesion, migration and invasion ability of gastric cancer (GC) and its possible mechanism. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed for cell viability and adhesion ability of MGC-803 cells. Cell scratch repair experiments were conducted for the determination of migration ability while transwell assay for cell invasion ability. Western blot analysis and real-time polymerase chain reaction assay were used for the detection of protein and mRNA expressions. Fifty per cent inhibitory concentration of Shikonin on MGC-803 cells was 1.854 μm. Shikonin (1 μm) inhibited significantly the adhesion, invasion and migratory ability of MGC-803 cells. Interestingly, Shikonin in the presence or absence of anti-Toll-like receptor 2 (TLR2) antibody (2 μg) and nuclear factor-kappa B (NF-κB) inhibitor MG-132 (10 μm) could decrease these ability of MGC-803 cells markedly, as well as the expression levels of matrix metalloproteinases (MMP)-2, MMP-7, TLR2 and p65 NF-κB. In addition, the co-incubation of Shikonin and anti-TLR2/MG-132 has a significant stronger activity than anti-TLR2 or MG-132 alone. The results indicated that Shikonin could suppress the cell viability, adhesion, invasion and migratory ability of MGC-803 cells through TLR2- or NF-κB-mediated pathway. Our findings provide novel information for the treatment of Shikonin on GC. © 2015 Royal Pharmaceutical Society.

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.

Parallel effect of 4-octylphenol and cyclic adenosine monophosphate (cAMP) alters steroidogenesis, cell viability and ROS production in mice Leydig cells.

PubMed

Jambor, Tomas; Greifova, Hana; Kovacik, Anton; Kovacikova, Eva; Tvrda, Eva; Forgacs, Zsolt; Massanyi, Peter; Lukac, Norbert

2018-05-01

Over the last decade, there is growing incidence of male reproductive malfunctions. It has been documented that numerous environmental contaminants, such as endocrine disruptors (EDs) may adversely affect the reproductive functions of humans as well as wildlife species. The aim of this in vitro study was to examine the effects of 4-octylphenol (4-OP) on the steroidogenesis in mice Leydig cells. We evaluated the impact of this endocrine disruptor on the cholesterol levels and hormone secretion in a primary culture. Subsequently, we determined the cell viability and generation of reactive oxygen species (ROS) following 4-OP treatment. Isolated mice Leydig cells were cultured in the presence of different 4-OP concentrations (0.04-5.0 μg/mL) and 1 mM cyclic adenosine-monophosphate during 44 h. Cholesterol levels were determined from the culture medium using photometry. Quantification of steroid secretion was performed by enzyme-linked immunosorbent assay. The cell viability was assessed using the metabolic activity assay, while ROS production was assessed by the chemiluminescence technique. Slightly increased cholesterol levels were recorded following exposure to the whole applied range of 4-OP, without significant changes (P>0.05). In contrast, the secretion of steroid hormones, specifically dehydroepiandrosterone, androstenedione, and testosterone was decreased following exposure to 4-OP. Experimental doses of 4-OP did not affect cell viability significantly; however a moderate decrease was recorded following the higher doses (2.5 and 5.0 μg/mL) of 4-OP. Furthermore, relative treatment of 4-OP (5.0 μg/mL) caused a significant (P < 0.001) ROS overproduction in the exposed cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

GPER mediates enhanced cell viability and motility via non-genomic signaling induced by 17β-estradiol in triple-negative breast cancer cells.

PubMed

Yu, Tenghua; Liu, Manran; Luo, Haojun; Wu, Chengyi; Tang, Xi; Tang, Shifu; Hu, Ping; Yan, Yuzhao; Wang, Zhiliang; Tu, Gang

2014-09-01

Triple-negative breast cancer (TNBC) is an aggressive breast cancer with a generally poor prognosis. Due to lack of specific targets for its treatment, an efficient therapy is needed. G protein-coupled estrogen receptor (GPER), a novel estrogen receptor, has been reported to be expressed in TNBC tissues. In this study, we investigated the effects of blocking non-genomic signaling mediated by the estrogen/GPER pathway on cell viability and motility in the TNBC cells. GPER was strongly expressed in the TNBC cell lines MDA-MB-468 and MDA-MB-436, and the estrogen-mediated non-genomic ERK signaling activated by GPER was involved in cell viability and motility of TNBC cells. Treatment with 17β-estradiol (E2), the GPER-specific agonist G-1 and tamoxifen (TAM) led to rapid activation of p-ERK1/2, but not p-Akt. Moreover, estrogen/GPER/ERK signaling was involved in increasing cell growth, survival, and migration/invasion by upregulating expression of cyclinA, cyclinD1, Bcl-2, and c-fos associated with the cell cycle, proliferation, and apoptosis. Immunohistochemical analysis of TNBC specimens showed a significantly different staining of p-ERK1/2 between GPER-positive tissues (58/66, 87.9%) and GPER-negative tissues (13/30, 43.3%). The positivity of GPER and p-ERK1/2 displayed a strong association with large tumor size and poor clinical stage, indicating that GPER/ERK signaling might also contribute to tumor progression in TNBC patients which corresponded with in vitro experimental data. Our findings suggest that inhibition of estrogen/GPER/ERK signaling represents a novel targeted therapy in TNBC. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Fluorescence-based visualization of autophagic activity predicts mouse embryo viability

NASA Astrophysics Data System (ADS)

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Kito, Seiji; Minami, Naojiro; Kubota, Toshiro; Sato, Ken; Kokubo, Toshiaki

2014-03-01

Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality.

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.

Viability and proliferation potential of adipose-derived stem cells following labeling with a positron-emitting radiotracer.

PubMed

Elhami, Esmat; Goertzen, Andrew L; Xiang, Bo; Deng, Jixian; Stillwell, Chris; Mzengeza, Shadreck; Arora, Rakesh C; Freed, Darren; Tian, Ganghong

2011-07-01

Adipose-derived stem cells (ASCs) have promising potential in regenerative medicine and cell therapy. Our objective is to examine the biological function of the labeled stem cells following labeling with a readily available positron emission tomography (PET) tracer, (18)F-fluoro-2-deoxy-D: -glucose (FDG). In this work we characterize labeling efficiency through assessment of FDG uptake and retention by the ASCs and the effect of FDG on cell viability, proliferation, transdifferentiation, and cell function in vitro using rat ASCs. Samples of 10(5) ASCs (from visceral fat tissue) were labeled with concentrations of FDG (1-55 Bq/cell) in 0.75 ml culture medium. Label uptake and retention, as a function of labeling time, FDG concentration, and efflux period were measured to determine optimum cell labeling conditions. Cell viability, proliferation, DNA structure damage, cell differentiation, and other cell functions were examined. Non-labeled ASC samples were used as a control for all experimental groups. Labeled ASCs were injected via tail vein in several healthy rats and initial cell biodistribution was assessed. Our results showed that FDG uptake and retention by the stem cells did not depend on FDG concentration but on labeling and efflux periods and glucose content of the labeling and efflux media. Cell viability, transdifferentiation, and cell function were not greatly affected. DNA damage due to FDG radioactivity was acute, but reversible; cells managed to repair the damage and continue with cell cycles. Over all, FDG (up to 25 Bq/cell) did not impose severe cytotoxicity in rat ASCs. Initial biodistribution of the FDG-labeled ASCs was 80% + retention in the lungs. In the delayed whole-body images (2-3 h postinjection) there was some activity distribution resembling typical FDG uptake patterns. For in vivo cell tracking studies with PET tracers, the parameter of interest is the amount of radiotracer that is present in the cells being labeled and consequent

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.

Anticancer effects of phenoxazine derivatives revealed by inhibition of cell growth and viability, disregulation of cell cycle, and apoptosis induction in HTLV-1-positive leukemia cells.

PubMed

Miyano-Kurosaki, Naoko; Ikegami, Kou; Kurosaki, Kunihiko; Endo, Takahiko; Aoyagi, Hoshimi; Hanami, Mari; Yasumoto, Jun; Tomoda, Akio

2009-05-01

Adult T-cell leukemia (ATL) is a malignant tumor of human CD4(+) T cells infected with a human retrovirus, T lymphotropic virus type-1 (HTLV-1). The aim of the present study was to investigate the apoptotic effects of phenoxazines, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha,8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-aminophenoxazine-3-one (Phx-3) on a T cell leukemia cell line from ATL patients, MT-1 cells; HTLV-1 transformed T-cell lines, HUT-102 cells and MT-2 cells; and an HTLV-1-negative rat sarcoma cell line, XC cells. Among these phenoxazines, Phx-3 at concentrations of less than 10 microg/ml extensively inhibited growth and cell viability; arrested cell cycles at sub G(0)/G(1) phase; and augmented apoptosis of MT-1, HUT-102, and MT-2 cells. However, these phenoxazines did not affect the cell viability of an HTLV-1-negative rat sarcoma cell line, XC cells, and phytohemaggutinin-activated human peripheral blood mononuclear cells, although they markedly inhibited the growth of these cells. The transmission of HTLV-1 from HTLV-1-positive cells (MT-2 cells) to HTLV-1-negative cells (XC cells) was considered to be prevented by Phx-1, Phx-2, or Phx-3 because the syncytium formation between these cells was inhibited markedly in the presence of these phenoxazines. The present results suggest that Phx-1, Phx-2, and, in particular, Phx-3 may be useful as therapeutic agents against ATL, which is extremely refractory to current therapies.

The Effect of Sericin from Various Extraction Methods on Cell Viability and Collagen Production

PubMed Central

Aramwit, Pornanong; Kanokpanont, Sorada; Nakpheng, Titpawan; Srichana, Teerapol

2010-01-01

Silk sericin (SS) can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 μg/mL after 24 h incubation, but SS obtained from some extraction methods can be toxic at higher concentrations. Heat-degraded SS was the least toxic to cells and activated the highest collagen production, while urea-extracted SS showed the lowest cell viability and collagen production. SS from urea extraction was severely harmful to cells at concentrations higher than 100 μg/mL. SS from all extraction methods could still promote collagen production in a concentration-dependent manner, even at high concentrations that are toxic to cells. PMID:20559510

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.

Assessment of bacterial endospore viability with fluorescent dyes.

PubMed

Laflamme, C; Lavigne, S; Ho, J; Duchaine, C

2004-01-01

To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0.02). It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

Direct effects of phenformin on metabolism/bioenergetics and viability of SH-SY5Y neuroblastoma cells.

PubMed

Geoghegan, Fintan; Chadderton, Naomi; Farrar, G Jane; Zisterer, Daniela M; Porter, Richard K

2017-11-01

Phenformin, a member of the biguanides class of drugs, has been reported to be efficacious in cancer treatment. The focus of the current study was to establish whether there were direct effects of phenformin on the metabolism and bioenergetics of neuroblastoma SH-SY5Y cancer cells. Cell viability was assessed using the alamar blue assay, flow cytometry analysis using propidium iodide and annexin V stain and poly (ADP-ribose) polymerase analysis. Cellular and mitochondrial oxygen consumption was determined using a Seahorse Bioscience Flux analyser and an Oroboros Oxygraph respirometer. Cells were transfected using electroporation and permeabilized for in situ mitochondrial functional analysis using digitonin. Standard protocols were used for immunoblotting and proteins were separated on denaturing gels. Phenformin was effective in reducing the viability of SH-SY5Y cells, causing G 1 cell cycle arrest and inducing apoptosis. Bioenergetic analysis demonstrated that phenformin significantly decreased oxygen consumption in a dose- and time-dependent manner. The sensitivity of oxygen consumption in SH-SY5Y cells to phenformin was circumvented by the expression of NADH-quinone oxidoreductase 1, a ubiquinone oxidoreductase, suggesting that complex I may be a target of phenformin. As a result of this inhibition, adenosine monophosphate protein kinase is activated and acetyl-coenzyme A carboxylase is inhibited. To the best of our knowledge, the current study is the first to demonstrate the efficacy and underlying mechanism by which phenformin directly effects the survival of neuroblastoma cancer cells.

Direct effects of phenformin on metabolism/bioenergetics and viability of SH-SY5Y neuroblastoma cells

PubMed Central

Geoghegan, Fintan; Chadderton, Naomi; Farrar, G. Jane; Zisterer, Daniela M.; Porter, Richard K.

2017-01-01

Phenformin, a member of the biguanides class of drugs, has been reported to be efficacious in cancer treatment. The focus of the current study was to establish whether there were direct effects of phenformin on the metabolism and bioenergetics of neuroblastoma SH-SY5Y cancer cells. Cell viability was assessed using the alamar blue assay, flow cytometry analysis using propidium iodide and annexin V stain and poly (ADP-ribose) polymerase analysis. Cellular and mitochondrial oxygen consumption was determined using a Seahorse Bioscience Flux analyser and an Oroboros Oxygraph respirometer. Cells were transfected using electroporation and permeabilized for in situ mitochondrial functional analysis using digitonin. Standard protocols were used for immunoblotting and proteins were separated on denaturing gels. Phenformin was effective in reducing the viability of SH-SY5Y cells, causing G1 cell cycle arrest and inducing apoptosis. Bioenergetic analysis demonstrated that phenformin significantly decreased oxygen consumption in a dose- and time-dependent manner. The sensitivity of oxygen consumption in SH-SY5Y cells to phenformin was circumvented by the expression of NADH-quinone oxidoreductase 1, a ubiquinone oxidoreductase, suggesting that complex I may be a target of phenformin. As a result of this inhibition, adenosine monophosphate protein kinase is activated and acetyl-coenzyme A carboxylase is inhibited. To the best of our knowledge, the current study is the first to demonstrate the efficacy and underlying mechanism by which phenformin directly effects the survival of neuroblastoma cancer cells. PMID:29113281

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

In Vitro Electrochemical Corrosion and Cell Viability Studies on Nickel-Free Stainless Steel Orthopedic Implants

PubMed Central

Salahinejad, Erfan; Hadianfard, Mohammad Jafar; Macdonald, Digby Donald; Sharifi-Asl, Samin; Mozafari, Masoud; Walker, Kenneth J.; Rad, Armin Tahmasbi; Madihally, Sundararajan V.; Tayebi, Lobat

2013-01-01

The corrosion and cell viability behaviors of nanostructured, nickel-free stainless steel implants were studied and compared with AISI 316L. The electrochemical studies were conducted by potentiodynamic polarization and electrochemical impedance spectroscopic measurements in a simulated body fluid. Cytocompatibility was also evaluated by the adhesion behavior of adult human stem cells on the surface of the samples. According to the results, the electrochemical behavior is affected by a compromise among the specimen's structural characteristics, comprising composition, density, and grain size. The cell viability is interpreted by considering the results of the electrochemical impedance spectroscopic experiments. PMID:23630603

In vitro electrochemical corrosion and cell viability studies on nickel-free stainless steel orthopedic implants.

PubMed

Salahinejad, Erfan; Hadianfard, Mohammad Jafar; Macdonald, Digby Donald; Sharifi-Asl, Samin; Mozafari, Masoud; Walker, Kenneth J; Rad, Armin Tahmasbi; Madihally, Sundararajan V; Tayebi, Lobat

2013-01-01

The corrosion and cell viability behaviors of nanostructured, nickel-free stainless steel implants were studied and compared with AISI 316L. The electrochemical studies were conducted by potentiodynamic polarization and electrochemical impedance spectroscopic measurements in a simulated body fluid. Cytocompatibility was also evaluated by the adhesion behavior of adult human stem cells on the surface of the samples. According to the results, the electrochemical behavior is affected by a compromise among the specimen's structural characteristics, comprising composition, density, and grain size. The cell viability is interpreted by considering the results of the electrochemical impedance spectroscopic experiments.

Autogenous bone chips: influence of a new piezoelectric device (Piezosurgery) on chip morphology, cell viability and differentiation.

PubMed

Chiriac, G; Herten, M; Schwarz, F; Rothamel, D; Becker, J

2005-09-01

The aim of the present study was to investigate the influence of a new piezoelectric device, designed for harvesting autogenous bone chips from intra-oral sites, on chip morphology, cell viability and differentiation. A total of 69 samples of cortical bone chips were randomly gained by either (1) a piezoelectric device (PS), or (2) conventional rotating drills (RD). Shape and size of the bone chips were compared by means of morphometrical analysis. Outgrowing osteoblasts were identified by means of alkaline phosphatase activity (AP), immunhistochemical staining for osteocalcin (OC) synthesis and reverse transcriptase-polymerase chain reaction phenotyping. In 88.9% of the RD and 87.9% of the PS specimens, an outgrowth of adherent cells nearby the bone chips was observed after 6-19 days. Confluence of cells was reached after 4 weeks. Positive staining for AP and OC identified the cells as osteoblasts. The morphometrical analysis revealed a statistically significant more voluminous size of the particles collected with PS than RD. Within the limits of the present study, it may be concluded that both the harvesting methods are not different from each other concerning their detrimental effect on viability and differentiation of cells growing out of autogenous bone chips derived from intra-oral cortical sites.

Inhibition of CXCL12/CXCR4 autocrine/paracrine loop reduces viability of human glioblastoma stem-like cells affecting self-renewal activity.

PubMed

Gatti, Monica; Pattarozzi, Alessandra; Bajetto, Adriana; Würth, Roberto; Daga, Antonio; Fiaschi, Pietro; Zona, Gianluigi; Florio, Tullio; Barbieri, Federica

2013-12-15

Cancer stem cells (CSCs) or tumor initiating cells (TICs) drive glioblastoma (GBM) development, invasiveness and drug resistance. Distinct molecular pathways might regulate CSC biology as compared to cells in the bulk tumor mass, representing potential therapeutic targets. Chemokine CXCL12 and its receptor CXCR4 control proliferation, invasion and angiogenesis in GBM cell lines and primary cultures, but little is known about their activity in GBM CSCs. We demonstrate that CSCs, isolated from five human GBMs, express CXCR4 and release CXCL12 in vitro, although different levels of expression and secretion were observed in individual cultures, as expected for the heterogeneity of GBMs. CXCL12 treatment induced Akt-mediated significant pro-survival and self-renewal activities, while proliferation was induced at low extent. The role of CXCR4 signaling in CSC survival and self-renewal was further demonstrated using the CXCR4 antagonist AMD3100 that reduced self-renewal and survival with greater efficacy in the cultures that released higher CXCL12 amounts. The specificity of CXCL12 in sustaining CSC survival was demonstrated by the lack of AMD3100-dependent inhibition of viability in differentiated cells derived from the same GBMs. These findings, although performed on a limited number of tumor samples, suggest that the CXCL12/CXCR4 interaction mediates survival and self-renewal in GBM CSCs with high selectivity, thus emerging as a candidate system responsible for maintenance of cancer progenitors, and providing survival benefits to the tumor. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Effects of inorganic nanoparticles on viability and catabolic activities of Agrobacterium sp. PH-08 during biodegradation of dibenzofuran.

