A rapid method for measuring intracellular pH using BCECF-AM.

PubMed

Ozkan, Pinar; Mutharasan, Raj

2002-08-15

A rapid intracellular pH (pH(i)) measurement method based on initial rate of increase of fluorescence ratio of 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein upon dye addition to a cell suspension in growth medium is reported. A dye transport model that describes dye concentration and fluorescence values in intracellular and extracellular spaces provides the mathematical basis for the approach. Experimental results of ammonium chloride challenge response of the two suspension cells, Spodoptera frugiperda and Chinese hamster ovary (CHO) cells, successfully compared with results obtained using traditional perfusion method. Since the cell suspension does not require any preparation, measurement of pH(i) can be completed in about 1 min minimizing any potential errors due to dye leakage.

Enhanced Mulberroside A Production from Cell Suspension and Root Cultures of Morus alba Using Elicitation.

PubMed

Komaikul, Jukrapun; Kitisripanya, Tharita; Tanaka, Hiroyuki; Sritularak, Boonchoo; Putalun, Waraporn

2015-07-01

Morus alba L. has been used in Asian traditional medicine as an anti-inflammatory, anti-asthmatic, anthelmintic and as a whitening agent in cosmetic products. Mulberroside A is the major active compound from M. alba root bark. In this study, cell suspension and root cultures of M. alba were established, and the effect of the elicitors on the enhancement of mulberroside A production in M. alba was investigated. The cell suspension and root cultures of M. alba were exposed to elicitors and then mulberroside A contents were determined by an indirect competitive ELISA method. High levels of mulberroside A were obtained by addition of 100 and 200 μM salicylic acid with 24 h exposure time in cell suspension cultures (37.9 ± 1.5 and 34.0 ± 4.7 mg/g dry wt., respectively). Furthermore, addition of yeast extract at 2 mg/mL with 24 h exposure time can significantly increase mulberroside A contents from both cell suspension (3.2-fold) and root cultures (6.6-fold). Mulberroside A contents from both cell suspension and root cultures after treatment with elicitors are similar or higher than those found in the intact root and root bark of several years old M. alba. These results indicate that mulberry tissue cultures using the elicitation method are interesting alternative sources for mulberroside A production.

Eliminating malignant cells from cryopreserved ovarian tissue is possible in leukaemia patients.

PubMed

Soares, Michelle; Saussoy, Pascale; Maskens, Mathilde; Reul, Hélène; Amorim, Christiani A; Donnez, Jacques; Dolmans, Marie-Madeleine

2017-07-01

Reimplantation of cryopreserved ovarian tissue (OT) can successfully restore ovarian function in young cancer patients after gonadotoxic treatment. However, for patients with leukaemia, there is a risk of malignant cell transmission. Our objective was to evaluate minimal disseminated disease in OT from leukaemia patients and test a follicle isolation technique to obtain disease-free follicle suspensions. Cryopreserved OT from 12 leukaemia patients was thawed and analysed by histology and long-term xenografting in immunosuppressed mice. In 10 patients, follicles were isolated from OT, and polymerase chain reaction (PCR) was performed on tissue, digested ovarian suspensions and isolated follicle suspensions to investigate leukaemic cell presence. Mean patient age was 17·1 years. An average of 3·2 follicles were isolated per mm² of cortex. Xenografting of OT induced leukaemic masses in 2/12 mice. PCR identified leukaemic cell presence in 66% of OT. Malignant cells were also detected in digested ovarian suspensions. However, none of the follicle samples (>2300 follicles tested) showed any malignant cell presence after washing. This study demonstrates that it is possible to recover large numbers of viable follicles from cryopreserved OT of leukaemia patients. All isolated and washed follicle suspensions tested negative for leukaemic cells, giving leukaemia patients genuine hope of fertility restoration. © 2017 John Wiley & Sons Ltd.

[Flow cytometry in datecting lymph node micrometastasis in colorectal cancer].

PubMed

Sun, Q; Ding, Y; Zhang, J

2001-01-25

To study the methodology and significance of flow cytometry in detecting lymph node micrometastasis of colorectal cancer. One hundred sixty-two cellular suspensions were prepared with lymph nodes which were resected radically on 25 patients with colorectal cancer and in which no cancer cells were found by HE staining. Different concentrations of cultured Lovo colorectal cancer cells were added into the celular suspension prepared from lymph node tissue of persons without colorectal cancer in order to prepare a control model. Dual staining with CK/FTTC and PI was made to the sedimetns from those 2 kinds of suspension. Flow cytometry was used to detect cancer cells. An ideal correlation was obtained between the detection value and the theoretical value of cancer cells in the specimen suspensions and control models (r = 0.097 6) with a sensitivity rate of 10/10(5). Cancer cells were detected from 7 out of the 25 patients and 30 of the 162 cellular suspensions. The detection rate was correlated with the size and infiltrating depth of the cancer. Flow cytometry is a reliable, rapid, and quantitative method for detecting lymph node micrometastasis in colorectal cancer.