PubMed

Le, Thao Thanh; Murugesan, Kumarasamy; Kim, Eun-Ju; Chang, Yoon-Seok

2014-09-01

This study investigated the cytotoxicity, genotoxicity, and growth inhibition effects of four different inorganic nanoparticles (NPs) such as aluminum (nAl), iron (nFe), nickel (nNi), and zinc (nZn) on a dibenzofuran (DF) degrading bacterium Agrobacterium sp. PH-08. NP (0-1,000 mg L(-1)) -treated bacterial cells were assessed for cytotoxicity, genotoxicity, growth and biodegradation activities at biochemical and molecular levels. In an aqueous system, the bacterial cells treated with nAl, nZn and nNi at 500 mg L(-1) showed significant reduction in cell viability (30-93.6 %, p < 0.05), while nFe had no significant inhibition on bacterial cell viability. In the presence of nAl, nZn and nNi, the cells exhibited elevated levels of reactive oxygen species (ROS), DNA damage and cell death. Furthermore, NP exposure showed significant (p < 0.05) impairment in DF and catechol biodegradation activities. The reduction in DF biodegradation was ranged about 71.7-91.6 % with single NPs treatments while reached up to 96.3 % with a mixture of NPs. Molecular and biochemical investigations also clearly revealed that NP exposure drastically affected the catechol-2,3-dioxygenase activities and its gene (c23o) expression. However, no significant inhibition was observed in nFe treatment. The bacterial extracellular polymeric materials and by-products from DF degradation can be assumed as key factors in diminishing the toxic effects of NPs, especially for nFe. This study clearly demonstrates the impact of single and mixed NPs on the microbial catabolism of xenobiotic-degrading bacteria at biochemical and molecular levels. This is the first study on estimating the impact of mixed NPs on microbial biodegradation.

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.

The effect of Aloe vera gel on viability of dental pulp stem cells.

PubMed

Sholehvar, Fatemeh; Mehrabani, Davood; Yaghmaei, Parichehr; Vahdati, Akbar

2016-10-01

Dental pulp stem cells (DPSCs) can play a prominent role in tissue regeneration. Aloe vera L. (Liliaceae) contains the polysaccharide of acemannan that was shown to be a trigger factor for cell proliferation, differentiation, mineralization, and dentin formation. This study sought to determine the viability of DPSCs in Aloe vera in comparison with Hank's balanced salt solution (HBSS). Twelve rabbits underwent anesthesia, and their incisor teeth were extracted; the pulp tissue was removed, chopped, treated with collagenase and plated in culture flasks. DPSCs from passage 3 were cultured in 24-well plates, and after 3 days, the culture media changed to 10, 25, 50, and 100% concentrations of Aloe vera at intervals of 45 and 90 min and 3 and 6 h. Distilled water was used as negative and HBSS as positive control for comparison. The cell morphology, viability, population doubling time (PDT), and growth kinetics were evaluated. RT-PCR was carried out for characterization and karyotyping for chromosomal stability. Aloe vera showed a significant higher viability than HBSS (74.74%). The 50% Aloe vera showed higher viability (97.73%) than other concentrations. PDT in 50% concentration was 35.1 h and for HBSS was 49.5 h. DPSCs were spindle shaped and were positive for CD73 and negative for CD34 and CD45. Karyotyping was normal. Aloe vera as an inexpensive and available herb can improve survival of avulsed or broken teeth in emergency cases as a transfer media. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Oxygen Delivery from Hyperbarically Loaded Microtanks Extends Cell Viability in Anoxic Environments

PubMed Central

Cook, Colin A.; Hahn, Kathryn C.; Morrissette-McAlmon, Justin B.F.; Grayson, Warren L.

2016-01-01

Oxygen diffusion limitations within nascent tissue engineered (TE) grafts lead to the development of hypoxic regions, cell death, and graft failure. Previous efforts have been made to deliver oxygen within TE scaffolds, including peroxide-doping, perfluorocarbons, and hyperbaric oxygen therapy, to mitigate these effects and help maintain post transplantation cell viability, but these have suffered from significant drawbacks. Here we present a novel approach utilizing polymeric hollow-core microspheres that can be hyperbarically loaded with oxygen and subsequently provide prolonged oxygen delivery. These oxygen carriers are termed, microtanks. With an interest in orthopedic applications, we combined microtanks within polycaprolactone to form solid phase constructs with oxygen delivery capabilities. The mathematical laws governing oxygen delivery from microtank-loaded constructs are developed along with empirical validation. Constructs achieved periods of oxygen delivery out to 6 days, which was shown to prolong the survival of human adipose derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) as well as to enhance their cellular morphology under anoxic conditions. The results of this study suggest the microtank approach may be a feasible means of maintaining cell viability in TE scaffolds during the critical period of vascularization in vivo. PMID:25818444

Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells

NASA Astrophysics Data System (ADS)

Fan, Rong; Emery, Travis; Zhang, Yongguo; Xia, Yuxuan; Sun, Jun; Wan, Jiandi

2016-06-01

During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm2), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis.

Antiproliferative Activity of Egg Yolk Peptides in Human Colon Cancer Cells.

PubMed

Yousr, Marwa N; Aloqbi, Akram A; Omar, Ulfat M; Howell, Nazlin K

2017-01-01

Egg yolk peptides were successfully prepared from egg yolk protein by-products after lecithin extraction. Defatted egg yolk protein was hydrolyzed with pepsin and pancreatin and purified by gel filtration to produce egg yolk gel filtration fraction (EYGF-33) with antiproliferative activity. The highlight of this study was that the peptide EYGF-33 (1.0 mg/ml) significantly inhibits cell viability of colon cancer cells (Caco-2) with no inhibitory effects on the viability of human colon epithelial normal cells (HCEC) after 48 h. Reduced cell viability can be explained by cell cycle arrest in the S-phase in which DNA replication normally takes place. EYGF-33 significantly enhanced the production of superoxide anions in the mitochondria of Caco-2 cells; this could activate a mitochondrial apoptotic pathway leading to typical Poly Adenosine diphosphate-ribose polymerase (PARP) cleavage as observed in the Western blot result. The induction of apoptotic cell death by EYGF-33 was supported by the externalization of phosphatidylserine (PS). However, further elucidation of the mechanism of EYGF-33-mediated apoptosis would provide further support for its use as a potential therapeutic and chemopreventive agent.

Autocrine VEGF–VEGFR2–Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth

PubMed Central

Hamerlik, Petra; Lathia, Justin D.; Rasmussen, Rikke; Wu, Qiulian; Bartkova, Jirina; Lee, MyungHee; Moudry, Pavel; Bartek, Jiri; Fischer, Walter; Lukas, Jiri

2012-01-01

Although vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) is traditionally regarded as an endothelial cell protein, evidence suggests that VEGFRs may be expressed by cancer cells. Glioblastoma multiforme (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with the humanized VEGF antibody bevacizumab reduces GBM tumor growth; however, the clinical benefits are transient and invariably followed by tumor recurrence. In this study, we show that VEGFR2 is preferentially expressed on the cell surface of the CD133+ human glioma stem-like cells (GSCs), whose viability, self-renewal, and tumorigenicity rely, at least in part, on signaling through the VEGF-VEGFR2–Neuropilin-1 (NRP1) axis. We find that the limited impact of bevacizumab-mediated VEGF blockage may reflect ongoing autocrine signaling through VEGF–VEGFR2–NRP1, which is associated with VEGFR2–NRP1 recycling and a pool of active VEGFR2 within a cytosolic compartment of a subset of human GBM cells. Whereas bevacizumab failed to inhibit prosurvival effects of VEGFR2-mediated signaling, GSC viability under unperturbed or radiation-evoked stress conditions was attenuated by direct inhibition of VEGFR2 tyrosine kinase activity and/or shRNA-mediated knockdown of VEGFR2 or NRP1. We propose that direct inhibition of VEGFR2 kinase may block the highly dynamic VEGF–VEGFR2–NRP1 pathway and inspire a GBM treatment strategy to complement the currently prevalent ligand neutralization approach. PMID:22393126

The impact of α-Lipoic acid on cell viability and expression of nephrin and ZNF580 in normal human podocytes.

PubMed

Leppert, Ulrike; Gillespie, Allan; Orphal, Miriam; Böhme, Karen; Plum, Claudia; Nagorsen, Kaj; Berkholz, Janine; Kreutz, Reinhold; Eisenreich, Andreas

2017-09-05

Human podocytes (hPC) are essential for maintaining normal kidney function and dysfunction or loss of hPC play a pivotal role in the manifestation and progression of chronic kidney diseases including diabetic nephropathy. Previously, α-Lipoic acid (α-LA), a licensed drug for treatment of diabetic neuropathy, was shown to exhibit protective effects on diabetic nephropathy in vivo. However, the effect of α-LA on hPC under non-diabetic conditions is unknown. Therefore, we analyzed the impact of α-LA on cell viability and expression of nephrin and zinc finger protein 580 (ZNF580) in normal hPC in vitro. Protein analyses were done via Western blot techniques. Cell viability was determined using a functional assay. hPC viability was dynamically modulated via α-LA stimulation in a concentration-dependent manner. This was associated with reduced nephrin and ZNF580 expression and increased nephrin phosphorylation in normal hPC. Moreover, α-LA reduced nephrin and ZNF580 protein expression via 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) inhibition. These data demonstrate that low α-LA had no negative influence on hPC viability, whereas, high α-LA concentrations induced cytotoxic effects on normal hPC and reduced nephrin and ZNF580 expression via NF-κB inhibition. These data provide first novel information about potential cytotoxic effects of α-LA on hPC under non-diabetic conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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

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.

The C2238/αANP variant is a negative modulator of both viability and function of coronary artery smooth muscle cells.

PubMed

Rubattu, Speranza; Marchitti, Simona; Bianchi, Franca; Di Castro, Sara; Stanzione, Rosita; Cotugno, Maria; Bozzao, Cristina; Sciarretta, Sebastiano; Volpe, Massimo

2014-01-01

Abnormalities of vascular smooth muscle cells (VSMCs) contribute to development of vascular disease. Atrial natriuretic peptide (ANP) exerts important effects on VSMCs. A common ANP molecular variant (T2238C/αANP) has recently emerged as a novel vascular risk factor. We aimed at identifying effects of CC2238/αANP on viability, migration and motility in coronary artery SMCs, and the underlying signaling pathways. Cells were exposed to either TT2238/αANP or CC2238/αANP. At the end of treatment, cell viability, migration and motility were evaluated, along with changes in oxidative stress pathway (ROS levels, NADPH and eNOS expression), on Akt phosphorylation and miR21 expression levels. CC2238/αANP reduced cell vitality, increased apoptosis and necrosis, increased oxidative stress levels, suppressed miR21 expression along with consistent changes of its molecular targets (PDCD4, PTEN, Bcl2) and of phosphorylated Akt levels. As a result of increased oxidative stress, CC2238/αANP markedly stimulated cell migration and increased cell contraction. NPR-C gene silencing with specific siRNAs restored cell viability, miR21 expression, and reduced oxidative stress induced by CC2238/αANP. The cAMP/PKA/CREB pathway, driven by NPR-C activation, significantly contributed to both miR21 and phosphoAkt reduction upon CC2238/αANP. miR21 overexpression by mimic-hsa-miR21 rescued the cellular damage dependent on CC2238/αANP. CC2238/αANP negatively modulates viability through NPR-C/cAMP/PKA/CREB/miR21 signaling pathway, and it augments oxidative stress leading to increased migratory and vasoconstrictor effects in coronary artery SMCs. These novel findings further support a damaging role of this common αANP variant on vessel wall and its potential contribution to acute coronary events.

Effect of heat stress and recovery on viability, oxidative damage, and heat shock protein expression in hepatic cells of grass carp (Ctenopharyngodon idellus).

PubMed

Cui, Yanting; Liu, Bo; Xie, Jun; Xu, Pao; Habte-Tsion, H-Michael; Zhang, Yuanyuan

2014-06-01

In this study, we investigated the effects of hyperthermia and recovery on cell viability, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA), total antioxidant capacity (T-AOC), and heat shock protein (HSP60, 70, and 90) mRNA expression in the hepatic cells of the grass carp, Ctenopharyngodon idellus. Triplicate groups of cultured cells were exposed to 30, 32, or 34 °C for 0.5 h and then immediately incubated at 27 °C in 5 % CO2 for 6, 12, 24, or 48 h. Hyperthermia stress greatly reduced cell viability and increased LDH release. Cell damage declined after recovery. Hyperthermia stress increased the lipid peroxide levels and reduced the antioxidant capacity (e.g., reduced SOD and T-AOC) of the cells. However, oxidative damage declined as the recovery period increased, and the levels of MDA, SOD, and T-AOC were restored. After cells were exposed to 32 °C, the expression of HSP60 after recovery for 1, 2, and 4 h (P < 0.05), the expression of HSP70 after recovery for 0.5 and 1 h (P < 0.01), and the expression of HSP90 throughout recovery were significantly higher (P < 0.01) than the prestress levels. During the recovery period, the variations in HSP gene expression reflected the transition period from a state of cellular growth to one of the cellular repairs. In conclusion, hyperthermia depresses cell viability, induces oxidative damage, and increases HSP expression, which plays an important role during hyperthermic stress in grass carp hepatic cells.

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

Cell Viability and Functionality of Probiotic Bacteria in Dairy Products

PubMed Central

Vinderola, Gabriel; Binetti, Ana; Burns, Patricia; Reinheimer, Jorge

2011-01-01

Probiotic bacteria, according to the definition adopted by the World Health Organization in 2002, are live microorganisms, which when administered in adequate amounts confer a health benefit to the host. Recent studies show that the same probiotic strain produced and/or preserved under different storage conditions, may present different responses regarding their susceptibility to the adverse conditions of the gastrointestinal tract, its capacity to adhere to the intestinal epithelium, or its immunomodulating capacity, the functionality being affected without changes in cell viability. This could imply that the control of cell viability is not always enough to guarantee the functionality (probiotic capacity) of a strain. Therefore, a new challenge arises for food technologists and microbiologists when it comes to designing and monitoring probiotic food: to be able to monitor the functionality of a probiotic microorganism throughout all the stages the strain goes through from the moment it is produced and included in the food vehicle, until the moment of consumption. Conventional methodological tools or others still to be developed must be used. The application of cell membrane functionality markers, the use of tests of resistance to intestinal barriers, the study of surface properties and the application of in vivo models come together as complementary tools to assess the actual capacity of a probiotic organism in a specific food, to exert functional effects regardless of the number of viable cells present at the moment of consumption. PMID:21833320

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.

The influence of micronutrients in cell culture: a reflection on viability and genomic stability.

PubMed

Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Machado, Miriana; Bordin, Diana Lilian; Bergter, Lothar; Prá, Daniel; Henriques, João Antonio Pêgas

2013-01-01

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.

Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer

PubMed Central

Poff, AM; Ari, C; Arnold, P; Seyfried, TN; D’Agostino, DP

2014-01-01

Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM-M3 cells cultured in the presence and absence of β-hydroxybutyrate (βHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase-tagged syngeneic VM-M3 cells. Mice were fed a standard diet supplemented with either 1,3-butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies βHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood βHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM-M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM-M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use. PMID:24615175

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.

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

Photo-activated disinfection based on indocyanine green against cell viability and biofilm formation of Porphyromonas gingivalis.

PubMed

Pourhajibagher, Maryam; Chiniforush, Nasim; Ghorbanzadeh, Roghayeh; Bahador, Abbas

2017-03-01

Photo-activated disinfection (PAD) is a novel treatment approach, in which bacteria in the root canal system may be exposed to sub-lethal doses of PAD. Such exposure can affect bacterial survival and virulence features, such as biofilm formation ability. The aim of this study was to evaluate the effects of sub-lethal doses of PAD (sPAD) using indocyanine green (ICG) on load and biofilm formation ability of Porphyromonas gingivalis as an anaerobic bacterium associated with endodontic infection. The anti-bacterial and anti-biofilm potential of sPAD against P. gingivalis at sub-lethal doses of ICG as a photosensitizer and using 810nm wavelength of diode laser light via colony forming unit and crystal violet assays, respectively, was determined. High concentrations of ICG and light irradiation time significantly reduced bacteria. High doses of sPAD markedly reduced the number of bacteria and the formation of biofilm, up to 30.4% and 25.1%, respectively. High doses of sPAD affected cell viability and the biofilm formation ability of P. gingivalis; lower doses did not. Thus, selection of appropriate PAD dosage should be considered for the successful treatment of endodontic in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation.

PubMed

Mizuno, Mitsuru; Katano, Hisako; Otabe, Koji; Komori, Keiichiro; Kohno, Yuji; Fujii, Shizuka; Ozeki, Nobutake; Horie, Masafumi; Tsuji, Kunikazu; Koga, Hideyuki; Muneta, Takeshi; Sekiya, Ichiro

2017-06-13

In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 μL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide - and annexin V - cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. The viability and chondrogenic potential of synovial MSCs were

Low-intensity pulsed ultrasound promotes Schwann cell viability and proliferation via the GSK-3β/β-catenin signaling pathway

PubMed Central

Ren, Cong; Chen, Xiaohui; Du, Ning; Geng, Shuo; Hu, Yingying; Liu, Xin; Wu, Xianxian; Lin, Yuan; Bai, Xue; Yin, Wenzhe; Cheng, Shi; Yang, Lei; Zhang, Yong

2018-01-01

Background: It has been reported that ultrasound enhances peripheral nerve regeneration, but the mechanism remains elusive. Low-intensity pulsed ultrasound (LIPUS) has been reported to enhance proliferation and alter protein production in various types of cells. In this study, we detected the effects of LIPUS on Schwann cells. Material and methods: Schwann cells were separated from new natal Sprague-Dawley rat sciatic nerves and were cultured and purified. The Schwann cells were treated by LIPUS for 10 minutes every day, with an intensity of 27.37 mW/cm2. After treatment for 5 days, MTT, EdU staining, and flow cytometry were performed to examine cell viability and proliferation. Neurotrophic factors, including FGF, NGF, BDNF, and GDNF, were measured by western blot and real-time PCR. GSK-3β, p-GSK-3β, β-catenin and Cyclin D1 protein levels were detected using a western blot analysis. The expression of Cyclin D1 was also detected by immunofluorescence. Results: MTT and EdU staining showed that LIPUS increased the Schwann cells viability and proliferation. Compared to the control group, LIPUS increased the expression of growth factors and neurotrophic factors, including FGF, NGF, BDNF, GDNF, and Cyclin D1. Meanwhile, GSK-3β activity was inhibited in the LIPUS group as demonstrated by the increased level of p-GSK-3β and the ratio of the p-GSK-3β/GSK-3β level. The mRNA and protein expressions of β-catenin were increased in the LIPUS group. However, SB216763, a GSK-3β inhibitor, reversed the effects of LIPUS on Schwann cells. Conclusion: LIPUS promotes Schwann cell viability and proliferation by increasing Cyclin D1 expression via enhancing the GSK-3β/β-catenin signaling pathway.

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

Purine analogue ENERGI-F706 induces apoptosis of 786-O renal carcinoma cells via 5'-adenosine monophosphate-activated protein kinase activation.

PubMed

Hsu, Chao-Yu; Lin, Chun-Hsiang; Lin, Jiun-Tsai; Cheng, Yi-Fang; Chen, Han-Min; Kao, Shao-Hsuan

2015-09-01

Purine compounds are known to activate 5'-adenosine monophosphate-activated protein kinase (AMPK), which has important roles in treatments for renal cell carcinoma. The present study was aimed to investigate the effects of the purine analogue ENERGI‑F706 on the human renal carcinoma cell line 786‑O and the underlying mechanisms. The results revealed that ENERGI‑F706 (0.2‑0.6 mg/ml) significantly decreased the cell viability to up to 36.4±2.4% of that of the control. Compared to 786‑O cells, ENERGI‑F706 exerted less suppressive effects on the viability of the human non‑tumorigenic renal cell line HK‑2. Flow cytometric analysis showed that ENERGI‑F706 contributed to cell cycle arrest at S‑phase and triggered apoptosis of 786‑O cells. Immunoblot analysis revealed that anti‑apoptotic B‑cell lymphoma 2 (Bcl‑2) levels were reduced and pro‑apoptotic Bcl‑2‑associated X protein levels were diminished. In addition, activation of caspase‑9, caspase‑3 and poly(adenosine diphosphate ribose) polymerase (PARP) was promoted in 786‑O cells in response to ENERGI‑F706. Effects of ENERGI‑F706 on AMPK cascades were investigated and the results showed that ENERGI‑F706 enhanced phosphorylation of AMPKα (T172) and p53 (S15), a downstream target of AMPK. In addition, the AMPK activation, p53 (S15) phosphorylation, reduction of Bcl‑2, cleavage of caspase‑3 and PARP as well as suppressed cell viability induced by ENERGI‑F706 were reversed in the presence of AMPK inhibitor compound C (dorsomorphin). In conclusion, the findings of the present study revealed that ENERGI‑F706 significantly suppressed the viability of 786‑O cells via induction of cell cycle arrest and apoptosis, attributing to AMPK and p53 activation and subsequent cell cycle regulatory and apoptotic signaling. It was therefore indicated that ENERGI‑F706 may be suitable for the treatment of renal cell carcinoma.

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.

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.

The addition of albumin improves Schwann cells viability in nerve cryopreservation.

PubMed

González Porto, Sara Alicia; Domenech, Nieves; González Rodríguez, Alba; Avellaneda Oviedo, Edgar Mauricio; Blanco, Francisco J; Arufe Gonda, María C; Álvarez Jorge, Ángel; Sánchez Ibañez, Jacinto; Rendal Vázquez, Esther

2018-04-26

The purpose of the current study was to establish a valid protocol for nerve cryopreservation, and to evaluate if the addition of albumin supposed any advantage in the procedure. We compared a traditional cryopreservation method that uses dimethyl sulfoxide (DMSO) as cryoprotectant, to an alternative method that uses DMSO and albumin. Six Wistar Lewis rats were used to obtain twelve 20 mm fragments of sciatic nerve. In the first group, six fragments were cryopreserved in 199 media with 10% DMSO, with a temperature decreasing rate of 1 °C per minute. In the second group, six fragments were cryopreserved adding 4% human albumin. The unfreezing process consisted of sequential washings with saline in the first group, and saline and 20% albumin in the second group at 37 °C until the crioprotectant was removed. Structural evaluation was performed through histological analysis and electronic microscopy. The viability was assessed with the calcein-AM (CAM) and 4',6-diamino-2-fenilindol (DAPI) staining. Histological results showed a correct preservation of peripheral nerve architecture and no significant differences were found between the two groups. However, Schwann cells viability showed in the CAM-DAPI staining was significantly superior in the albumin group. The viability of Schwann cells was significantly increased when albumin was added to the nerve cryopreservation protocol. However, no significant structural differences were found between groups. Further studies need to be performed to assess the cryopreserved nerve functionality using this new method.

Respective effects of oxygen and energy substrate deprivation on beta cell viability.

PubMed

Lablanche, Sandrine; Cottet-Rousselle, Cécile; Argaud, Laurent; Laporte, Camille; Lamarche, Frédéric; Richard, Marie-Jeanne; Berney, Thierry; Benhamou, Pierre-Yves; Fontaine, Eric

2015-01-01

Deficit in oxygen and energetic substrates delivery is a key factor in islet loss during islet transplantation. Permeability transition pore (PTP) is a mitochondrial channel involved in cell death. We have studied the respective effects of oxygen and energy substrate deprivation on beta cell viability as well as the involvement of oxidative stress and PTP opening. Energy substrate deprivation for 1h followed by incubation in normal conditions led to a cyclosporin A (CsA)-sensitive-PTP-opening in INS-1 cells and human islets. Such a procedure dramatically decreased INS-1 cells viability except when transient removal of energy substrates was performed in anoxia, in the presence of antioxidant N-acetylcysteine (NAC) or when CsA or metformin inhibited PTP opening. Superoxide production increased during removal of energy substrates and increased again when normal energy substrates were restored. NAC, anoxia or metformin prevented the two phases of oxidative stress while CsA prevented the second one only. Hypoxia or anoxia alone did not induce oxidative stress, PTP opening or cell death. In conclusion, energy substrate deprivation leads to an oxidative stress followed by PTP opening, triggering beta cell death. Pharmacological prevention of PTP opening during islet transplantation may be a suitable option to improve islet survival and graft success. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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.

Fasting inhibits hepatic stellate cells activation and potentiates anti-cancer activity of Sorafenib in hepatocellular cancer cells.

PubMed

Lo Re, Oriana; Panebianco, Concetta; Porto, Stefania; Cervi, Carlo; Rappa, Francesca; Di Biase, Stefano; Caraglia, Michele; Pazienza, Valerio; Vinciguerra, Manlio

2018-02-01

Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. A 24 hr fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. A 24 hr fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation, and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use. © 2017 Wiley Periodicals, Inc.

The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

PubMed Central

Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Bordin, Diana Lilian; Prá, Daniel; Pêgas Henriques, João Antonio

2013-01-01

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed. PMID:23781504

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.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

PubMed

Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

2015-08-21

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

How plasma induced oxidation, oxygenation, and de-oxygenation influences viability of skin cells

NASA Astrophysics Data System (ADS)

Oh, Jun-Seok; Strudwick, Xanthe; Short, Robert D.; Ogawa, Kotaro; Hatta, Akimitsu; Furuta, Hiroshi; Gaur, Nishtha; Hong, Sung-Ha; Cowin, Allison J.; Fukuhara, Hideo; Inoue, Keiji; Ito, Masafumi; Charles, Christine; Boswell, Roderick W.; Bradley, James W.; Graves, David B.; Szili, Endre J.

2016-11-01

The effect of oxidation, oxygenation, and de-oxygenation arising from He gas jet and He plasma jet treatments on the viability of skin cells cultured in vitro has been investigated. He gas jet treatment de-oxygenated cell culture medium in a process referred to as "sparging." He plasma jet treatments oxidized, as well as oxygenated or de-oxygenated cell culture medium depending on the dissolved oxygen concentration at the time of treatment. He gas and plasma jets were shown to have beneficial or deleterious effects on skin cells depending on the concentration of dissolved oxygen and other oxidative molecules at the time of treatment. Different combinations of treatments with He gas and plasma jets can be used to modulate the concentrations of dissolved oxygen and other oxidative molecules to influence cell viability. This study highlights the importance of a priori knowledge of the concentration of dissolved oxygen at the time of plasma jet treatment, given the potential for significant impact on the biological or medical outcome. Monitoring and controlling the dynamic changes in dissolved oxygen is essential in order to develop effective strategies for the use of cold atmospheric plasma jets in biology and medicine.

Measuring tendon properties in mdx mice: cell viability and viscoelastic characteristics.

PubMed

Rizzuto, E; Musarò, A; Catizone, A; Del Prete, Z

2009-10-16

Muscular dystrophy is a genetic disorder of skeletal muscle characterized by progressive muscle weakness. Here we assessed whether muscle wasting affects cell viability and mechanical properties of extensor digitorum longus (EDL) and of tibialis anterior (TA) tendons from mdx dystrophic mice compared to wild type (WT) mice. mdx mice represent the classical animal model for human Duchenne muscular dystrophy, and show several signs of the pathology, including a decrease in specific force and an increase of fibrotic index. Cell viability of tendons was evaluated by histological analysis, and viscoelastic properties have been assessed by a rapid measurement protocol that allowed us to compute, at the same time, tissue complex compliance for all the frequencies of interest. Confocal microscopy and mechanical properties measurements revealed that mdx tendons, compared to WT ones, have an increase in the number of dead cells and a significant reduction in tissue elasticity for all the frequencies that were tested. These findings indicate a reduced quality of the tissue. Moreover, mdx tendons have an increase in the viscous response, indicating that during dynamic loading, they dissipate more energy compared to WT. Our results demonstrate that muscular dystrophy involves not only muscle wasting, but also alteration in the viscoelastic properties of tendons, suggesting a paracrine effect of altered skeletal muscle on tendinous tissue.

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

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

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.

Release kinetics and cell viability of ibuprofen nanocrystals produced by melt-emulsification.

PubMed

Fernandes, A R; Dias-Ferreira, J; Cabral, C; Garcia, M L; Souto, E B

2018-06-01

The clinical use of poorly water-soluble drugs has become a big challenge in pharmaceutical development due to the compromised bioavailability of the drugs in vivo. Nanocrystals have been proposed as a formulation strategy to improve the dissolution properties of these drugs. The benefits of using nanocrystals in drug delivery, when compared to other nanoparticles, are related to their production facilities, simple structure, and suitability for a variety of administration routes. High pressure homogenization (HPH) is the most promising production process, which can be employed at low or high temperatures. Ibuprofen nanocrystals with a mean size below 175 nm, and polydispersity below 0.18, have been produced by melt-emulsification, followed by HPH. Two nanocrystal formulations, differing on the surfactant composition, have been produced, their in vitro ibuprofen release tested in Franz diffusion cells and adjusted to several kinetic models (zero order, first order, Higuchi, Hixson-Crowell, Korsmeyer-Peppas, Baker-Lonsdale and Weibull model). Cell viability was assessed at 3, 6 and 24 h of incubation on human epithelial colorectal cells (Caco-2) by AlamarBlue ® colorimetric assay. For both formulations, Caco-2 cells viability was dependent on the drug concentration and time of exposure. Copyright © 2018 Elsevier B.V. All rights reserved.

6-Gingerol inhibits osteosarcoma cell proliferation through apoptosis and AMPK activation.

PubMed

Fan, Jingzhang; Yang, Xin; Bi, Zhenggang

2015-02-01

6-Gingerol, a major component of ginger, is demonstrated to possess a variety of pharmacological activities. Despite demonstration of its anti-cancer activity, the exact mechanism underlying the effects of 6-gingerol against sarcoma remains sketchy. In the present study, we investigated the anti-cancer effects of 6-gingerol on osteosarcoma cells. MTT assay was performed to determine cell viability. Phosphorylation and protein levels were determined by immunoblotting. Cell cycle was determined using flow cytometry. Quantitative polymerase chain reaction was employed to determine the changes in the messenger RNA (mRNA) expression of genes. Treatment with 6-gingerol resulted in a significant decrease in the viability of osteosarcoma cells in a dose-dependent fashion. In parallel, the number of cells arrested at the sub-G1 cell cycle phase was significantly increased. The results showed that 6-gingerol induced activation of caspase cascades and regulated cellular levels of Bcl2 and Bax. Moreover, 6-gingerol activated AMP-activated protein kinase (AMPK) signaling associated with the apoptotic pathways. Our findings suggest that 6-gingerol suppresses the growth of osteosarcoma cells. The anti-cancer activity is attributed to the activation of apoptotic signaling and the inhibition of anti-apoptotic signaling incorporating with 6-gingerol-induced AMPK activation. The study identifies a new molecular mechanism by which AMPK is involved in anti-cancer effects of 6-gingerol.

In vitro effect of 4-nonylphenol on human chorionic gonadotropin (hCG) stimulated hormone secretion, cell viability and reactive oxygen species generation in mice Leydig cells.

PubMed

Jambor, Tomáš; Tvrdá, Eva; Tušimová, Eva; Kováčik, Anton; Bistáková, Jana; Forgács, Zsolt; Lukáč, Norbert

2017-03-01

Nonylphenol is considered an endocrine disruptor and has been reported to affect male reproductive functions. In our in vitro study, we evaluated the effects of 4-nonylphenol (4-NP) on cholesterol levels, hormone formation and viability in cultured Leydig cells from adult ICR male mice. We also determined the potential impact of 4-NP on generation of reactive oxygen species (ROS) after 44 h of cultivation. The cells were cultured with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 μg/mL of 4-NP in the present of 1 IU/mL human chorionic gonadotropin (hCG) and compared to the control. The quantity of cholesterol was determined from culture medium using photometry. Determination of hormone production was performed by enzyme-linked immunosorbent assay. Metabolic activity assay was used for quantification of cell viability. The chemiluminescence technique, which uses a luminometer to measure reactive oxygen species, was employed. Applied doses of 4-NP (0.04-5.0 μg/mL) slight increase cholesterol levels and decrease production of dehydroepiandrosterone after 44 h of cultivation, but not significantly. Incubation of 4-NP treated cells with hCG significantly (P < 0.001) inhibited androstenedione, but not testosterone, formation at the highest concentration (5.0 μg/mL). The viability was significantly (P < 0.05); (P < 0.001) increased at 1.0; 2.5 and 5.0 μg/mL of 4-NP after 44 h treatment. Furthermore, 44 h treatment of 4-NP (0.04-5.0 μg/mL) caused significant (P < 0.001) intracellular accumulation of ROS in exposed cells. Taken together, the results of our in vitro study reported herein is consistent with the conclusion that 4-nonylphenol is able to influence hormonal profile, cell viability and generate ROS. Copyright © 2017 Elsevier Ltd. All rights reserved.

The effect of UV-filters on the viability of neuroblastoma (SH-SY5Y) cell line.

PubMed

Broniowska, Żaneta; Pomierny, Bartosz; Smaga, Irena; Filip, Małgorzata; Budziszewska, Bogusława

2016-05-01

Topical application of cosmetic products, containing ultraviolet filters (UV filters) are recommended as a protection against sunburns and in order to reduce the risk of skin cancer. However, some UV filters can be absorbed through skin and by consuming contaminated food. Among the chemical UV filters, benzophenone-3 (BP-3), 3-(4-methylbenzylidene)camphor (4-MBC) and 2-ethylhexyl-4-methoxycinnamate (OMC) are absorbed through the skin to the greatest extent. So far, these lipophilic compounds were demonstrated to influence the gonadal and thyroid hormone function, but their effect on central nervous system cells has not been investigated, yet. In the present study, we investigated the effect of some UV filters on cell viability and caspase-3 activity in SH-SY5Y cells. It has been found that benzophenone-2 (BP-2), BP-3, 4-methylbenzophenone (4-MBP) and OMC present in the culture medium for 72h in high concentration (10(-5) and 10(-4)M) and 4-MBC only 10(-4)M produced a significant cytotoxic effect, as determined both by the MTT reduction test and LDH release assay. In contrast to necrotic changes, all tested UV filters increased caspase-3 activity in much lower concentrations (from 10(-8) to 10(-7)M). Proapoptotic properties of the test compounds were positively verified by Hoechst staining. The obtained results indicated that UV filters adversely affected the viability of nerve cells, most likely by enhancing the process of apoptosis. The most potent effect was exerted by BP-3 and 4-MBC and at concentrations that may be reached in vivo. Since human exposure to UV filters is significant these compound should be taken into consideration as one of the possible factors involved in pathogenesis of neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Low osmolality and shear stress during liposuction impair cell viability in autologous fat grafting.

PubMed

Ismail, T; Bürgin, J; Todorov, A; Osinga, R; Menzi, N; Largo, R D; Haug, M; Martin, I; Scherberich, A; Schaefer, D J

2017-05-01

Liposuction and subsequent autologous fat grafting have become essential techniques for fat augmentation in plastic surgery. However, standard harvesting techniques that ensure the survival of adipocytes and stromal vascular fraction (SVF) cells and thus preserve the transplanted fat volume are lacking. In particular, the effect of different parameters of the tumescent solution has not been studied in this context. We hypothesized that the osmolality of the tumescent solution could have a significant effect on the survival of adipocytes and SVF cells. We developed two distinct in vitro models based on freshly harvested excision fat from patients undergoing surgical treatment. First, we investigated the effect of osmolality by incubating excision fat in different tumescent solutions and analyzed the total cell survival and the differentiation potential of SVF cells. Vital whole-mount staining, isolation yield of SVF cells, clonogenicity, and osteogenic and adipogenic differentiation capacities were analyzed. Second, we addressed the additional effect of mechanical stress by simulating a liposuction on pieces of excision fat after incubation with the tumescent solutions. Osmolality of the tumescent solution by itself did not have a significant effect on adipocyte and SVF viability or SVF differentiation. However, when osmolality was combined with liposuction, a significant trend toward lower viability and more lipid droplets with lower osmolality was observed. Especially, SVF viability was significantly lower after liposuction with a hypotonic (150 mOsm/kg) solution. This study demonstrates the considerable effect of osmolality during liposuction and may lead to the development of "cell-protective" tumescent solutions. Copyright © 2017 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Thymoquinone Induces Cell Death in Human Squamous Carcinoma Cells via Caspase Activation-Dependent Apoptosis and LC3-II Activation-Dependent Autophagy

PubMed Central

Yu, Cheng-Chia; Lai, Yi-Yeh; Chen, Pei-Ni

2014-01-01

Background Thymoquinone (TQ), an active component of Nigella sativa or black cumin, elicits cytotoxic effects on various cancer cell lines. However, the anti-cancer effects of TQ on head and neck squamous cell carcinoma (HNSCC) remain unclear. Methodology/Principal Findings In this study, TQ elicited a strong cytotoxic effect on SASVO3, a highly malignant HNSCC cell line. The mechanisms of this cytotoxic effect were concentration dependent. TQ also induced apoptotic cell death in SASVO3 cells as indicated by an increase in Bax expression and caspase-9 activation. Apoptosis was possibly caspase-9 dependent because the exposure of cells to a caspase-9 inhibitor partially prevented cell death. The exposed cells also showed increased levels of autophagic vacuoles and LC3-II proteins, which are specific autophagy markers. Cell viability assay results further revealed that bafilomycin-A1, an autophagy inhibitor, enhanced TQ cytotoxicity; by comparison, Annexin V and propidium-iodide staining assay results showed that this inhibitor did not promote apoptosis. TQ treatment also increased the accumulation of autophagosomes. Using a lentivirus-shRNA system for LC3 silencing, we found that cell viability was eradicated in autophagy-defective cells. An in vivo BALB/c nude mouse xenograft model further showed that TQ administered by oral gavage reduced tumor growth via induced autophagy and apoptosis. Conclusions These findings indicated that TQ induced cell death in oral cancer cells via two distinct anti-neoplastic activities that can induce apoptosis and autophagy. Therefore, TQ is a promising candidate in phytochemical-based, mechanistic, and pathway-targeted cancer prevention strategies. PMID:25000169

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.