Transition to organized behavior on suspensions of concentrated bacteria

NASA Astrophysics Data System (ADS)

Ganguly, Sujoy; Cisneros, Luis; Kessler, John; Goldstein, Raymond

2008-11-01

Concentrated populations of the swimming bacterium Bacillus subtilis develop a collective phase, the Zooming BioNematic, that exhibits large-scale coherence analogous to the molecular alignment of nematic liquid crystals. Bacterial suspensions were prepared in order to experimentally measure the transition to organized behavior as a function of the cell number concentration. PIV analysis was used to obtain cell velocities and define an order parameter in order to characterize the dynamics of the system.

Chitosan mediated enhancement of hydrolysable tannin in Phyllanthus debilis Klein ex Willd via plant cell suspension culture.

PubMed

V, Malayaman; N, Sisubalan; R P, Senthilkumar; S, Sheik Mohamed; R, Ranjithkumar; M, Ghouse Basha

2017-11-01

Phyllanthus debilis Klein ex Willd. is wild medicinal plant used in the traditional system of medicine. This plant has been actively used for hepatoprotection and to cure many diseases including jaundice and so on; which leads to complete extinction of this particular species. Therefore, the chitosan mediated cost effective cell suspension method has been developed for the production of hydrolysable tannin. The hydrolysable tannins are the main therapeutically active constituents with antioxidant, anticancer, and antimicrobial properties. An in vitro cell suspension culture was optimized by adding chitosan for production of hydrolysable tannin. According to the growth kinetics, a maximum biomass of 4.46±0.06g fresh cell weight and 1.33±0.04g dry cell weight were obtained from the optimal suspension medium consisted of MS medium+0.5mgL -1 BAP+1.5mgL -1 NAA. Chitosan was treated at the stationary phase which leads to the highest accumulation of hydrolysable tannin compared to the untreated control. Hydrolysable tannin was observed and compared using HPLC at the Rt of 4.91 in both chitosan treated and untreated cells. This is the first ever report where use of chitosan has been done to enhance the production of the hydrolysable tannin in P. debilis using cell suspension culture technique. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes.

PubMed

van Pelt, A M; Morena, A R; van Dissel-Emiliani, F M; Boitani, C; Gaemers, I C; de Rooij, D G; Stefanini, M

1996-08-01

A method for isolating A spermatogonia from the adult vitamin A-deficient (VAD) rat testis is described. After removal, the testes were decapsulated and tubules were dissected. An enzymatic digestion with collagenase, hyaluronidase, and trypsin was performed first to eliminate most of the interstitial cells. A second digestion with collagenase and hyaluronidase was performed to obtain a cell suspension with a high number of A spermatogonia. The cell suspension was further enriched with A spermatogonia by preplating on peanut agglutinin and separating on a discontinuous Percoll gradient. By this procedure, purification of the suspension to 70-90% A spermatogonia was obtained. In the seminiferous tubules of the VAD rats, only Sertoli cells, A spermatogonia, and some preleptotene spermatocytes are present. In our rats, the A spermatogonia are almost all arrested in the G1 phase of the cell cycle before the S phase of A1 spermatogonia, and presumably before their differentiation into A1 spermatogonia. After administration of vitamin A, spermatogenesis starts synchronously from these A spermatogonia. The isolation of these synchronized A spermatogonia opens ways to investigate the regulation of differentiation and proliferation of A spermatogonia and the biochemical characteristics of the subsequent types of A spermatogonia.

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis

PubMed Central

Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

2015-01-01

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here we report the use of stirred suspension bioreactors to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: stirred suspension bioreactor followed by differential plating. After 66 hours of culture, germ cell enrichment in stirred suspension bioreactors provided 7.3±1.0 fold (n=9), differential plating 9.8±2.4 fold (n=6) and combination of both methods resulted in 9.1±0.3 fold enrichment of germ cells from the initial germ cell population (n=3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the stirred suspension bioreactor allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability due to handling. PMID:25877677

Synchronization of Somatic Embryogenesis in Date Palm Suspension Culture Using Abscisic Acid.

PubMed

Alwael, Hussain A; Naik, Poornananda M; Al-Khayri, Jameel M

2017-01-01

Somatic embryogenesis is considered the most effective method for commercial propagation of date palm. However, the limitation of obtaining synchronized development of somatic embryos remains an impediment. The synchronization of somatic embryo development is ideal for the applications to produce artificial seeds. Abscisic acid (ABA) is associated with stress response and influences in vitro growth and development. This chapter describes an effective method to achieve synchronized development of somatic embryos in date palm cell suspension culture. Among the ABA concentrations tested (0, 1, 10, 50, 100 μM), the best synchronized growth was obtained in response to 50-100 μM. Here we provide a comprehensive protocol for in vitro plant regeneration of date palm starting with shoot-tip explant, callus initiation and growth, cell suspension establishment, embryogenesis synchronization with ABA treatment, somatic embryo germination, and rooting as well as acclimatized plantlet establishment.

Water-Based Suspensions of Iron Oxide Nanoparticles with Electrostatic or Steric Stabilization by Chitosan: Fabrication, Characterization and Biocompatibility

PubMed Central

Litvinova, Larisa S.; Safronov, Alexander P.; Schupletsova, Valeria V.; Tyukova, Irina S.; Khaziakhmatova, Olga G.; Slepchenko, Galina B.; Yurova, Kristina A.; Cherempey, Elena G.; Kulesh, Nikita A.; Andrade, Ricardo; Beketov, Igor V.; Khlusov, Igor A.

2017-01-01

Present day biomedical applications, including magnetic biosensing, demand better understanding of the interactions between living systems and magnetic nanoparticles (MNPs). In this work spherical MNPs of maghemite were obtained by a highly productive laser target evaporation technique. XRD analysis confirmed the inverse spinel structure of the MNPs (space group Fd-3m). The ensemble obeyed a lognormal size distribution with the median value 26.8 nm and dispersion 0.362. Stabilized water-based suspensions were fabricated using electrostatic or steric stabilization by the natural polymer chitosan. The encapsulation of the MNPs by chitosan makes them resistant to the unfavorable factors for colloidal stability typically present in physiological conditions such as pH and high ionic force. Controlled amounts of suspensions were used for in vitro experiments with human blood mononuclear leukocytes (HBMLs) in order to study their morphofunctional response. For sake of comparison the results obtained in the present study were analyzed together with our previous results of the study of similar suspensions with human mesenchymal stem cells. Suspensions with and without chitosan enhanced the secretion of cytokines by a 24-h culture of HBMLs compared to a control without MNPs. At a dose of 2.3, the MTD of chitosan promotes the stimulating effect of MNPs on cells. In the dose range of MNPs 10–1000 MTD, chitosan “inhibits” cellular secretory activity compared to MNPs without chitosan. Both suspensions did not caused cell death by necrosis, hence, the secretion of cytokines is due to the enhancement of the functional activity of HBMLs. Increased accumulation of MNP with chitosan in the cell fraction at 100 MTD for 24 h exposure, may be due to fixation of chitosan on the outer membrane of HBMLs. The discussed results can be used for an addressed design of cell delivery/removal incorporating multiple activities because of cell capability to avoid phagocytosis by immune cells. They are also promising for the field of biosensor development for the detection of magnetic labels. PMID:29137198

Evaluation of Simulated Microgravity Environments Induced by Diamagnetic Levitation of Plant Cell Suspension Cultures

NASA Astrophysics Data System (ADS)

Kamal, Khaled Y.; Herranz, Raúl; van Loon, Jack J. W. A.; Christianen, Peter C. M.; Medina, F. Javier

2016-06-01

Ground-Based Facilities (GBF) are essetial tools to understand the physical and biological effects of the absence of gravity and they are necessary to prepare and complement space experiments. It has been shown previously that a real microgravity environment induces the dissociation of cell proliferation from cell growth in seedling root meristems, which are limited populations of proliferating cells. Plant cell cultures are large and homogeneous populations of proliferating cells, so that they are a convenient model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of the Arabidopsis thaliana cell line MM2d were exposed to four altered gravity and magnetic field environments in a magnetic levitation facility for 3 hours, including two simulated microgravity and Mars-like gravity levels obtained with different magnetic field intensities. Samples were processed either by quick freezing, to be used in flow cytometry for cell cycle studies, or by chemical fixation for microscopy techniques to measure parameters of the nucleolus. Although the trend of the results was the same as those obtained in real microgravity on meristems (increased cell proliferation and decreased cell growth), we provide a technical discussion in the context of validation of proper conditions to achieve true cell levitation inside a levitating droplet. We conclude that the use of magnetic levitation as a simulated microgravity GBF for cell suspension cultures is not recommended.