Influence of power ultrasound on the main quality properties and cell viability of osmotic dehydrated cranberries.

PubMed

Nowacka, Malgorzata; Fijalkowska, Aleksandra; Wiktor, Artur; Dadan, Magdalena; Tylewicz, Urszula; Dalla Rosa, Marco; Witrowa-Rajchert, Dorota

2018-02-01

The aim of the study was to investigate the effect of ultrasound treatment in two osmotic solutions, carried out at different time, on some physical properties, antioxidant activity and cell survival of cranberries. Ultrasound treatment was conducted at 21kHz for 30 and 60min in liquid medium: 61.5% sucrose solution and 30% sucrose solution with 0.1% steviol glycosides addition. Some samples before the ultrasound treatment were subjected to cutting or blanching. The results showed that dry matter content and concentration of the dissolved substances increased during ultrasound treatment in osmotic solution, however higher value was observed for treatment in 61.5% sucrose solution and for longer time. Water activity and volume of cranberries did not change after the ultrasonic treatment. Combined treatment led to colour and antioxidant activity alterations as well. A cell viability of whole and cut samples decreased after 60min of osmotic treatment and completely lost in the blanched samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids.

PubMed

Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2017-05-01

Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×10 5 (group A) or 8×10 5 (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular

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.

Ebselen alters mitochondrial physiology and reduces viability of rat hippocampal astrocytes.

PubMed

Santofimia-Castaño, Patricia; Salido, Ginés M; González, Antonio

2013-04-01

The seleno-organic compound and radical scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) have been extensively employed as an anti-inflammatory and neuroprotective compound. However, its glutathione peroxidase activity at the expense of cellular thiols groups could underlie certain deleterious actions of the compound on cell physiology. In this study, we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular viability, the intracellular free-Ca(2+) concentration ([Ca(2+)]c), the mitochondrial free-Ca(2+) concentration ([Ca(2+)]m), and mitochondrial membrane potential (ψm) were analyzed. The caspase-3 activity was also assayed. Our results show that cell viability was reduced by treatment of cells with ebselen, depending on the concentration employed. In the presence of ebselen, we observed an initial transient increase in [Ca(2+)]c that was then followed by a progressive increase to an elevated plateau. We also observed a transient increase in [Ca(2+)]m in the presence of ebselen that returned toward a value over the prestimulation level. The compound induced depolarization of ψm and altered the permeability of the mitochondrial membrane. Additionally, a disruption of the mitochondrial network was observed. Finally, we did not detect changes in caspase-3 activation in response to ebselen treatment. Collectively, these data support the likelihood of ebselen, depending on the concentration employed, reduces viability of rat hippocampal astrocytes via its action on the mitochondrial activity. These may be early effects that do not involve caspase-3 activation. We conclude that, depending on the concentration used, ebselen might exert deleterious actions on astrocyte physiology that could compromise cell function.

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.

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

Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

PubMed

Schapire, Arnaldo L; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A

2008-12-01

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.

Cannabinoids synergize with carfilzomib, reducing multiple myeloma cells viability and migration.

PubMed

Nabissi, Massimo; Morelli, Maria Beatrice; Offidani, Massimo; Amantini, Consuelo; Gentili, Silvia; Soriani, Alessandra; Cardinali, Claudio; Leoni, Pietro; Santoni, Giorgio

2016-11-22

Several studies showed a potential anti-tumor role for cannabinoids, by modulating cell signaling pathways involved in cancer cell proliferation, chemo-resistance and migration. Cannabidiol (CBD) was previously noted in multiple myeloma (MM), both alone and in synergy with the proteasome inhibitor bortezomib, to induce cell death. In other type of human cancers, the combination of CBD with Δ9-tetrahydrocannabinol (THC) was found to act synergistically with other chemotherapeutic drugs suggesting their use in combination therapy. In the current study, we evaluated the effects of THC alone and in combination with CBD in MM cell lines. We found that CBD and THC, mainly in combination, were able to reduce cell viability by inducing autophagic-dependent necrosis. Moreover, we showed that the CBD-THC combination was able to reduce MM cells migration by down-regulating expression of the chemokine receptor CXCR4 and of the CD147 plasma membrane glycoprotein. Furthermore, since the immuno-proteasome is considered a new target in MM and also since carfilzomib (CFZ) is a new promising immuno-proteasome inhibitor that creates irreversible adducts with the β5i subunit of immuno-proteasome, we evaluated the effect of CBD and THC in regulating the expression of the β5i subunit and their effect in combination with CFZ. Herein, we also found that the CBD and THC combination is able to reduce expression of the β5i subunit as well as to act in synergy with CFZ to increase MM cell death and inhibits cell migration. In summary, these results proved that this combination exerts strong anti-myeloma activities.

Comparative Effect Between Laser and Radiofrequency Heating of RGD-Gold Nanospheres on MCF7 Cell Viability.

PubMed

Sánchez-Hernández, Lidia; Ferro-Flores, Guillermina; Jiménez-Mancilla, Nallely P; Luna-Gutiérrez, Myrna A; Santos-Cuevas, Clara L; Ocampo-García, Blanca E; Azorín-Vega, Erika; Isaac-Olivé, Keila

2015-12-01

Gold nanoparticles conjugated to cyclo-[Arg-Gly-Asp-D-Phe-Lys(Cys)] peptides (AuNP-c[RGDfK(C)]) have been reported as systems with specific cell internalization in breast cancer cells. AuNPs have also been proposed as localized heat sources for cancer treatment using laser irradiation or radiofrequency (RF). The aim of this research was to analyze, based on the Mie theory, the AuNP-c[RGDfK(C)] absorption cross-sections (C(abs)) of low-frequency electromagnetic waves (13.56 MHz, λ = 22 m) and optical frequency waves (laser at λ = 532 nm) and to compare their effect on MCF7 cell viability as thermal conversion sources in AuNPs (20 nm) located inside cells. Cell viability was assessed in MCF7 cells treated with AuNP-c[RGDfK(C)] or water after exposure to the RF field (200 W, 100 V/cm) or laser irradiation (Irradiance 0.65 W/cm2). In both cases (RF and laser) the presence of nanoparticles in cells caused a significant increase in the temperature of the medium (RF: AT = 29.9 ± 1.7 degrees C for AuNP compared to ΔT = 13.0 ± 1.4 degrees C for water; laser: ΔT = 13.5 ± 0.7 degrees C for AuNP compared to 3.3 ± 0.5 degrees C for water). Although RF induced a higher increase in the temperature of the medium with nanoparticles, the largest effect on the cell viability was produced by laser when nanoparticles were located inside the cells (8.7?0.7% for laser compared to 19.4 ± 0.9% for RF). The differences obtained in C(abs) values (laser: 3.7 x 10- (16) m2; RF: 7.9 x 10-(23) m2) and the observed effect on MFC7 cell viability support two mechanisms previously proposed "wave energy absorption by AuNPs" when laser is used as a thermal conversion source, and "attenuation of the wave passing through the AuNP suspension" when RF is applied. The AuNP-c[RGDfK(C)] nanosystem shows suitable properties to improve hyperthermia treatments under laser irradiation due to a larger heat release inside cells.

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

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

PubMed Central

Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

2015-01-01

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

The relationship between viability and intracellular pH in the yeast Saccharomyces cerevisiae.

PubMed Central

Imai, T; Ohno, T

1995-01-01

The relationship between viability (cell proliferation activity) and intracellular pH in the yeast Saccharomyces cerevisiae was investigated by using cells that had been deactivated by low-temperature storage, ethanol treatment, or heat treatment. The intracellular pH was measured with a microscopic image processor or a spectrofluorophotometer. At first, the intracellular pH measurements of individual cells were compared with slide culture results by microscopic image processing. A clear correlation existed between the proliferation activity and intracellular pH. Moreover, by spectrofluorophotometry analysis, it was found that there was a relationship between the viability and intracellular pH of brewing yeast under conditions of low external pH (n = 15, r = 0.960, P = 0.001). This relationship was also observed in baker's yeast (n = 13, r = 0.950, P = 0.001). On the other hand, when the fluorescein staining method was used in these experiments, the relationship between viability and staining percentage was not observed. From these results, intracellular pH was found to be a sensitive factor for estimating yeast physiology. The possible role of cell deterioration is also discussed. PMID:7486996

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.

Divergent branches of mitochondrial signaling regulate specific genes and the viability of specialized cell types of differentiated yeast colonies.

PubMed

Podholová, Kristýna; Plocek, Vítězslav; Rešetárová, Stanislava; Kučerová, Helena; Hlaváček, Otakar; Váchová, Libuše; Palková, Zdena

2016-03-29

Mitochondrial retrograde signaling mediates communication from altered mitochondria to the nucleus and is involved in many normal and pathophysiological changes, including cell metabolic reprogramming linked to cancer development and progression in mammals. The major mitochondrial retrograde pathway described in yeast includes three activators, Rtg1p, Rtg2p and Rtg3p, and repressors, Mks1p and Bmh1p/Bmh2p. Using differentiated yeast colonies, we show that Mks1p-Rtg pathway regulation is complex and includes three branches that divergently regulate the properties and fate of three specifically localized cell subpopulations via signals from differently altered mitochondria. The newly identified RTG pathway-regulated genes ATO1/ATO2 are expressed in colonial upper (U) cells, the cells with active TORC1 that metabolically resemble tumor cells, while CIT2 is a typical target induced in one subpopulation of starving lower (L) cells. The viability of the second L cell subpopulation is strictly dependent on RTG signaling. Additional co-activators of Rtg1p-Rtg3p specific to particular gene targets of each branch are required to regulate cell differentiation.

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.

Ultrasound-microbubble mediated cavitation of plant cells: effects on morphology and viability.

PubMed

Qin, Peng; Xu, Lin; Zhong, Wenjing; Yu, Alfred C H

2012-06-01

The interaction between ultrasound pulses and microbubbles is known to generate acoustic cavitation that may puncture biological cells. This work presents new experimental findings on the bioeffects of ultrasound-microbubble mediated cavitation in plant cells with emphasis on direct observations of morphological impact and analysis of viability trends in tobacco BY-2 cells that are widely studied in higher plant physiology. The tobacco cell suspensions were exposed to 1 MHz ultrasound pulses in the presence of 1% v/v microbubbles (10% duty cycle; 1 kHz pulse repetition frequency; 70 mm between probe and cells; 1-min exposure time). Few bioeffects were observed at low peak negative pressures (<0.4 MPa) where stable cavitation presumably occurred. In contrast, at 0.9 MPa peak negative pressure (with more inertial cavitation activities according to our passive cavitation detection results), random pores were found on tobacco cell wall (observed via scanning electron microscopy) and enhanced exogenous uptake into the cytoplasm was evident (noted in our fluorescein isothiocyanate dextran uptake analysis). Also, instant lysis was observed in 23.4% of cells (found using trypan blue staining) and programmed cell death was seen in 23.3% of population after 12 h (determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]). These bioeffects generally correspond in trend with those for mammalian cells. This raises the possibility of developing ultrasound-microbubble mediated cavitation into a targeted gene transfection paradigm for plant cells and, conversely, adopting plant cells as experimental test-beds for sonoporation-based gene therapy in mammalian cells. Copyright © 2012 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

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

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.

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 Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

PubMed Central

Varghese, Divya S.; Parween, Shama; Ardah, Mustafa T.; Emerald, Bright Starling

2017-01-01

Human embryonic stem cells (hESCs) are being used extensively in array of studies to understand different mechanisms such as early human embryogenesis, drug toxicity testing, disease modeling, and cell replacement therapy. The protocols for the directed differentiation of hESCs towards specific cell types often require long-term cell cultures. To avoid bacterial contamination, these protocols include addition of antibiotics such as pen-strep and gentamicin. Although aminoglycosides, streptomycin, and gentamicin have been shown to cause cytotoxicity in various animal models, the effect of these antibiotics on hESCs is not clear. In this study, we found that antibiotics, pen-strep, and gentamicin did not affect hESC cell viability or expression of pluripotency markers. However, during directed differentiation towards neural and hepatic fate, significant cell death was noted through the activation of caspase cascade. Also, the expression of neural progenitor markers Pax6, Emx2, Otx2, and Pou3f2 was significantly reduced suggesting that gentamicin may adversely affect early embryonic neurogenesis whereas no effect was seen on the expression of endoderm or hepatic markers during differentiation. Our results suggest that the use of antibiotics in cell culture media for the maintenance and differentiation of hESCs needs thorough investigation before use to avoid erroneous results. PMID:29147115

Cell viability of mycorrhiza helper bacteria solid inoculant in different carrier material

NASA Astrophysics Data System (ADS)

Asyiah, Iis Nur; Hindersah, Reginawanti; Harni, Rita

2018-02-01

Roots of food crops are colonized by nonpathogenic mycorrhizal fungi which show natural ability to control plant pathogen. Mycorrhizal establishment in plant roots is affected by rhizobacteria, known as mycorrhiza helper bacteria (MHB), which has synergetic effects on mycorrhizal associations. Laboratory experiment has been conducted to assess the best carrier material to develop well-qualified MHB of Pseudomonas diminuta and Bacillus subtilis solid inoculant. Carrier materials were 100 mesh organic matter of agricultural waste. Different spore concentration of both bacterial liquid inoculants were grown on three kinds of 100-mesh organic matter and stored at room temperature up to 90 days. Cell viability of both MHB were counted by serial dilution plate method by using specific medium. The results showed that sugar cane baggase ash was the best carrier material to maintain cell viability for both MHB. However, the population of Pseudomonas diminuta and Bacillus subtilis in sugar cane baggase ash were slightly decreased after 90 days. The use of sugarcane baggase ash for solid MHB inoculant development could be suggested.

Effects of indirubin and isatin on cell viability, mutagenicity, genotoxicity and BAX/ERCC1 gene expression.

PubMed

Fogaça, Manoela Viar; Cândido-Bacani, Priscila de Matos; Benicio, Lucas Milanez; Zapata, Lara Martinelli; Cardoso, Priscilla de Freitas; de Oliveira, Marcelo Tempesta; Calvo, Tamara Regina; Varanda, Eliana Aparecida; Vilegas, Wagner; de Syllos Cólus, Ilce Mara

2017-12-01

Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood. We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes. HeLa and/or CHO-K1 cell lines were tested (3 or 24 h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72 h) tests after treatment with IRN (0.1 to 200 μM) or ISA (0.5 to 50 μM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24 h) by gavage (50, 100 and 150 mg/kg determined from the LD 50 - 1 g/kg b.w.) and submitted to comet assay in vivo. IRN reduced the viability of CHO-K1 (24 h; 5 to 200 μM) and HeLa cells (10 to 200 μM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10 μM; HeLa: 5 and 10 μM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24 h) at all doses tested. IRN (100 and 150 mg/kg) also induced genotoxicity in vivo (4 h). IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.

Effects of hydrostatic pressure and supercritical carbon dioxide on the viability of Botryococcus braunii algae cells.

PubMed

Yildiz-Ozturk, Ece; Ilhan-Ayisigi, Esra; Togtema, Arnoud; Gouveia, Joao; Yesil-Celiktas, Ozlem

2018-05-01

In bio-based industries, Botryococcus braunii is identified as a potential resource for production of hydrocarbons having a wide range of applications in chemical and biopolymer industries. For a sustainable production platform, the algae cultivation should be integrated with downstream processes. Ideally the algae are not harvested, but the product is isolated while cultivation and growth is continued especially if the doubling time is slow. Consequently, hydrocarbons can be extracted while keeping the algae viable. In this study, the effects of pressure on the viability of B. braunii cells were tested hydrostatically and under supercritical CO 2 conditions. Viability was determined by light microscopy, methylene blue uptake and by re-cultivation of the algae after treatments to follow the growth. It was concluded that supercritical CO 2 was lethal to the algae, whereas hydrostatic pressure treatments up to 150 bar have not affected cell viability and recultivation was successful. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synergistic effects of hydrogen peroxide and ethanol on cell viability loss in PC12 cells by increase in mitochondrial permeability transition.

PubMed

Lee, Chung Soo; Kim, Yun Jeong; Ko, Hyun Hee; Han, Eun Sook

2005-07-15

The promoting effect of ethanol against the cytotoxicity of hydrogen peroxide (H2O2) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with H2O2 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. In PC12 cells and dopaminergic neuroblastoma SH-SY5Y cells, the promoting effect of ethanol on the H2O2-induced cell death was increased with exposure time. Ethanol promoted the nuclear damage, change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to H2O2 in PC12 cells. Catalase, carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the H2O2 and ethanol-induced mitochondrial dysfunction and cell injury. The results show that the ethanol treatment promotes the cytotoxicity of H2O2 against PC12 cells. Ethanol may enhance the H2O2-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that ethanol as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by oxidants.

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.

Investigation of cell viability and morphology in 3D bio-printed alginate constructs with tunable stiffness.