Proper selection of 1 g controls in simulated microgravity research as illustrated with clinorotated plant cell suspension cultures

NASA Astrophysics Data System (ADS)

Kamal, Khaled Y.; Hemmersbach, Ruth; Medina, F. Javier; Herranz, Raúl

2015-04-01

Understanding the physical and biological effects of the absence of gravity is necessary to conduct operations on space environments. It has been previously shown that the microgravity environment induces the dissociation of cell proliferation from cell growth in young seedling root meristems, but this source material is limited to few cells in each row of meristematic layers. Plant cell cultures, composed by a large and homogeneous population of proliferating cells, are an ideal model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of Arabidopsis thaliana cell line (MM2d) were exposed to 2D-clinorotation in a pipette clinostat for 3.5 or 14 h, respectively, and were then processed either by quick freezing, to be used in flow cytometry, or by chemical fixation, for microscopy techniques. After long-term clinorotation, the proportion of cells in G1 phase was increased and the nucleolus area, as revealed by immunofluorescence staining with anti-nucleolin, was decreased. Despite the compatibility of these results with those obtained in real microgravity on seedling meristems, we provide a technical discussion in the context of clinorotation and proper 1 g controls with respect to suspension cultures. Standard 1 g procedure of sustaining the cell suspension is achieved by continuously shaking. Thus, we compare the mechanical forces acting on cells in clinorotated samples, in a control static sample and in the standard 1 g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1 g controls.

Transgenic Russian wildrye (Psathyrostachys juncea) plants obtained by biolistic transformation of embryogenic suspension cells.

PubMed

Wang, Z-Y; Bell, J; Lehmann, D

2004-07-01

Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates. We are interested in developing biotechnological methods to improve this monocot forage species. Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric beta-glucuronidase (gusA) gene was co-transformed with hph. Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin. Plants were regenerated from 45% of the hygromycin resistant calli. Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed. Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues. Fertile transgenic plants were obtained after two winters of vernalization under field conditions. This is the first report on the generation of transgenic plants in Russian wildrye.

Magnetically driven, acoustically tracked, translating-ball rheometer for small, opaque samples

NASA Astrophysics Data System (ADS)

Tran-Son-Tay, R.; Beaty, B. B.; Acker, D. N.; Hochmuth, R. M.

1988-08-01

A new rheometer has been designed to measure the rheological properties at low Reynolds number of microliter quantities of opaque suspensions. The rheometer uses a falling-ball technique to measure steady-state viscosity and a vertically oscillating, magnetically driven ball for viscoelastic measurements. The motion of the ball is tracked by ultrasound echo location, in which sound waves are transmitted and received by an ultrasound transducer mounted at the base of the tube. Concentrated suspensions of red blood cells are used as opaque test samples. The results obtained are in good agreement with those reported in the literature. The data confirm the fact that a concentrated suspension of red blood cells behaves as a shear thinning material and that the energy stored by the suspension during an oscillatory cycle increases with frequency. Testing of the rheometer is also made by using a Newtonian silicone oil. Viscosity measurements obtained with both the falling- and oscillating-ball methods are consistent and are within 2% of the value of 47.3±0.5 cP given by the Cannon-Fenske viscometer. However, it is found that the oscillating-ball technique gives the largest standard deviation, 6%, as opposed to 2% for the falling-ball technique.

[A study of the aggregation of human red blood cells induced by picric acid].

PubMed

Sheremet'ev, Iu A; Sheremet'eva, A V; Lednev, A V

2005-01-01

The effect of picric acid on the aggregation of human erythrocytes was studied. It was shown that the addition of picric acid to a suspension of washed erythrocytes leads to a decrease in pH of medium to 1.5-2 and the formation of echinocytes. Stirring the suspension of echinocytes at low pH values results in a strong aggregation of cells. Increasing the pH value to 7.4 leads to a desaggregation of echinocytes. It was found that picric acid does not induce the aggregation of cells fixed by glutaraldehyde. A substantial decrease in the aggegation of spheric erythrocytes obtained after heating the cells at 50 degrees C was observed.

In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes

PubMed Central

Bestoso, Federica; Ottaggio, Laura; Armirotti, Andrea; Balbi, Alessandro; Damonte, Gianluca; Degan, Paolo; Mazzei, Mauro; Cavalli, Francesca; Ledda, Bernardetta; Miele, Mariangela

2006-01-01

Background Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species) were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established. Results Calli were successfully induced from stems, leaves and seeds grown in various hormone concentrations and combinations. The most suitable callus to establish suspension cell cultures was obtained from seeds. Media recovered from suspension cell cultures contained taxanes, and showed antiproliferative activity on human tumour cells. Taxol, 10-deacetyltaxol and 10-deacetylbaccatin III were the main taxanes identified. The level of Taxol recovered from the media of hazel cultures was similar to that found in yew cultures. Moreover, the production of taxanes in hazel cell cultures increased when elicitors were used. Conclusion Here we show that hazel cell cultures produce Taxol and taxanes under controlled conditions. This result suggests that hazel possesses the enzymes for Taxol production, which until now was considered to be a pathway particular to Taxus genus. The main benefit of producing taxanes through hazel cell cultures is that hazel is widely available, grows at a much faster rate in vivo, and is easier to cultivate in vitro than yew. In addition, the production of callus directly from hazel seeds shortens the culture time and minimizes the probability of contamination. Therefore, hazel could become a commercial source of Taxol and taxanes, both to be used as new therapeutic agents or as new precursors for Taxol semi-synthesis. PMID:17150090

The effects of erythrocyte deformability upon hematocrit assessed by the conductance method.

PubMed

Hayashi, Yoshihito; Katsumoto, Yoichi; Oshige, Ikuya; Omori, Shinji; Yasuda, Akio; Asami, Koji

2009-04-21

A comparative study of centrifugation and conductance methods for the estimation of cell volume fraction (phi) was performed to examine whether the strong forces exerted upon erythrocytes during centrifugation affect their volume, and the results are discussed in terms of erythrocyte deformability. Rabbit erythrocytes of four shapes (spherocytes, echinocytes, stomatocyte-like enlarged erythrocytes and discocytes) were prepared by controlling the pH of the suspending media. The packed cell volumes of the suspensions were measured by standard hematocrit determination methods using centrifugation in capillary tubes. Simultaneously, the same suspensions and their supernatants were used in dielectric spectroscopy measurements, and the low-frequency limits of their conductivities were used for the numerical estimation of phi. The hematocrit values of spherocytes and echinocytes were markedly less than the volume fractions obtained by the conductance method. Namely, the centrifugation reduced the cell volume. For enlarged erythrocytes and discocytes, however, the reduction of cell volume was not observed. These findings showed that phi obtained by the centrifugation method can be greatly affected by the deformability of the cells, but the level of the effect depends on the cell types. Consequently, phi obtained by the centrifugation method should be carefully interpreted.

Glycyrrhiza glabra (Linn.) and Lavandula officinalis (L.) cell suspension cultures-based biotransformation of β-artemether.

PubMed

Patel, Suman; Gaur, Rashmi; Upadhyaya, Mohita; Mathur, Archana; Mathur, Ajay K; Bhakuni, Rajendra S

2011-07-01

The biotransformation of β-artemether (1) by cell suspension cultures of Glycyrrhiza glabra and Lavandula officinalis is reported here for the first time. The major biotransformed product appeared as a grayish-blue color spot on thin-layer chromatography (TLC) with transparent crystal-like texture. Based on its infrared (IR) and (1)H nuclear magnetic resonance (NMR) spectra, the product was characterized as a tetrahydrofuran (THF)-acetate derivative (2). The highest conversion efficiencies of 57 and 60% were obtained when 8-9-day-old cell suspensions of G. glabra and L. officinalis were respectively fed with 4-7 mg of compound 1 in 40 ml of medium per culture and the cells were harvested after 2-5 days of incubation. The addition of compound 1 at the beginning of the culture cycle caused severe growth depression in a dose-dependent manner, resulting in poor bioconversion efficiency of ~25% at 2-5 mg/culture dose only.

Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

PubMed

Walther, Christa G; Whitfield, Robert; James, David C

2016-04-01

The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

Stem Cell Niche is Partially Lost during Follicular Plucking: A Preliminary Pilot Study