PubMed

Shi, Pujiang; Laude, Augustinus; Yeong, Wai Yee

2017-04-01

In this article, mouse fibroblast cells (L929) were seeded on 2%, 5%, and 10% alginate hydrogels, and they were also bio-printed with 2%, 5%, and 10% alginate solutions individually to form constructs. The elastic and viscous moduli of alginate solutions, their interior structure and stiffness, interactions of cells and alginate, cell viability, migration and morphology were investigated by rheometer, MTT assay, scanning electron microscope (SEM), and fluorescent microscopy. The three types of bio-printed scaffolds of distinctive stiffness were prepared, and the seeded cells showed robust viability either on the alginate hydrogel surfaces or in the 3D bio-printed constructs. Majority of the proliferated cells in the 3D bio-printed constructs weakly attached to the surrounding alginate matrix. The concentration of alginate solution and hydrogel stiffness influenced cell migration and morphology, moreover the cells formed spheroids in the bio-printed 10% alginate hydrogel construct. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1009-1018, 2017. © 2017 Wiley Periodicals, Inc.

Bee venom induced cytogenetic damage and decreased cell viability in human white blood cells after treatment in vitro: a multi-biomarker approach.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2011-09-01

The aim of this study was to evaluate cytogenotoxic effects of bee venom to human lymphocytes and take a look into the mechanisms behind them. Bee venom was tested in concentrations ranging from 0.1μg/ml to 20μg/ml over different lengths of time. Cell viability, type of the cell death, and morphological alterations were evaluated using phase-contrast and fluorescent microscopy in addition to DNA diffusion assay, whereas cytogenotoxic effects were assessed with the micronucleus test. DNA damage and its relation to oxidative stress were evaluated combining the standard alkaline and the Fpg-modified comet assay. Our results showed lower cell viability, morphological cell alterations, cytogenotoxicity, and dominantly necrotic type of cell death in human lymphocytes after treatment with bee venom. All the effects were time- and dose-dependent. These results provide an insight into the effects of bee venom on the cell structure that could be relevant for therapeutic purposes. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of Various Concentrations of Antibiotics on Osteogenic Cell Viability and Activity

DTIC Science & Technology

2011-07-01

antibiotic-supple- mented bone allografts in the guinea pig . J Oral Maxillofac Surg 42:631–636. 32. McKeeMD,WildLM,SchemitschEH, et al. 2002. Theuse of an...and Antibiotic Treatments Human osteoblasts (Promocell, Heidelberg, Germany) were maintained in media consisting of alpha-MEM containing 10% fetal calf...that when antibiotics are extremely toxic to cells, subtle differences between metabolic activity and overt cell death are not discernable

Effect of silica nanoparticles with variable size and surface functionalization on human endothelial cell viability and angiogenic activity

NASA Astrophysics Data System (ADS)

Guarnieri, Daniela; Malvindi, Maria Ada; Belli, Valentina; Pompa, Pier Paolo; Netti, Paolo

2014-02-01

Silica nanoparticles could be promising delivery vehicles for drug targeting or gene therapy. However, few studies have been undertaken to determine the biological behavior effects of silica nanoparticles on primary endothelial cells. Here we investigated uptake, cytotoxicity and angiogenic properties of silica nanoparticle with positive and negative surface charge and sizes ranging from 25 to 115 nm in primary human umbilical vein endothelial cells. Dynamic light scattering measurements and nanoparticle tracking analysis were used to estimate the dispersion status of nanoparticles in cell culture media, which was a key aspect to understand the results of the in vitro cellular uptake experiments. Nanoparticles were taken up by primary endothelial cells in a size-dependent manner according to their degree of agglomeration occurring after transfer in cell culture media. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake, compared to negatively charged nanoparticles. However, this effect was contrasted by the tendency of particles to form agglomerates, leading to lower internalization efficiency. Silica nanoparticle uptake did not affect cell viability and cell membrane integrity. More interestingly, positively and negatively charged 25 nm nanoparticles did not influence capillary-like tube formation and angiogenic sprouting, compared to controls. Considering the increasing interest in nanomaterials for several biomedical applications, a careful study of nanoparticle-endothelial cells interactions is of high relevance to assess possible risks associated to silica nanoparticle exposure and their possible applications in nanomedicine as safe and effective nanocarriers for vascular transport of therapeutic agents.

Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements.

PubMed

Bhatia, Sujata K; Yetter, Ann B

2008-08-01

Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.

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.

The effect of TRAIL molecule on cell viability in in vitro beta cell culture.

PubMed

Tekmen, I; Ozyurt, D; Pekçetin, C; Buldan, Z

2007-06-01

Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disorder triggered by autoreactive T cells directed to pancreas beta-cell antigens. In this disorder, more than 90% of beta cells are destroyed. Cell death may be mediated via soluble or membrane-bound cell death ligands. One of these ligands may be tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF-alpha superfamily. In the present study, we examined whether TRAIL had cytotoxic effects on adult rat pancreas beta cell cultures and INS1-E rat insulinoma cell line cultures or not. In this study, cell destruction models were built with TRAIL concentrations of 10, 100 and 1000 ng. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for evaluating cell viability. It was detected that cell cultures with TRAIL added showed no differences statistically when compared with control cultures containing no toxic additions. These results showed that TRAIL did not have significant cytotoxic effects on pancreas beta cell culture and INS-1E rat insulinoma cell line cultures. Detection of the expression of TRAIL receptors and natural apoptosis inhibitor proteins will be favourable to investigate the resistance mechanisms to TRAIL-induced cell death in this cell culture system.

Influence of shear stress and size on viability of endothelial cells exposed to gold nanoparticles

NASA Astrophysics Data System (ADS)

Fede, C.; Albertin, Giovanna; Petrelli, L.; De Caro, R.; Fortunati, I.; Weber, V.; Ferrante, Camilla

2017-09-01

Screening nanoparticle toxicity directly on cell culture can be a fast and cheap technique. Nevertheless, to obtain results in accordance with those observed in live animals, the conditions in which cells are cultivated should resemble the one encountered in live systems. Microfluidic devices offer the possibility to satisfy this requirement, in particular with endothelial cell lines, because they are capable to reproduce the flowing media and shear stress experienced by these cell lines in vivo. In this work, we exploit a microfluidic device to observe how human umbilical vein endothelial cells (HUVEC) viability changes when subject to a continuous flow of culture medium, in which spherical citrate-stabilized gold nanoparticles of different sizes and at varying doses are investigated. For comparison, the same experiments are also run in multiwells where the cells do not experience the shear stress induced by the flowing medium. We discuss the results considering the influence of mode of exposure and nanoparticle size (24 and 13 nm). We observed that gold nanoparticles show a lower toxicity under flow conditions with respect to static and the HUVEC viability decreases as the nanoparticle surface area per unit volume increases, regardless of size.

Effects of four commonly used UV filters on the growth, cell viability and oxidative stress responses of the Tetrahymena thermophila.

PubMed

Gao, Li; Yuan, Tao; Zhou, Chuanqi; Cheng, Peng; Bai, Qifeng; Ao, Junjie; Wang, Wenhua; Zhang, Haimou

2013-11-01

UV filters are increasingly used in sunscreens and other personal care products. Although their residues have been widely identified in aquatic environment, little is known about the influences of UV filters to protozoan. The growth inhibition effects, cell viability and oxidative stress responses of four commonly used UV filters, 2-ethylhexyl 4-methoxycinnamate (EHMC), benzophenone-3 (BP-3), 4-methyl-benzylidene camphor (4-MBC) and octocrylene (OC), to protozoan Tetrahymena thermophila were investigated in this study. The 24-h EC50 values with 95% confidence intervals for BP-3 and 4-MBC were 7.544 (6.561-8.675) mg L(-1) and 5.125 (4.874-5.388) mg L(-1), respectively. EHMC and OC did not inhibit the growth of T. thermophila after 24h exposure at the tested concentrations. The results of cell viability assays with propidium iodide (PI) staining were consistent with that of the growth inhibition tests. As for BP-3 and 4-MBC, the relatively higher concentrations, i.e. of 10.0 and 15.0 mg L(-1), could lead to the cell membranes impairment after 4h exposure. With the increase of the exposure time to 6h, their adverse effects on cell viability of T. thermophila were observed at the relatively lower concentration groups (1.0 mg L(-1) and 5.0 mg L(-1)). In addition, it is noticeable that at environmentally relevant concentration (1.0 μg L(-1)), BP-3 and 4-MBC could lead to the significant increase of catalase (CAT) activities of the T. thermophila cells. Especially for the BP-3, the oxidative injuries were further confirmed by the reduction of glutathione (GSH) content. It is imperative to further investigate the additive action of UV filters and seek other sensitive endpoint, especially at environmentally relevant concentration. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells.

PubMed

Wang, Yan-hong; Li, Ke-jin; Mao, Li; Hu, Xin; Zhao, Wen-jie; Hu, An; Lian, Hong-zhen; Zheng, Wei-juan

2013-09-01

As a non-toxic metal to humans, zinc is essential for cell proliferation, differentiation, regulation of DNA synthesis, genomic stability and mitosis. Zinc homeostasis in cells, which is crucial for normal cellular functioning, is maintained by various protein families including ZnT (zinc transporter/SLC30A) and ZIP (Zrt-, Irt-like proteins/SLC39A) that decrease and increase cytosolic zinc availability, respectively. In this study, we investigated the influences of a specific concentration range of ZnSO4 on cell cycle and apoptosis by flow cytometry, and cell viability by MTT method in MDAMB231, HepG2 and 293 T cell lines. Fluorescent sensors NBD-TPEA and the counterstain for nuclei Hoechst 33342 were used to stain the treated cells for observing the localisation and amount of Zn(2+) via laser scanning confocal microscope. It was found that the influence manners of ZnSO4 on cell cycle, apoptosis and cell viability in various cell lines were different and corresponding to the changes of Zn(2+) content of the three cell lines, respectively. The significant increase on intracelluar zinc content of MDAMB231 cells resulted in cell death, G1 and G2/M cell cycle arrest and increased apoptotic fraction. Additionally, the mRNA expression levels of ZnT and ZIP families in the three cell lines, when treated with high concentration of ZnSO4, increased and decreased corresponding to their functions, respectively.

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.

A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.

PubMed

Mitsuki, Yu-Ya; Yamamoto, Takuya; Mizukoshi, Fuminori; Momota, Masatoshi; Terahara, Kazutaka; Yoshimura, Kazuhisa; Harada, Shigeyoshi; Tsunetsugu-Yokota, Yasuko

2016-05-01

Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability. Copyright © 2016 Elsevier B.V. All rights reserved.

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

PubMed Central

Ortiz-Espín, Ana; Locato, Vittoria; Camejo, Daymi; Schiermeyer, Andreas; De Gara, Laura; Sevilla, Francisca; Jiménez, Ana

2015-01-01

Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. Conclusions A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment.

PubMed

Ortiz-Espín, Ana; Locato, Vittoria; Camejo, Daymi; Schiermeyer, Andreas; De Gara, Laura; Sevilla, Francisca; Jiménez, Ana

2015-09-01

Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative

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

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.

Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality.

PubMed

Angel, Stephanie; von Briesen, Hagen; Oh, Young-Joo; Baller, Marko K; Zimmermann, Heiko; Germann, Anja

2016-12-01

Cryopreservation of biological materials such as cells, tissues, and organs is a prevailing topic of high importance. It is employed not only in many research fields but also in the clinical area. Cryopreservation is of great importance for reproductive medicine and clinical studies, as well as for the development of vaccines. Peripheral blood mononuclear cells (PBMCs) are commonly used in vaccine research where comparable and reliable results between different research institutions and laboratories are of high importance. Whereas freezing and thawing processes are well studied, controlled, and standardized, storage conditions are often disregarded. To close this gap, we investigated the influence of suboptimal storage conditions during low-temperature storage on PBMC viability, recovery, and T cell functionality. For this purpose, PBMCs were isolated and exposed with help of a robotic system in a low-temperature environment from 0 up to 350 temperature fluctuation cycles in steps of 50 cycles to simulate storage conditions in large biorepositories with sample storage, removal, and sorting functions. After the simulation, the viability, recovery, and T cell functionality were analyzed to determine the number of temperature rises, which ultimately lead to significant cell damage. All studied parameters decreased with increasing number of temperature cycles. Sometimes after as little as only 50 temperature cycles, a significant effect was observed. These results are very important for all fields in which cell cryopreservation is employed, particularly for clinical and multicenter studies wherein the comparability and reproducibility of results play a crucial role. To obtain reliable results and to maintain the quality of the cells, not only the freezing and thawing processes but also the storage conditions should be controlled and standardized, and any deviations should be documented.

Effects of a discoloration-resistant calcium aluminosilicate cement on the viability and proliferation of undifferentiated human dental pulp stem cells.

PubMed

Niu, Li-na; Watson, Devon; Thames, Kyle; Primus, Carolyn M; Bergeron, Brian E; Jiao, Kai; Bortoluzzi, Eduardo A; Cutler, Christopher W; Chen, Ji-hua; Pashley, David H; Tay, Franklin R

2015-11-30

Discoloration-resistant calcium aluminosilicate cement has been formulated to overcome the timely problem of tooth discoloration reported in the clinical application of bismuth oxide-containing hydraulic cements. The present study examined the effects of this experimental cement (Quick-Set2) on the viability and proliferation of human dental pulp stem cells (hDPSCs) by comparing the cellular responses with commercially available calcium silicate cement (white mineral trioxide aggregate; WMTA) after different aging periods. Cell viability and proliferation were examined using assays that examined plasma membrane integrity, leakage of cytosolic enzyme, caspase-3 activity for early apoptosis, oxidative stress, mitochondrial metabolic activity and intracellular DNA content. Results of the six assays indicated that both Quick-Set2 and WMTA were initially cytotoxic to hDPSCs after setting for 24 h, with Quick-Set2 being comparatively less cytotoxic than WMTA at this stage. After two aging cycles, the cytotoxicity profiles of the two hydraulic cements were not significantly different and were much less cytotoxic than the positive control (zinc oxide-eugenol cement). Based on these results, it is envisaged that any potential beneficial effect of the discoloration-resistant calcium aluminosilicate cement on osteogenesis by differentiated hDPSCs is more likely to be revealed after outward diffusion and removal of its cytotoxic components.

Effects of a discoloration-resistant calcium aluminosilicate cement on the viability and proliferation of undifferentiated human dental pulp stem cells

PubMed Central

Niu, Li-na; Watson, Devon; Thames, Kyle; Primus, Carolyn M.; Bergeron, Brian E.; Jiao, Kai; Bortoluzzi, Eduardo A.; Cutler, Christopher W.; Chen, Ji-hua; Pashley, David H.; Tay, Franklin R.

2015-01-01

Discoloration-resistant calcium aluminosilicate cement has been formulated to overcome the timely problem of tooth discoloration reported in the clinical application of bismuth oxide-containing hydraulic cements. The present study examined the effects of this experimental cement (Quick-Set2) on the viability and proliferation of human dental pulp stem cells (hDPSCs) by comparing the cellular responses with commercially available calcium silicate cement (white mineral trioxide aggregate; WMTA) after different aging periods. Cell viability and proliferation were examined using assays that examined plasma membrane integrity, leakage of cytosolic enzyme, caspase-3 activity for early apoptosis, oxidative stress, mitochondrial metabolic activity and intracellular DNA content. Results of the six assays indicated that both Quick-Set2 and WMTA were initially cytotoxic to hDPSCs after setting for 24 h, with Quick-Set2 being comparatively less cytotoxic than WMTA at this stage. After two aging cycles, the cytotoxicity profiles of the two hydraulic cements were not significantly different and were much less cytotoxic than the positive control (zinc oxide–eugenol cement). Based on these results, it is envisaged that any potential beneficial effect of the discoloration-resistant calcium aluminosilicate cement on osteogenesis by differentiated hDPSCs is more likely to be revealed after outward diffusion and removal of its cytotoxic components. PMID:26617338

Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

PubMed

Hua, Sui Sheng T; Hernlem, Bradley J; Yokoyama, Wallace; Sarreal, Siov Bouy L

2015-05-01

Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.

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.

Chorein interacts with α-tubulin and histone deacetylase 6, and overexpression preserves cell viability during nutrient deprivation in human embryonic kidney 293 cells.

PubMed

Sasaki, Natsuki; Nakamura, Masayuki; Kodama, Akiko; Urata, Yuka; Shiokawa, Nari; Hayashi, Takehiro; Sano, Akira

2016-11-01

The autophagy pathway has recently been implicated in several neurodegenerative diseases. Recently, it was reported that chorein-depleted cells showed accumulation of autophagic markers and impaired autophagic flux. Here, we demonstrate that chorein overexpression preserves cell viability from starvation-induced cell death in human embryonic kidney 293 (HEK293) cells. Subsequent coimmunoprecipitation and reverse coimmunoprecipitation assays using extracts from chorein that stably overexpressed HEK293 cells revealed that chorein interacts with α-tubulin and histone deacetylase 6, a known α-tubulin deacetylater and central component of basal autophagy. Indeed, acetylated α-tubulin immunoreactivity was significantly decreased in chorein that stably overexpressed HEK293 cells. These results suggest that chorein/histone deacetylase 6/α-tubulin interactions may play an important role in starvation-induced cell stress, and their disruption may be one of the molecular pathogenic mechanisms of chorea-acanthocytosis.-Sasaki, N., Nakamura, M., Kodama, A., Urata, Y., Shiokawa, N., Hayashi, T., Sano, A. Chorein interacts with α-tubulin and histone deacetylase 6, and overexpression preserves cell viability during nutrient deprivation in human embryonic kidney 293 cells. © FASEB.

Concentrations of and application protocols for hydrogen peroxide bleaching gels: effects on pulp cell viability and whitening efficacy.

PubMed

Soares, Diana Gabriela; Basso, Fernanda Gonçalves; Hebling, Josimeri; de Souza Costa, Carlos Alberto

2014-02-01

To assess the whitening effectiveness and the trans-enamel/trans-dentinal toxicity of experimental tooth-bleaching protocols on pulp cells. Enamel/dentine discs individually adapted to trans-well devices were placed on cultured odontoblast-like cells (MDPC-23) or human dental pulp cells (HDPCs). The following groups were formed: G1 - no treatment (control); G2 to G4 - 35% H2O2, 3 × 15, 1 × 15, and 1 × 5 min, respectively; and G5 to G7 - 17.5% H2O2, 3 × 15, 1 × 15, and 1 × 5 min, respectively. Cell viability and morphology were evaluated immediately after bleaching (T1) and 72 h thereafter (T2). Oxidative stress and cell membrane damage were also assessed (T1). The amount of H2O2 in culture medium was quantified (Mann-Whitney; α=5%) and colour change (ΔE) of enamel was analysed after 3 sessions (Tukey's test; α=5%). Cell viability reduction, H2O2 diffusion, cell morphology alteration, oxidative stress, and cell membrane damage occurred in a concentration-/time-dependent fashion. The cell viability reduction was significant in all groups for HDPCs and only for G2, G3, and G5 in MDPC-23 cells compared with G1. Significant cell viability and morphology recovery were observed in all groups at T2, except for G2 in HDPCs. The highest ΔE value was found in G2. However, all groups presented significant ΔE increases compared with G1. Shortening the contact time of a 35%-H2O2 gel for 5 min, or reducing its concentration to 17.5% and applying it for 45, 15, or 5 min produce gradual tooth colour change associated with reduced trans-enamel and trans-dentinal cytotoxicity to pulp cells. The experimental protocols tested in the present study provided significant tooth-bleaching improvement associated with decreased toxicity to pulp cells, which may be an interesting alternative to be tested in clinical situations intended to reduce tooth sensitivity and pulp damage. Copyright © 2013 Elsevier Ltd. All rights reserved.

Morphological variability, lectin binding and Na+,K+-activated adenosine triphosphatase activity of isolated Müller (glial) cells from the rabbit retina.

PubMed

Reichenbach, A; Dettmer, D; Brückner, G; Neumann, M; Birkenmeyer, G

1985-03-22

Rabbit retinal Müller cells were isolated by means of papaine and mechanical dissociation. These cells were shown to have a well preserved morphology and to preserve viability for many hours. Intense wheat germ agglutinin binding occurs on the photoreceptor side of Müller cells, especially in the microvillous region. Rabbit retinal Müller cells have a Na+,K+-activated adenosine triphosphatase activity in the same order of magnitude as brain astroglial cells.

Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro

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

Tiwari, Kirti Kumar; Chu, Chun; Couroucli, Xanthi

Highlights: • Caffeine at 0.05 mM decreases oxidative stress in hyperoxia. • Caffeine at 1 mM decreases cell viability, increases oxidative stress in hyperoxia. • Caffeine at 1 but not 0.05 mM, abrogates hyperoxia-induced G2/M arrest. - Abstract: Caffeine is used to prevent bronchopulmonary dysplasia (BPD) in premature neonates. Hyperoxia contributes to the development of BPD, inhibits cell proliferation and decreases cell survival. The mechanisms responsible for the protective effect of caffeine in pulmonary oxygen toxicity remain largely unknown. A549 and MLE 12 pulmonary epithelial cells were exposed to hyperoxia or maintained in room air, in the presence of differentmore » concentrations (0, 0.05, 0.1 and 1 mM) of caffeine. Caffeine had a differential concentration-specific effect on cell cycle progression, oxidative stress and viability, with 1 mM concentration being deleterious and 0.05 mM being protective. Reactive oxygen species (ROS) generation during hyperoxia was modulated by caffeine in a similar concentration-specific manner. Caffeine at 1 mM, but not at the 0.05 mM concentration decreased the G2 arrest in these cells. Taken together this study shows the novel funding that caffeine has a concentration-specific effect on cell cycle regulation, ROS generation, and cell survival in hyperoxic conditions.« less

Impact of ZnO and Ag Nanoparticles on Bacterial Growth and Viability

NASA Astrophysics Data System (ADS)

Olson, M. S.; Digiovanni, K. A.

2007-12-01

Hundreds of consumer products containing nanomaterials are currently available in the U.S., including computers, clothing, cosmetics, sports equipment, medical devices and product packaging. Metallic nanoparticles can be embedded in or coated on product surfaces to provide antimicrobial, deodorizing, and stain- resistant properties. Although these products have the potential to provide significant benefit to the user, the impact of these products on the environment remains largely unknown. The purpose of this project is to study the effect of metallic nanoparticles released to the environment on bacterial growth and viability. Inhibition of bacterial growth was tested by adding doses of suspended ZnO and Ag nanoparticles into luria broth prior to inoculation of Escherichia coli cells. ZnO particles (approximately 40 nm) were obtained commercially and Ag particles (12-14 nm) were fabricated by reduction of silver nitrate with sodium borohydride. Toxicity assays were performed to test the viability of E. coli cells exposed to both ZnO and Ag nanoparticles using the LIVE/DEAD BacLight bacterial viability kit (Invitrogen). Live cells stain green whereas cells with compromised membranes that are considered dead or dying stain red. Cells were first grown, stained, and exposed to varying doses of metallic nanoparticles, and then bacterial viability was measured hourly using fluorescence microscopy. Results indicate that both ZnO and Ag nanoparticles inhibit the growth of E. coli in liquid media. Preliminary results from toxicity assays confirm the toxic effect of ZnO and Ag nanoparticles on active cell cultures. Calculated death rates resulting from analyses of toxicity studies will be presented.

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation.

PubMed

Yoshitani, Kazuhiro; Kido, Akira; Honoki, Kanya; Akahane, Manabu; Fujii, Hiromasa; Tanaka, Yasuhito

2011-07-15

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation. Copyright © 2011 Elsevier GmbH. 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.

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.

In vitro and in vivo inhibition of tumor cell viability by combined dihydroartemisinin and doxorubicin treatment, and the underlying mechanism

PubMed Central

Tai, Xiang; Cai, Xiao-Bei; Zhang, Zhang; Wei, Rui

2016-01-01

The natural extract artemisinin and its derivatives have good anticancer activity. The present study aimed to investigate the in vitro inhibitory effects of combined dihydroartemisinin (DHA) and doxorubicin (DOX) treatment on a variety of tumor cell lines (HeLa, OVCAR-3, MCF-7, PC-3 and A549), as well as the underlying mechanisms. In addition, the in vivo effects of DHA and DOX were evaluated using a mouse HeLa tumor model. The HeLa, OVCAR-3, MCF-7, PC-3 and A549 cells were treated with a combination of DHA and DOX, and the effect on cell viability was detected by Cell Counting kit-8. The cells were observed under a fluorescence microscope after staining with Hoechst 33258 dye to observe morphological changes in the nuclei in order to determine whether the cells in the treatment group exhibited apoptosis. Apoptosis of the cells was further detected by flow cytometry, and statistical analysis was performed. The specific inhibitors of caspase-3, −8 and −9 were used to determine the intrinsic and extrinsic pathways of cell apoptosis. The cervical cancer HeLa cells treated with the combination of DHA and DOX showed up to a 91.5% decrease in viability, which was higher than that of the same cells treated with DHA or DOX alone at the same concentration, respectively (P<0.01). The optimal concentrations of the drugs used in combination were DHA at 10 µg/ml and DOX at 10 µg/ml. DHA + DOX also had a significant inhibitory effect on the ovarian cancer (OVCAR-3), breast cancer (MCF-7), lung cancer (A549) and prostate cancer (PC-3) cells. The images observed under fluorescence microscope after Hoechst 33258 staining showed marked pyknosis in the cells treated with DHA + DOX, similar to that when treated with DHA or DOX alone, which is typical in apoptosis. As determined by flow cytometry, the apoptotic rate of the cells treated with DHA + DOX at optimal concentrations was up to 90%, which was significantly higher than that of the cells treated with DHA or DOX alone at

In vitro and in vivo inhibition of tumor cell viability by combined dihydroartemisinin and doxorubicin treatment, and the underlying mechanism.

PubMed

Tai, Xiang; Cai, Xiao-Bei; Zhang, Zhang; Wei, Rui

2016-11-01

The natural extract artemisinin and its derivatives have good anticancer activity. The present study aimed to investigate the in vitro inhibitory effects of combined dihydroartemisinin (DHA) and doxorubicin (DOX) treatment on a variety of tumor cell lines (HeLa, OVCAR-3, MCF-7, PC-3 and A549), as well as the underlying mechanisms. In addition, the in vivo effects of DHA and DOX were evaluated using a mouse HeLa tumor model. The HeLa, OVCAR-3, MCF-7, PC-3 and A549 cells were treated with a combination of DHA and DOX, and the effect on cell viability was detected by Cell Counting kit-8. The cells were observed under a fluorescence microscope after staining with Hoechst 33258 dye to observe morphological changes in the nuclei in order to determine whether the cells in the treatment group exhibited apoptosis. Apoptosis of the cells was further detected by flow cytometry, and statistical analysis was performed. The specific inhibitors of caspase-3, -8 and -9 were used to determine the intrinsic and extrinsic pathways of cell apoptosis. The cervical cancer HeLa cells treated with the combination of DHA and DOX showed up to a 91.5% decrease in viability, which was higher than that of the same cells treated with DHA or DOX alone at the same concentration, respectively (P<0.01). The optimal concentrations of the drugs used in combination were DHA at 10 µg/ml and DOX at 10 µg/ml. DHA + DOX also had a significant inhibitory effect on the ovarian cancer (OVCAR-3), breast cancer (MCF-7), lung cancer (A549) and prostate cancer (PC-3) cells. The images observed under fluorescence microscope after Hoechst 33258 staining showed marked pyknosis in the cells treated with DHA + DOX, similar to that when treated with DHA or DOX alone, which is typical in apoptosis. As determined by flow cytometry, the apoptotic rate of the cells treated with DHA + DOX at optimal concentrations was up to 90%, which was significantly higher than that of the cells treated with DHA or DOX alone at the

In vitro effects on biofilm viability and antibacterial and antiadherent activities of silymarin.

PubMed

Evren, Ebru; Yurtcu, Erkan

2015-07-01

Limited treatment options in infectious diseases caused by resistant microorganisms created the need to search new approaches. Several herbal extracts are studied for their enormous therapeutic potential. Silymarin extract, from Silybum marianum (milk thistle), is an old and a new remedy for this goal. The purpose of this study is to evaluate the antibacterial and antiadherent effects of silymarin besides biofilm viability activity on standard bacterial strains. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), antiadherent/antibiofilm activity, and effects on biofilm viability of silymarin were evaluated against standard bacterial strains. MIC values were observed between 60 and >241 μg/mL (0.25->1 mmol/L). Gram-positive bacteria were inhibited at concentrations between 60 and 120 μg/mL. Gram-negative bacteria were not inhibited by the silymarin concentrations included in this study. MBC values for Gram-positive bacteria were greater than 241 μg/mL. Adherence/biofilm formations were decreased to 15 μg/mL silymarin concentration when compared with silymarin-untreated group. Silymarin reduced the biofilm viabilities to 13 and 46 % at 1 and 0.5 mmol/L concentrations, respectively. We demonstrated that silymarin shows antibacterial and antiadherent/antibiofilm activity against certain standard bacterial strains which may be beneficial when used as a dietary supplement or a drug.

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.

Effect of Procyanidin-rich Extract from Natural Cocoa Powder on Cellular Viability, Cell Cycle Progression, and Chemoresistance in Human Epithelial Ovarian Carcinoma Cell Lines

PubMed Central

Taparia, Shruti; Khanna, Aparna

2016-01-01

Background: Over the last 400 years, cocoa and chocolate have been described as having potential medicinal value, being consumed as a beverage or eaten as food. Concentration–dependant, antiproliferation, and cytotoxic effects of some of their polyphenolic constituents have been demonstrated against various cancers. Such an effect remains to be demonstrated in ovarian cancer Objective: To investigate the effect of cocoa procyanidins against ovarian cancer in vitro using OAW42 and OVCAR3 cell lines. Materials and Methods: Cocoa procyanidins were extracted and enriched from non alkalized cocoa powder. The polyphenolic content and antioxidant activity were determined. Effect on cell viability was determined after the treatment with ≤1000 μg/mL cocoa procyanidin-rich extract on OAW42 and OVCAR3 and normal human dermal fibroblasts. Similarly, chemosensitization effect was determined by pretreating cancer cell lines with extract followed by doxorubicin hydrochloride treatment. The effect of treatment on cell cycle and P-glycoprotein (P-gp) expression was determined using flow cytometry. Results: The cocoa extract showed high polyphenolic content and antioxidant activity. Treatment with extract caused cytotoxicity and chemosensitization in OAW42 and OVCAR3 cell lines. Normal dermal fibroblasts showed an increase in cell viability post treatment with extract. Treatment with extract affected the cell cycle and an increasing percentage of cells in hypodiploid sub-G1/G0 phase was observed. Treatment of OVCAR3 with the extract caused reduction of P-gp expression. Conclusion: Cocoa procyanidins were found to be selectively cytotoxic against epithelial ovarian cancer, interfered with the normal cell cycle and sensitized cells to subsequent chemotherapeutic treatment. Chemosensitization was found to be associated with P-gp reduction in OVCAR3 cells. SUMMARY Among the naturally occurring flavonoids, procyanidins have been shown to be effective against cancersNon alkalized

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.

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.

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.

Correlation between dielectric property by dielectrophoretic levitation and growth activity of cells exposed to electric field.

PubMed

Hakoda, Masaru; Hirota, Yusuke

2013-09-01

The purpose of this study is to develop a system analyzing cell activity by the dielectrophoresis method. Our previous studies revealed a correlation between the growth activity and dielectric property (Re[K(ω)]) of mouse hybridoma 3-2H3 cells using dielectrophoretic levitation. Furthermore, it was clarified that the differentiation activity of many stem cells could be evaluated by the Re[K(ω)] without differentiation induction. In this paper, 3-2H3 cells exposed to an alternating current (AC) electric field or a direct current (DC) electric field were cultivated, and the influence of damage by the electric field on the growth activity of the cells was examined. To evaluate the activity of the cells by measuring the Re[K(ω)], the correlation between the growth activity and the Re[K(ω)] of the cells exposed to the electric field was examined. The relations between the cell viability, growth activity, and Re[K(ω)] in the cells exposed to the AC electric field were obtained. The growth activity of the cells exposed to the AC electric field could be evaluated by the Re[K(ω)]. Furthermore, it was found that the adverse effects of the electric field on the cell viability and the growth activity were smaller in the AC electric field than the DC electric field.

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.

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.

Mobile phone electromagnetic radiation activates MAPK signaling and regulates viability in Drosophila.

PubMed

Lee, Kyu-Sun; Choi, Jong-Soon; Hong, Sae-Yong; Son, Tae-Ho; Yu, Kweon

2008-07-01

Mobile phones are widely used in the modern world. However, biological effects of electromagnetic radiation produced by mobile phones are largely unknown. In this report, we show biological effects of the mobile phone 835 MHz electromagnetic field (EMF) in the Drosophila model system. When flies were exposed to the specific absorption rate (SAR) 1.6 W/kg, which is the proposed exposure limit by the American National Standards Institute (ANSI), more than 90% of the flies were viable even after the 30 h exposure. However, in the SAR 4.0 W/kg strong EMF exposure, viability dropped from the 12 h exposure. These EMF exposures triggered stress response and increased the production of reactive oxygen species. The EMF exposures also activated extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling, but not p38 kinase signaling. Interestingly, SAR 1.6 W/kg activated mainly ERK signaling and expression of an anti-apoptotic gene, whereas SAR 4.0 W/kg strongly activated JNK signaling and expression of apoptotic genes. In addition, SAR 4.0 W/kg amplified the number of apoptotic cells in the fly brain. These findings demonstrate that the exposure limit on electromagnetic radiation proposed by ANSI triggered ERK-survival signaling but the strong electromagnetic radiation activated JNK-apoptotic signaling in Drosophila.

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

PubMed

Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

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.

Enrichment and Viability Inhibition of Circulating Tumor Cells on a Dual Acid-Responsive Composite Nanofiber Film.

PubMed

Wang, Wenqian; Cheng, Yaya; Li, Yansheng; Zhou, Hao; Xu, Li-Ping; Wen, Yongqiang; Zhao, Liang; Zhang, Xueji

2017-04-06

The formation and metastatic colonization of circulating tumor cells (CTCs) are responsible for the vast majority of cancer-related deaths. Over the last decade, drug-delivery systems (DDSs) have rapidly developed with the emergence of nanotechnology; however, most reported tumor-targeting DDSs are able to deliver drugs only to solid tumor cells and not CTCs. Herein, a novel DDS comprising a composite nanofiber film was constructed to inhibit the viability of CTCs. In this system, gold nanoparticles (Au NPs) were functionalized with doxorubicin (DOX) through an acid-responsive cleavable linker to obtain Au-DOX NPs. Then, the Au-DOX NPs were mixed in a solution of an acid-responsive polymer {i.e., poly[2-(dimethylamino)ethyl methacrylate]} to synthesize the nanofiber film through electrospinning technology. After that, the nanofiber film was modified with a specific antibody (i.e., anti-EpCAM) to enrich the concentration of CTCs on the film. Finally, the Au-DOX NPs were released from the nanofiber film, and they consequently inhibited the viability of CTCs by delivering DOX to the enriched CTCs. This composite nanofiber film was able to decrease the viability of CTCs significantly in the suspended and fluid states, and it is expected to limit the migration and proliferation of tumor cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Edaravone enhances the viability of ischemia/reperfusion flaps.

PubMed

Zhang, Dong-Yi; Kang, Shen-Song; Zhang, Zheng-Wen; Wu, Rui

2017-02-01

The purpose of the experiment was to study the efficacy of edaravone in enhancing flap viability after ischemia/reperfusion (IR) and its mechanism. Forty-eight adult male SD rats were randomly divided into 3 groups: control group (n=16), IR group (n=16), and edaravone-treated IR group (n=16). An island flap at left lower abdomen (6.0 cm×3.0 cm in size), fed by the superficial epigastric artery and vein, was created in each rat of all the three groups. The arterial blood flow of flaps in IR group and edaravone-treated IR group was blocked for 10 h, and then the blood perfusion was restored. From 15 min before reperfusion, rats in the edaravone-treated IR group were intraperitoneally injected with edaravone (10 mg/kg), once every 12 h, for 3 days. Rats in the IR group and control group were intraperitoneally injected with saline, with the same method and frequency as the rats in the edaravone-treated IR group. In IR group and edaravone-treated IR group, samples of flaps were harvested after reperfusion of the flaps for 24 h. In the control group, samples of flaps were harvested 34 h after creation of the flaps. The content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were determined, and changes in organizational structure and infiltration of inflammatory cells were observed by hematoxylin-eosin (HE) staining, apoptotic cells of vascular wall were marked by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and the apoptotic rate of cells in vascular wall was calculated. The ultrastructural changes of vascular endothelial cells were observed by transmission electron microscopy (TEM). Seven days after the operation, we calculated the flap viability of each group, and marked vessels of flaps by immunohistochemical staining for calculating the average number of subcutaneous vessels. The results showed that the content of MDA, the number of multicore inflammatory cells and apoptotic rate of cells in vascular wall

Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

NASA Astrophysics Data System (ADS)

Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

2017-04-01

Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

Freezing behavior of adherent neuron-like cells and morphological change and viability of post-thaw cells.