PubMed Central

Kumar, Anil; Gupta, Somesh; Mohanty, Sujata; Bhargava, Balram; Airan, Balram

2013-01-01

Background: Clinical hair transplant studies have revealed that follicular unit extraction (FUE) is superior in terms of stable hair growth in comparison to follicular plucking (FP). Various reasons have been cited for this clinical outcome. FUE and FP are employed to obtain the hair follicle units for hair transplant and recently for cell based therapies in vitiligo. However, there is no scientific data available on the comparison of stem cell fraction in the cell suspension obtained by FUE and FP. Therefore, we undertook this study to compare the percentage of stem cells in the hair follicle obtained by FUE and FP. Objective: The purpose of the following study is to evaluate the quantitative stem cell pool in the hair follicle obtained by FUE and FP. Materials and Methods: A total of 3 human subjects were enrolled with age groups of 17-25 years. Both methods of tissue harvest: FUE and FP; were employed on each subject. There was no vitiligo lesion on the scalp in any of the patients. Hair follicles were incubated with trypsin-EDTA solution at 37°C for 90 min to separate outer root sheath cells. The cell suspension was passed through a 70 μm cell strainer; filtrate was centrifuged to obtain the cell pellet. Cells were labeled with cluster of differentiation (CD200) antibody and acquired with flowcytometry. Results: The mean percentage of CD200 positive cells in FUE and FP method come out to be 8.43 and 1.63 respectively (P = 0.0152). Conclusion: FUE is a better method of the hair follicle harvesting for cell based applications as the stem cell fraction is significantly higher in comparison to FP. PMID:24403776

Neuropharmacological and neuroprotective activities of some metabolites produced by cell suspension culture of Waltheria americana Linn.

PubMed

Mundo, Jorge; Villeda-Hernández, Juana; Herrera-Ruiz, Maribel; Gutiérrez, María Del Carmen; Arellano-García, Jesús; León-Rivera, Ismael; Perea-Arango, Irene

2017-10-01

Waltheria americana is a plant used in Mexican traditional medicine to treat some nervous system disorders. The aims of the present study were to isolate and determine the neuropharmacological and neurprotective activities of metabolites produced by a cell suspension culture of Waltheria americana. Submerged cultivation of W. americana cells provided biomass. A methanol-soluble extract (WAsc) was obtained from biomass. WAsc was fractionated yielding the chromatographic fractions 4WAsc-H 2 O and WAsc-CH 2 Cl 2 . For the determination of anticonvulsant activity in vivo, seizures were induced in mice by pentylenetetrazol (PTZ). Neuropharmacological activities (release of gamma amino butyric acid (GABA) and neuroprotection) of chromatographic fractions were determined by in vitro histological analysis of brain sections of mice post mortem. Fraction 4WAsc-H 2 O (containing saccharides) did not produce neuronal damage, neurodegeneration, interstitial tissue edema, astrocytic activation, nor cell death. Pretreatment of animals with 4WAsc-H 2 O and WAsc-CH 2 Cl 2 from W. americana cell suspensions induced an increase in: GABA release, seizure latency, survival time, neuroprotection, and a decrease in the degree of severity of tonic/tonic-clonic convulsions, preventing PTZ-induced death of up to 100% of animals of study. Bioactive compounds produced in suspension cell culture of W. americana produce neuroprotective and neuropharmacological activities associated with the GABAergic neurotransmission system. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Increasing anthraquinone production by overexpression of 1-deoxy-D: -xylulose-5-phosphate synthase in transgenic cell suspension cultures of Morinda citrifolia.

PubMed

Quevedo, Carla; Perassolo, María; Alechine, Eugenia; Corach, Daniel; Giulietti, Ana María; Talou, Julián Rodriguez

2010-07-01

A Morinda citrifolia cell line was obtained by overexpresion of 1-deoxy-D: -xylulose 5-phosphate synthase (DXS) from Catharanthus roseus, a key enzyme of the metabolic pathway of anthraquinones (AQs). This cell line increased AQs production by about 24% compared to the control cell line. This transgenic cell line which carries dxs cDNA isolated from Catharanthus roseus, was achieved by direct transformation of cell suspension cultures of M. citrifolia using a hypervirulent Agrobacterium tumefaciens strain. The effects of the overexpression of the dxs gene also resulted in increased levels of dxs mRNA transcripts and DXS activity compared to the control cell line. In addition, total phenolics and phenylalanine ammonia-lyase activity were evaluated and were significantly higher in the transgenic line than in controls.

Enhanced production of phenolic acids in cell suspension culture of Salvia leriifolia Benth. using growth regulators and sucrose.