PubMed

Uemura, Makoto; Ishiguro, Hiroshi

2015-04-01

Freezing of nerve cells forming a neuronal network has largely been neglected, despite the fact that the cryopreservation of nerve cells benefits the study of cells in the areas of medicine and poison screening. Freezing of nerve cells is also attractive for studying cell morphology because of the characteristic long, thread-like neurites extending from the cell body. In the present study, freezing of neuron-like cells adhering to the substrate (differentiated PC12 cells), in physiological saline, was investigated in order to understand the fundamental freezing and thawing characteristics of nerve cells with neurites. The microscopic freezing behavior of cells under different cooling rates was observed. Next, the post-thaw morphological changes in the cells, including the cytoskeleton, were investigated and post-thaw cell viability was evaluated by dye exclusion using propidium iodide. Two categories of morphological changes, beading and shortening of the neurites, were found and quantified. Also, the morphological changes of neurites due to osmotic stress from sodium chloride were studied to gain a better understanding of causation. The results showed that morphological changes and cell death were promoted with a decrease in end temperature during freezing. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells

PubMed Central

Guon, Tae Eun; Chung, Ha Sook

2017-01-01

The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50–100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells. PMID:28789398

Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells.

PubMed

Guon, Tae Eun; Chung, Ha Sook

2017-08-01

The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50-100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles

PubMed Central

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel

2016-01-01

Summary Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle–cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN. PMID:27826507

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles.

PubMed

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel; Hilfiker, Andres

2016-01-01

Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle-cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

[Knockdown of PRDX6 in microglia reduces neuron viability after OGD/R injury].

PubMed

Tan, Li; Zhao, Yong; Jiang, Beibei; Yang, Bo; Zhang, Hui

2016-08-01

Objective To observe the effects of peroxiredoxin 6 (PRDX6) knockdown in the microglia on neuron viability after oxygen-glucose deprivation and reoxygenation (OGD/R). Methods Microglia was treated with lentivirus PRDX6-siRNA and Ca(2+)-independent phospholipase A2 (iPLA2) inhibitor, 1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol (MJ33). Twenty-four hours later, it was co-cultured with primary neuron to establish the microglia-neuron co-culture OGD/R model. According to the different treatment of microglia, the cells were divided into normal group, OGD/R group, negative control-siRNA treated OGD/R group, PRDX6-siRNA treated OGD/R group and PRDX6-siRNA combined with MJ33 treated OGD/R group. Western blot analysis and real-time quantitative PCR were respectively performed to detect PRDX6 protein and mRNA levels after knockdown of PRDX6 in microglia. The iPLA2 activity was measured by ELISA. MTS and lactate dehydrogenase (LDH) assay were used to measure neuron viability and cell damage. The oxidative stress level of neuron was determined by measuring superoxide dismutase (SOD) and malonaldehyde (MDA) content. Results In PRDX6-siRNA group, neuron viability was inhibited and oxidative stress damage was aggravated compared with OGD/R group. In PRDX6-siRNA combined with MJ33 group, cell viability was promoted and oxidative stress damage was alleviated compared with PRDX6-siRNA group. Conclusion PRDX6 in microglia protects neuron against OGD/R-induced injury, and iPLA2 activity has an effect on PRDX6.

[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.

Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

PubMed Central

Kirchner, Jasmin; Vissi, Emese; Gross, Sascha; Szoor, Balazs; Rudenko, Andrey; Alphey, Luke; White-Cooper, Helen

2008-01-01

Background Protein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β. Results URI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei. Conclusion Uri is the first PP1α specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development. PMID:18412953

Rosiglitazone induces the unfolded protein response, but has no significant effect on cell viability, in monocytic and vascular smooth muscle cells.

PubMed

Caddy, J; Isa, S; Mainwaring, L S; Adam, E; Roberts, A; Lang, D; Morris, R H K; Thomas, A W; Webb, R

2010-10-01

Given the safety concerns expressed over negative cardiovascular outcomes resulting from the clinical use of rosiglitazone, and the view that rosiglitazone exerts PPARγ-independent effects alongside its insulin-sensitising PPARγ-dependent effects, we hypothesised that rosiglitazone may trigger Unfolded Protein Responses (UPRs) due to disruptions in [Ca(2+)](i) homeostasis within two cardiovascular cell types: monocytic (MM6) and vascular smooth muscle (A7r5) cells. In microsomal samples derived from both cell types, pre-incubation with rosiglitazone rapidly (30min) brought about concentration-dependent PPARγ-independent inhibition of Ca(2+)ATPase activity (IC(50) ∼2μM). Fluo-3 fluorimetric data demonstrated in intact cells that 1h treatment with 1 or 10μM rosiglitazone caused Ca(2+) ions to leak into the cytoplasm. Gene expression analysis showed that within 4h of rosiglitazone exposure, the UPR transcription factor XBP-1 was activated (likely due to corresponding ER Ca(2+) depletion), and the UPR target genes BiP and SERCA2b were subsequently upregulated within 24-72h. After 72h 1 or 10μM rosiglitazone treatment, microsomal Ca(2+)ATPase activity increased to >2-fold of that seen in control microsomes, while [Ca(2+)](i) returned to basal, indicating that UPR-triggered SERCA2b upregulation was responsible for enhanced enzymatic Ca(2+) sequestration within the ER. This appeared to be sufficient to replenish ER Ca(2+) stores and restore normal cell physiology, as cell viability levels were not decreased due to rosiglitazone treatment throughout a 2-week study. Thus, incubation with 1-10μM rosiglitazone triggers the UPR, but does not prove cytotoxic, in cells of the cardiovascular system. This observation provides an important contribution to the current debate over the use of rosiglitazone in the clinical treatment of Type-2 Diabetes. Copyright © 2010 Elsevier Inc. All rights reserved.

Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease.

PubMed

Laprairie, Robert B; Bagher, Amina M; Kelly, Melanie E M; Denovan-Wright, Eileen M

2016-03-01

Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

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

Evaluation of cell viability and functionality in vessel-like bioprintable cell-laden tubular channels.

PubMed

Yu, Yin; Zhang, Yahui; Martin, James A; Ozbolat, Ibrahim T

2013-09-01

Organ printing is a novel concept recently introduced in developing artificial three-dimensional organs to bridge the gap between transplantation needs and organ shortage. One of the major challenges is inclusion of blood-vessellike channels between layers to support cell viability, postprinting functionality in terms of nutrient transport, and waste removal. In this research, we developed a novel and effective method to print tubular channels encapsulating cells in alginate to mimic the natural vascular system. An experimental investigation into the influence on cartilage progenitor cell (CPCs) survival, and the function of printing parameters during and after the printing process were presented. CPC functionality was evaluated by checking tissue-specific genetic marker expression and extracellular matrix production. Our results demonstrated the capability of direct fabrication of cell-laden tubular channels by our newly designed coaxial nozzle assembly and revealed that the bioprinting process could induce quantifiable cell death due to changes in dispensing pressure, coaxial nozzle geometry, and biomaterial concentration. Cells were able to recover during incubation, as well as to undergo differentiation with high-level cartilage-associated gene expression. These findings may not only help optimize our system but also can be applied to biomanufacturing of 3D functional cellular tissue engineering constructs for various organ systems.

Effect of alternative peritoneal dialysis solutions on cell viability, apoptosis/necrosis and cytokine expression in human monocytes.

PubMed

Plum, J; Lordnejad, M R; Grabensee, B

1998-07-01

Cellular function, cell viability and the cytokine network of human monocytes are influenced by the specific composition of peritoneal dialysis (PD) fluids. In an in vitro study using isolated human blood monocytes, we investigated the effect of peritoneal dialysates containing amino acids (Amino) or glucose polymer (Glu-poly) instead of glucose (Glu) as the osmotic agent, and bicarbonate (Bic) or PBS instead of lactate (Lac) as a buffer. The following parameters were studied: mitochondrial dehydrogenase activity (using the MTT assay), interleukin (IL)-6 and IL-8 release (ELISA) and cellular IL-6 mRNA expression after lipopolysaccharide (LPS) stimulation (using RT-PCR). FACS flow cytometry with annexin V and propidium iodide as markers and fluorescence microscopic methods were used to study the effects of the test fluids on cell necrosis and apoptosis. Glu/Lac pH 5.5 and Glu-poly/PBS pH 7.4 both significantly reduced mitochondrial dehydrogenase activity by more than 50% after 60 minutes of incubation (30.5 +/- 7.6%, 42.5 +/- 6.5%, referred to RPMI 1640 as 100%). Amino/Bic and Glu/Bic were both superior (Mtt assay > 63%). The rate of necrotic cells after 15 minutes of incubation measured by FACS was mostly increased with Glu/Lac pH 5.5 (29.9 +/- 4.0%). The rate of apoptotic cells, however, was not significantly different between the test solutions. The concentration of IL-6 in the supernatant of stimulated monocytes was highest with Glu/Bic (1023 +/- 278 pg/ml) and Amino/Bic (776 +/- 296 pg/ml) an lowest with Glu/lac pH 5.5 (46 +/- 22 pg/ml) and Glu-poly/PBS (32 +/- 13 pg/ml). IL-8 release from stimulated monocytes showed a similar pattern. Glu-poly/PBS showed a suppressive effect on IL-6 mRNA expression (ratio IL-6/beta-Actin, 0.4 +/- 0.25 vs. RPMI 1.5 +/- 3.6). Bicarbonate buffered solutions both with glucose or amino acids as osmotic agents were superior when regarding cell metabolism, viability and cytokine release, while lactate buffered solutions and Glu

Anticancer activity of Petroselinum sativum seed extracts on MCF-7 human breast cancer cells.

PubMed

Farshori, Nida Nayyar; Al-Sheddi, Ebtesam Saad; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2013-01-01

Pharmacological and preventive properties of Petroselinum sativum seed extracts are well known, but the anticancer activity of alcoholic extracts and oil of Petroselinum sativum seeds on human breast cancer cells have not been explored so far. Therefore, the present study was designed to investigate the cytotoxic activities of these extracts against MCF-7 cells. Cells were exposed to 10 to 1000 μg/ml of alcoholic seed extract (PSA) and seed oil (PSO) of Petroselinum sativum for 24 h. Post-treatment, percent cell viability was studied by 3-(4, 5-dimethylthiazol-2yl)-2, 5-biphenyl tetrazolium bromide (MTT) and neutral red uptake (NRU) assays, and cellular morphology by phase contrast inverted microscopy. The results showed that PSA and PSO significantly reduced cell viability, and altered the cellular morphology of MCF-7 cells in a concentration dependent manner. Concentrations of 50 μg/ml and above of PSA and 100 μg/ml and above of PSO were found to be cytotoxic in MCF-7 cells. Cell viability at 50, 100, 250, 500 and 1000 μg/ml of PSA was recorded as 81%, 57%, 33%, 8% and 5%, respectively, whereas at 100, 250, 500, and 1000 μg/ml of PSO values were 90%, 78%, 62%, and 8%, respectively by MTT assay. MCF-7 cells exposed to 250, 500 and 1000 μg/ml of PSA and PSO lost their typical morphology and appeared smaller in size. The data revealed that the treatment with PSA and PSO of Petroselinum sativum induced cell death in MCF-7 cells.

Classical and alternative activation of rat hepatic sinusoidal endothelial cells by inflammatory stimuli.

PubMed

Liu, Yinglin; Gardner, Carol R; Laskin, Jeffrey D; Laskin, Debra L

2013-02-01

The ability of rat hepatic sinusoidal endothelial cells (HSEC) to become activated in response to diverse inflammatory stimuli was analyzed. Whereas the classical macrophage activators, IFNγ and/or LPS upregulated expression of iNOS in HSEC, the alternative macrophage activators, IL-10 or IL-4+IL-13 upregulated arginase-1 and mannose receptor. Similar upregulation of iNOS and arginase-1 was observed in classically and alternatively activated Kupffer cells, respectively. Removal of inducing stimuli from the cells had no effect on expression of these markers, demonstrating that activation is persistent. Washing and incubation of IFNγ treated cells with IL-4+IL-13 resulted in decreased iNOS and increased arginase-1 expression, while washing and incubation of IL-4+IL-13 treated cells with IFNγ resulted in decreased arginase-1 and increased iNOS, indicating that classical and alternative activation of the cells is reversible. HSEC were more sensitive to phenotypic switching than Kupffer cells, suggesting greater functional plasticity. Hepatocyte viability and expression of PCNA, β-catenin and MMP-9 increased in the presence of alternatively activated HSEC. In contrast, the viability of hepatocytes pretreated for 2 h with 5 mM acetaminophen decreased in the presence of classically activated HSEC. These data demonstrate that activated HSEC can modulate hepatocyte responses following injury. The ability of hepatocytes to activate HSEC was also investigated. Co-culture of HSEC with acetaminophen-injured hepatocytes, but not control hepatocytes, increased the sensitivity of HSEC to classical and alternative activating stimuli. The capacity of HSEC to respond to phenotypic activators may represent an important mechanism by which they participate in inflammatory responses associated with hepatotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

Efficient 5'-3' DNA end resection by HerA and NurA is essential for cell viability in the crenarchaeon Sulfolobus islandicus.

PubMed

Huang, Qihong; Liu, Linlin; Liu, Junfeng; Ni, Jinfeng; She, Qunxin; Shen, Yulong

2015-02-14

ATPase/Helicases and nucleases play important roles in homologous recombination repair (HRR). Many of the mechanistic details relating to these enzymes and their function in this fundamental and complicated DNA repair process remain poorly understood in archaea. Here we employed Sulfolobus islandicus, a hyperthermophilic archaeon, as a model to investigate the in vivo functions of the ATPase/helicase HerA, the nuclease NurA, and their associated proteins Mre11 and Rad50. We revealed that each of the four genes in the same operon, mre11, rad50, herA, and nurA, are essential for cell viability by a mutant propagation assay. A genetic complementation assay with mutant proteins was combined with biochemical characterization demonstrating that the ATPase activity of HerA, the interaction between HerA and NurA, and the efficient 5'-3' DNA end resection activity of the HerA-NurA complex are essential for cell viability. NurA and two other putative HRR proteins: a PIN (PilT N-terminal)-domain containing ATPase and the Holliday junction resolvase Hjc, were co-purified with a chromosomally encoded N-His-HerA in vivo. The interactions of HerA with the ATPase and Hjc were further confirmed by in vitro pull down. Efficient 5'-3' DNA end resection activity of the HerA-NurA complex contributes to necessity of HerA and NurA in Sulfolobus, which is crucial to yield a 3'-overhang in HRR. HerA may have additional binding partners in cells besides NurA.

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.

In vitro Effects of Lemongrass Extract on Candida albicans Biofilms, Human Cells Viability, and Denture Surface

PubMed Central

Madeira, Petrus L. B.; Carvalho, Letícia T.; Paschoal, Marco A. B.; de Sousa, Eduardo M.; Moffa, Eduardo B.; da Silva, Marcos A. dos Santos; Tavarez, Rudys de Jesus Rodolfo; Gonçalves, Letícia M.

2016-01-01

The purpose of this study was to investigate whether immersion of a denture surface in lemongrass extract (LGE) has effects on C. albicans biofilms, human cell viability and denture surface. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were performed for LGE against C. albicans. For biofilm analysis, discs were fabricated using a denture acrylic resin with surface roughness standardization. C. albicans biofilms were developed on saliva-coated discs, and the effects of LGE at MIC, 5XMIC, and 10XMIC were investigated during biofilm formation and after biofilm maturation. Biofilms were investigated for cell counting, metabolic activity, and microscopic analysis. The cytotoxicity of different concentrations of LGE to peripheral blood mononuclear cells (PBMC) was analyzed using MTT. The effects of LGE on acrylic resin were verified by measuring changes in roughness, color and flexural strength after 28 days of immersion. Data were analyzed by ANOVA, followed by a Tukey test at a 5% significance level. The minimal concentration of LGE required to inhibit C. albicans growth was 0.625 mg/mL, while MFC was 2.5 mg/mL. The presence of LGE during biofilm development resulted in a reduction of cell counting (p < 0.05), which made the MIC sufficient to reduce approximately 90% of cells (p < 0.0001). The exposure of LGE after biofilm maturation also had a significant antifungal effect at all concentrations (p < 0.05). When compared to the control group, the exposure of PBMC to LGE at MIC resulted in similar viability (p > 0.05). There were no verified differences in color perception, roughness, or flexural strength after immersion in LGE at MIC compared to the control (p > 0.05). It could be concluded that immersion of the denture surface in LGE was effective in reducing C. albicans biofilms with no deleterious effects on acrylic properties at MIC. MIC was also an effective and safe concentration for use. PMID:27446818

In vitro Effects of Lemongrass Extract on Candida albicans Biofilms, Human Cells Viability, and Denture Surface.

PubMed

Madeira, Petrus L B; Carvalho, Letícia T; Paschoal, Marco A B; de Sousa, Eduardo M; Moffa, Eduardo B; da Silva, Marcos A Dos Santos; Tavarez, Rudys de Jesus Rodolfo; Gonçalves, Letícia M

2016-01-01

The purpose of this study was to investigate whether immersion of a denture surface in lemongrass extract (LGE) has effects on C. albicans biofilms, human cell viability and denture surface. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were performed for LGE against C. albicans. For biofilm analysis, discs were fabricated using a denture acrylic resin with surface roughness standardization. C. albicans biofilms were developed on saliva-coated discs, and the effects of LGE at MIC, 5XMIC, and 10XMIC were investigated during biofilm formation and after biofilm maturation. Biofilms were investigated for cell counting, metabolic activity, and microscopic analysis. The cytotoxicity of different concentrations of LGE to peripheral blood mononuclear cells (PBMC) was analyzed using MTT. The effects of LGE on acrylic resin were verified by measuring changes in roughness, color and flexural strength after 28 days of immersion. Data were analyzed by ANOVA, followed by a Tukey test at a 5% significance level. The minimal concentration of LGE required to inhibit C. albicans growth was 0.625 mg/mL, while MFC was 2.5 mg/mL. The presence of LGE during biofilm development resulted in a reduction of cell counting (p < 0.05), which made the MIC sufficient to reduce approximately 90% of cells (p < 0.0001). The exposure of LGE after biofilm maturation also had a significant antifungal effect at all concentrations (p < 0.05). When compared to the control group, the exposure of PBMC to LGE at MIC resulted in similar viability (p > 0.05). There were no verified differences in color perception, roughness, or flexural strength after immersion in LGE at MIC compared to the control (p > 0.05). It could be concluded that immersion of the denture surface in LGE was effective in reducing C. albicans biofilms with no deleterious effects on acrylic properties at MIC. MIC was also an effective and safe concentration for use.

Growth differentiation factor‑5 induces tenomodulin expression via phosphorylation of p38 and promotes viability of murine mesenchymal stem cells from compact bone.

PubMed

Qu, Yanlong; Zhou, Li; Lv, Bing; Wang, Chunlei; Li, Pengwei

2018-03-01

Growth differentiation factor (GDF)‑5 serves a role in tissue development and tenomodulin serves an important role in the development of tendons. The effects of GDF‑5 on mesenchymal stem cells (MSCs), particularly with regards to tendon bioengineering, are poorly understood. The present study aimed to investigate the effects of GDF‑5 on cell viability and tenomodulin expression in MSCs from murine compact bone. MSCs were isolated from murine compact bones and confirmed by flow cytometric analysis. In addition, the adipogenic, osteoblastic and chondrocyte differentiation capabilities of the MSCs were determined. MSCs were treated with GDF‑5 and the effects of GDF‑5 on MSC viability were determined. The mRNA and protein expression levels of tenomodulin were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. MSCs from murine compact bone were successfully isolated. GDF‑5 had optimal effects on cell viability at 100 ng/ml (+36.9% of control group without GDF‑5 treatment, P<0.01) and its effects peaked after 6 days of treatment (+56.6% of control group, P<0.001). Compared with the control group, treatment with 100 ng/ml GDF‑5 for 4 days enhanced the mRNA expression levels of tenomodulin (3.56±0.94 vs. 1.02±0.25; P<0.05). In addition, p38 was activated by GDF‑5, as determined by enhanced expression levels of phosphorylated p38 (p‑p38). The GDF‑5‑induced protein expression levels of p‑p38 and tenomodulin were markedly inhibited following treatment with SB203580, an inhibitor of p38 mitogen‑activated protein kinase. These results suggested that GDF‑5 treatment may increase tenomodulin protein expression via phosphorylation of p38 in MSCs from murine compact bone. These findings may aid the future development of tendon bioengineering.

Evaluation of graft cell viability-efficacy of piezoelectric versus manual bone scraper technique.

PubMed

Pekovits, Karin; Wildburger, Angelika; Payer, Michael; Hutter, Heinz; Jakse, Norbert; Dohr, Gottfried

2012-01-01

The aim of the present study was to compare the influence of 2 different bone scrapers with respect to graft quality. The study was conducted as a prospective, controlled experimental study of patients selected from the outpatient unit of the Department of Oral Surgery and Radiology (Dental Clinic, Medical University, Graz, Austria). Bone samples were obtained during routine lower third molar removal. Both a manual bone scraper (MS) and a piezoelectric device (PD) were used in directly adjacent regions in each case. As variables, the chip morphology, cell viability, and osteogenic differentiation were investigated. For statistical analysis, the Student t test and Fisher's exact test (P < .05) were applied. A total of 20 patients (12 women and 8 men, mean age 28.15 ± 5.8 years) were included in the study. A series of 40 bone samples was obtained during lower third molar removal. MS and PD enabled similar intraoral harvest of bone chips. In vitro outgrowth of adherent cells was found in 90% of the MS and 80% of the PD samples after 7 to 18 days, without statistical significance (P = .67). Similar cell viability of outgrowing cells in both groups was observed (94.7% ± 2.2% in the MS group and 94.1% ± 1.6% in the PD group). Reverse transcriptase-polymerase chain reaction analysis and the staining pattern verified osteopotent cells in both groups. Both manual and piezoelectric techniques are adequate harvesting technologies for limited intraoral augmentations. Our results did not show an advantage for the piezoelectric device. Copyright © 2012 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Effect of microemulsions on cell viability of human dermal fibroblasts

NASA Astrophysics Data System (ADS)

Li, Juyi; Mironava, Tatsiana; Simon, Marcia; Rafailovich, Miriam; Garti, Nissim

Microemulsions are optically clear, thermostable and isotropic mixture consisting of water, oil and surfactants. Their advantages of ease preparation, spontaneous formation, long-term stability and enhanced solubility of bioactive materials make them great potentials as vehicles in food and pharmaceutical applications. In this study, comparative in vitro cytotoxicity tests were performed to select a best formulation of microemulsion with the least toxicity for human dermal fibroblasts. Three different kinds of oils and six different kinds of surfactants were used to form microemulsions by different ratios. The effect of oil type and surfactant type as well as their proportions on cell proliferation and viability were tested.

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.

Importance of Donor Chondrocyte Viability for Osteochondral Allografts.

PubMed

Cook, James L; Stannard, James P; Stoker, Aaron M; Bozynski, Chantelle C; Kuroki, Keiichi; Cook, Cristi R; Pfeiffer, Ferris M

2016-05-01

Osteochondral allograft (OCA) transplantation provides a biological treatment option for functional restoration of large articular cartilage defects in multiple joints. While successful outcomes after OCA transplantation have been linked to viable donor chondrocytes, the importance of donor cell viability has not been comprehensively validated. To use a canine model to determine the importance of donor chondrocyte viability at the time of implantation with respect to functional success of femoral condylar OCAs based on radiographic, gross, cell viability, histologic, biochemical, and biomechanical outcome measures. Controlled laboratory study. After approval was obtained from the institutional animal care and use committee, adult female dogs (N = 16) were implanted with 8-mm cylindrical OCAs from male dogs in the lateral and medial femoral condyles of 1 knee. OCAs were preserved for 28 or 60 days after procurement, and chondrocyte viability was quantified before implantation. Two different storage media, temperatures, and time points were used to obtain a spectrum of percentage chondrocyte viability at the time of implantation. A successful outcome was defined as an OCA that was associated with graft integration, maintenance of hyaline cartilage, lack of associated cartilage disorder, and lack of fibrillation, fissuring, or fibrous tissue infiltration of the allograft based on subjective radiographic, gross, and histologic assessments at 6 months after implantation. Chondrocyte viability ranged from 23% to 99% at the time of implantation. All successful grafts had >70% chondrocyte viability at the time of implantation, and no graft with chondrocyte viability <70% was associated with a successful outcome. Live-dead stained sections and histologic findings with respect to cell morphological features suggested that successful grafts were consistently composed of viable chondrocytes in lacunae, while grafts that were not successful were composed of nonviable

Use of Cell Viability Assay Data Improves the Prediction Accuracy of Conventional Quantitative Structure–Activity Relationship Models of Animal Carcinogenicity

PubMed Central

Zhu, Hao; Rusyn, Ivan; Richard, Ann; Tropsha, Alexander

2008-01-01

Background To develop efficient approaches for rapid evaluation of chemical toxicity and human health risk of environmental compounds, the National Toxicology Program (NTP) in collaboration with the National Center for Chemical Genomics has initiated a project on high-throughput screening (HTS) of environmental chemicals. The first HTS results for a set of 1,408 compounds tested for their effects on cell viability in six different cell lines have recently become available via PubChem. Objectives We have explored these data in terms of their utility for predicting adverse health effects of the environmental agents. Methods and results Initially, the classification k nearest neighbor (kNN) quantitative structure–activity relationship (QSAR) modeling method was applied to the HTS data only, for a curated data set of 384 compounds. The resulting models had prediction accuracies for training, test (containing 275 compounds together), and external validation (109 compounds) sets as high as 89%, 71%, and 74%, respectively. We then asked if HTS results could be of value in predicting rodent carcinogenicity. We identified 383 compounds for which data were available from both the Berkeley Carcinogenic Potency Database and NTP–HTS studies. We found that compounds classified by HTS as “actives” in at least one cell line were likely to be rodent carcinogens (sensitivity 77%); however, HTS “inactives” were far less informative (specificity 46%). Using chemical descriptors only, kNN QSAR modeling resulted in 62.3% prediction accuracy for rodent carcinogenicity applied to this data set. Importantly, the prediction accuracy of the model was significantly improved (72.7%) when chemical descriptors were augmented by HTS data, which were regarded as biological descriptors. Conclusions Our studies suggest that combining NTP–HTS profiles with conventional chemical descriptors could considerably improve the predictive power of computational approaches in toxicology. PMID

Dihydroartemisinin Exerts Anti-Tumor Activity by Inducing Mitochondrion and Endoplasmic Reticulum Apoptosis and Autophagic Cell Death in Human Glioblastoma Cells

PubMed Central

Qu, Chengbin; Ma, Jun; Liu, Xiaobai; Xue, Yixue; Zheng, Jian; Liu, Libo; Liu, Jing; Li, Zhen; Zhang, Lei; Liu, Yunhui

2017-01-01

Glioblastoma (GBM) is the most advanced and aggressive form of gliomas. Dihydroartemisinin (DHA) has been shown to exhibit anti-tumor activity in various cancer cells. However, the effect and molecular mechanisms underlying its anti-tumor activity in human GBM cells remain to be elucidated. Our results proved that DHA treatment significantly reduced cell viability in a dose- and time-dependent manner by CCK-8 assay. Further investigation identified that the cell viability was rescued by pretreatment either with Z-VAD-FMK, 3-methyladenine (3-MA) or in combination. Moreover, DHA induced apoptosis of GBM cells through mitochondrial membrane depolarization, release of cytochrome c and activation of caspases-9. Enhanced expression of GRP78, CHOP and eIF2α and activation of caspase 12 were additionally confirmed that endoplasmic reticulum (ER) stress pathway of apoptosis was involved in the cytotoxicity of DHA. DHA-treated GBM cells exhibited the morphological and biochemical changes typical of autophagy. Co-treatment with chloroquine (CQ) significantly induced the above effects. Furthermore, ER stress and mitochondrial dysfunction were involved in the DHA-induced autophagy. Further study revealed that accumulation of reactive oxygen species (ROS) was attributed to the DHA induction of apoptosis and autophagy. The illustration of these molecular mechanisms will present a novel insight for the treatment of human GBM. PMID:29033794

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

PubMed Central

Schilz, Jodi R.; Reddy, K. J.; Nair, Sreejayan; Johnson, Thomas E.; Tjalkens, Ronald B.; Krueger, Kem P.; Clark, Suzanne

2015-01-01

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic however; this method could have a broader use assessing CuO-NP treatment in more neutral waters. PMID:26132311

The Effects of Cyclic Hydrostatic Pressure on Chondrogenesis and Viability of Human Adipose- and Bone Marrow-Derived Mesenchymal Stem Cells in Three-Dimensional Agarose Constructs

PubMed Central

Puetzer, Jennifer; Williams, John; Gillies, Allison; Bernacki, Susan

2013-01-01

This study investigates the effects of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3-D) agarose constructs maintained in a complete growth medium without soluble chondrogenic inducing factors. hASCs were seeded in 2% agarose hydrogels and exposed to 7.5 MPa CHP for 4 h per day at a frequency of 1 Hz for up to 21 days. On days 0, 7, 14, and 21, the expression levels of collagen II, Sox9, aggrecan, and cartilage oligomeric matrix protein (COMP) were examined by real-time reverse transcriptase–polymerase chain reaction analysis. Gene expression analysis found collagen II mRNA expression in only the CHP-loaded construct at day 14 and at no other time during the study. CHP-loaded hASCs exhibited upregulated mRNA expression of Sox9, aggrecan, and COMP at day 7 relative to unloaded controls, suggesting that CHP initiated chondrogenic differentiation of hASCs in a manner similar to human bone marrow-derived mesenchymal stem cells (hMSC). By day 14, however, loaded hASC constructs exhibited significantly lower mRNA expression of the chondrogenic markers than unloaded controls. Additionally, by day 21, the samples exhibited little measurable mRNA expression at all, suggesting a decreased viability. Histological analysis validated the lack of mRNA expression at day 21 for both the loaded and unloaded control samples with a visible decrease in the cell number and change in morphology. A comparative study with hASCs and hMSCs further examined long-term cell viability in 3-D agarose constructs of both cell types. Decreased cell metabolic activity was observed throughout the 21-day experimental period in both the CHP-loaded and control constructs of both hMSCs and hASCs, suggesting a decrease in cell metabolic activity, alluding to a decrease in cell viability. This suggests that a 2% agarose hydrogel may not optimally support hASC or hMSC viability in a complete growth medium in the

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.

A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms.

PubMed

Pérez, L M; Alvarez, B L; Codony, F; Fittipaldi, M; Adrados, B; Peñuela, G; Morató, J

2010-09-01

It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]Bis(4-methoxy)-6-nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.

A PTEN inhibitor displays preclinical activity against hepatocarcinoma cells

PubMed Central

Augello, Giuseppa; Puleio, Roberto; Emma, Maria Rita; Cusimano, Antonella; Loria, Guido R.; McCubrey, James A.; Montalto, Giuseppe; Cervello, Melchiorre

2016-01-01

ABSTRACT Phosphatase and tensin homolog (PTEN) gene is considered a tumor suppressor gene. However, PTEN mutations rarely occur in hepatocellular carcinoma (HCC), whereas heterozygosity of PTEN, resulting in reduced PTEN expression, has been observed in 32–44% of HCC patients. In the present study, we investigated the effects of the small molecule PTEN inhibitor VO-OHpic in HCC cells. VO-OHpic inhibited cell viability, cell proliferation and colony formation, and induced senescence-associated β-galactosidase activity in Hep3B (low PTEN expression) and to a lesser extent in PLC/PRF/5 (high PTEN expression) cells, but not in PTEN-negative SNU475 cells. VO-OHpic synergistically inhibited cell viability when combined with PI3K/mTOR and RAF/MEK/ERK pathway inhibitors, but only in Hep3B cells, and significantly inhibited tumor growth in nude mice bearing xenografts of Hep3B cells. Therefore, we demonstrated for the first time that VO-OHpic inhibited cell growth and induced senescence in HCC cells with low PTEN expression, and that the combination of VO-OHpic with PI3K/mTOR and RAF/MEK/ERK inhibitors resulted in a more effective tumor cell kill. Our findings, hence, provide proof-of-principle evidence that pharmacological inhibition of PTEN may represent a promising approach for HCC therapy in a subclass of patients with a low PTEN expression. PMID:26794644

The effect of automobile exhaust particulates on cell viability, plating efficiency and cell division of mammalian tissue culture cells.

PubMed

Seemayer, N H; Hadnagy, W; Tomingas, R

1987-03-01

Extract of particulate matter (EPM) of gasoline engine exhaust induced only a slight loss of cell viability of mouse macrophages (line IC-21) in vitro, while a strong dose-dependent reduction of plating efficiency of human cell line A-549 and of Syrian hamster line 14-1b occurred. Cytological investigations of exposed macrophages of line IC-21 revealed an increase in the mitotic index from 1.5% of control values up to 14.6% at the highest tested concentration of EPM. Mitotic arrest is based almost exclusively on C-type mitoses occurring dose-dependently in the presence of EPM. Results indicate disturbances of the spindle apparatus in the presence of EPM.

Assessing the Financial Viability of Academic Programmes

ERIC Educational Resources Information Center

Swift, Lynette

2012-01-01

This paper reviews and examines approaches to determining the financial viability of academic programmes as a critical component of assessing a programme's overall sustainability. Key to assessing the financial viability of a programme is understanding the teaching activities required to deliver the programme and the cost of those activities. A…

Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: effects on growth, viability, adherence and ultrastructure.

PubMed

Machado, M; Dinis, A M; Salgueiro, L; Custódio, José B A; Cavaleiro, C; Sousa, M C

2011-04-01

The present work evaluates the anti-Giardia activity of Syzygium aromaticum and its major compound eugenol. The effects were evaluated on parasite growth, adherence, viability and ultrastructure. S. aromaticum essential oil (IC(50)=134 μg/ml) and eugenol (IC(50)=101 μg/ml) inhibited the growth of G. lamblia. The essential oil inhibited trophozoites adherence since the first hour of incubation and was able to kill almost 50% of the parasites population in a time dependent manner. The eugenol inhibited G. lamblia trophozoites adherence since the third hour and not induce cell lyses. The main morphological alterations were modifications on the cell shape, presence of precipitates in the cytoplasm, autophagic vesicles, internalization of flagella and ventral disc, membrane blebs, and intracellular and nuclear clearing. Taken together, our findings lead us to propose that eugenol was responsible for the anti-giardial activity of the S. aromaticum essential oil and both have potential for use as therapeutic agents against giardiasis. Copyright © 2011 Elsevier Inc. All rights reserved.

Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo

PubMed Central

Lotti, Roberta; Palazzo, Elisabetta; Petrachi, Tiziana; Dallaglio, Katiuscia; Saltari, Annalisa; Truzzi, Francesca; Quadri, Marika; Puviani, Mario; Maiorana, Antonino; Marconi, Alessandra; Pincelli, Carlo

2016-01-01

Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β1-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in RasG12V-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development. PMID:26771605

Increase in the nitric oxide release without changes in cell viability of macrophages after laser therapy with 660 and 808 nm lasers.

PubMed

Silva, Igor Henrique Morais; de Andrade, Samantha Cardoso; de Faria, Andreza Barkokebas Santos; Fonsêca, Deborah Daniela Diniz; Gueiros, Luiz Alcino Monteiro; Carvalho, Alessandra Albuquerque Tavares; da Silva, Wylla Tatiana Ferreira; de Castro, Raul Manhães; Leão, Jair Carneiro

2016-12-01

The aim of this study was to evaluate the influence of low-level laser therapy (LLLT) with different parameters and wavelengths on nitric oxide (NO) release and cell viability. Irradiation was performed with Ga-Al-As laser, continuous mode and wavelengths of 660 and 808 nm at different energy and power densities. For each wavelength, powers of 30, 50, and 100 mW and times of 10, 30, and 60 s were used. NO release was measured using Griess reaction, and cell viability was evaluated by mitochondrial reduction of bromide 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to formazan. LLLT promoted statistically significant changes in NO release and MTT value only at the wavelength of 660 nm (p < 0.05). LLLT also promoted an increase in the NO release and cell viability when the energy densities 64 (p = 0.04) and 214 J/cm 2 (p = 0.012), respectively, were used. LLLT has a significant impact on NO release without affecting cell viability, but the significance of these findings in the inflammatory response needs to be further studied.