PubMed

Modarres, Masoomeh; Esmaeilzadeh Bahabadi, Sedigheh; Taghavizadeh Yazdi, Mohammad Ehsan

2018-04-01

Salvia leriifolia Benth. (Lamiaceae) is an endangered medicinal plant with hypoglycemic, anti-inflammatory and analgesic properties. Many of the beneficial effects of Salvia spp. are attributed to the phenolic compounds. In the present study, an efficient procedure has been developed for establishment of cell suspension culture of S. leriifolia as a strategy to obtain an in vitro phenolic acids producing cell line for the first time. The effect of growth regulators and various concentrations of sucrose have been analyzed, to optimize biomass growth and phenolic acids production. The callus used for this purpose was obtained from leaves of 15-day-old in vitro seedlings, on Murashige and Skoog (MS) basal medium supplemented with different hormone balances including benzylaminopurine (BAP) and indole butyric acid (IBA); 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN); naphthaleneacetic acid (NAA) and BAP. Modified MS medium supplemented with 5 mg/L BAP and 5 mg/L NAA was the optimal condition for callus formation with the highest induction rate (100%), the best callus growth and the highest phenolic acids content. No callus induction was observed in combinations of IBA and BAP. Cell suspension cultures were established by transferring 0.5 g of callus to 30 mL liquid MS medium supplemented with 5 mg/L BAP and 5 mg/L NAA. Dynamics of phenolic acids production has been investigated during the growth cycle of the suspension cultures. The maximum content of caffeic acid and salvianolic acid B were observed on the 15th day of the cultivation cycle while the highest amount of rosmarinic acid was observed on the first day. In response to various sucrose concentrations, cell cultures with 40 g/L sucrose not only produced the highest dry biomass but also the highest induction of caffeic acid and salvianolic acid B. The highest amount of rosmarinic acid was observed in media containing 50 g/L sucrose. These prepared cell suspension cultures provided a useful system for further enhanced production of phenolic acids at a large scale.

A new continuum model for suspensions of gyrotactic micro-organisms

NASA Technical Reports Server (NTRS)

Pedley, T. J.; Kessler, J. O.

1990-01-01

A new continuum model is formulated for dilute suspensions of swimming micro-organisms with asymmetric mass distributions. Account is taken of randomness in a cell's swimming direction, p, by postulating that the probability density function for p satisfies a Fokker-Planck equation analogous to that obtained for colloid suspensions in the presence of rotational Brownian motion. The deterministic torques on a cell, viscous and gravitational, are balanced by diffusion, represented by an isotropic rotary diffusivity Dr, which is unknown a priori, but presumably reflects stochastic influences on the cell's internal workings. When the Fokker-Planck equation is solved, macroscopic quantities such as the average cell velocity Vc, the particle diffusivity tensor D and the effective stress tensor sigma can be computed; Vc and D are required in the cell conservation equation, and sigma in the momentum equation. The Fokker-Planck equation contains two dimensionless parameters, lambda and epsilon; lambda is the ratio of the rotary diffusion time Dr-1 to the torque relaxation time B (balancing gravitational and viscous torques), while epsilon is a scale for the local vorticity or strain rate made dimensionless with B. In this paper we solve the Fokker-Planck equation exactly for epsilon = 0 (lambda arbitrary) and also obtain the first-order solution for small epsilon. Using experimental data on Vc and D obtained with the swimming alga, Chlamydomonas nivalis, in the absence of bulk flow, the epsilon = 0 results can be used to estimate the value of lambda for that species (lambda approximately 2.2; Dr approximately 0.13 s-1). The continuum model for small epsilon is then used to reanalyse the instability of a uniform suspension, previously investigated by Pedley, Hill & Kessler (1988). The only qualitatively different result is that there no longer seem to be circumstances in which disturbances with a non-zero vertical wavenumber are more unstable than purely horizontal disturbances. On the way, it is demonstrated that the only significant contribution to sigma, other than the basic Newtonian stress, is that derived from the stresslets associated with the cells' intrinsic swimming motions.

Development of suspension cell culture model to mimic circulating tumor cells

PubMed Central

Park, Ji Young; Jeong, Ae Lee; Joo, Hyun Jeong; Han, Sora; Kim, So-Hyun; Kim, Hye-Youn; Lim, Jong-Seok; Lee, Myeong-Sok; Choi, Hyung-Kyoon; Yang, Young

2018-01-01

Circulating tumor cells (CTCs) are essential for the establishment of distant metastasis. Numerous studies have characterized CTCs as metastatic precursors; however, the molecular nature of CTCs has not been completely revealed yet due to the low number of CTCs in the blood stream. As an alternative approach, we developed a long-term suspension cell culture model using human breast cancer cell lines to mimic CTCs. We found that more than 40 passaged suspension cells acquired the ability to enhance metastasis like cancer stem cells. To identify molecular changes acquired during the suspension cell culture, we analyzed metabolic and lipidomic profiles as well as transcriptome in MDA-MB-468 suspension cells. Glutamate and leucine levels increased in suspension cells, and cholesterol synthesis pathway was altered. The inhibition of glutamate metabolic pathway decreased the proliferation of suspension cells compared to that of adherent cells. In the lipidomic profile, PC species containing long chain and polyunsaturated fatty acids increased in suspension cells and these species could be authentic and specific biomarkers for highly metastatic cancers. As this CTC-mimicking suspension cell culture model may easily apply to various types of cancer, we suggest this model as a great tool to develop therapeutic targets and drugs to eradicate metastatic cancer cells. PMID:29416640

Stable transformation via particle bombardment in two different soybean regeneration systems.

PubMed

Sato, S; Newell, C; Kolacz, K; Tredo, L; Finer, J; Hinchee, M

1993-05-01

The Biolistics(®) particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for β-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.

Signal transduction in artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] callus and cell suspension cultures under nutritional stress.

PubMed

Lattanzio, Vincenzo; Caretto, Sofia; Linsalata, Vito; Colella, Giovanni; Mita, Giovanni

2018-06-01

Stimulated production of secondary phenolic metabolites and proline was studied by using cell cultures of artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] submitted to nutritional stress. Artichoke cell cultures accumulated phenolic secondary metabolites in a pattern similar to that seen in artichoke leaves and heads (capitula). This paper shows that both callus and cell suspension cultures under nutritional stress accumulated phenolic compounds and proline, at the same time their biomass production was negatively affected by nutrient deficiency. The results obtained strongly suggest that plant tissues respond to nutrient deprivation by a defensive costly mechanism, which determines the establishment of a mechanism of trade-off between growth and adaptive response. Furthermore, the results of this research suggest that perception of abiotic stress and increased phenolic metabolites are linked by a sequence of biochemical processes that also involves the intracellular free proline and the oxidative pentose phosphate pathway. The main conclusion of this paper is that, once calli and cell suspension cultures respond to nutrient deficiency, in acclimated cells the establishment of a negative correlation between primary metabolism (growth) and secondary metabolism (defence compounds) is observed. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

In vitro skin permeation and anti-atopic efficacy of lipid nanocarriers containing water soluble extracts of Houttuynia cordata.

PubMed

Kwon, Taek Kwan; Kim, Jin-Chul

2014-10-01

The aims of this work are to enhance the in vitro skin permeation of Houttuynia cordata (water-soluble extract of H. cordata; HCWSE) and to boost the efficacy of HCWSE against atopic dermatitis (AD) - like skin lesion in hairless mice using lipid nano-carriers (liposome and cubosome). HCWSE was obtained by a hot water extraction. Monoolein cubosomal suspension containing HCWSE and egg phosphatidylcholine liposomal suspension containing the same was prepared by a sonication and a film hydration method, respectively. The lipid nano-carriers, especially cubosome, enhanced the in vitro skin permeation of HCWSE. The inhibitory effects of HCWSE-containing lipid carrier suspensions on the development of 1-chloro-2,4-dinitrobenzene (DNCB)-induced AD-like skin lesion in hairless mice were investigated by observing appearance of skin surface, serum immunoglobulin E (IgE) level and cytokine expression. HCWSE-containing preparations suppressed IgE production and interleukin 4 expression, whereas they promoted interferon gamma expression. The order of lymphocyte (B-cell, Th1 cell and Th2 cell) modulating effect was HCWSE-containing cubosomal suspension > HCWSE-containing liposomal suspension > HCWSE solution in phosphate buffered saline, indicating that the cubosomal suspension, among the preparations, was the most efficacious in inhibiting the development of DNCB-induced AD-like skin lesion. It is believed that the cubosomal suspension containing HCWSE would be an efficacious preparation for the treatment of AD.

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

PubMed

Dardikman, Gili; Nygate, Yoav N; Barnea, Itay; Turko, Nir A; Singh, Gyanendra; Javidi, Barham; Shaked, Natan T

2018-03-01

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

Electrochemical Cell for Obtaining Oxygen from Carbon Dioxide Atmospheres

NASA Technical Reports Server (NTRS)

Hooker, Matthew; Rast, H. Edward; Rogers, Darren K.; Borja, Luis; Clark, Kevin; Fleming, Kimberly; Mcgurren, Michael; Oldaker, Tom; Sweet, Nanette

1989-01-01

To support human life on the Martian surface, an electrochemical device will be required to obtain oxygen from the carbon dioxide rich atmosphere. The electrolyte employed in such a device must be constructed from extremely thin, dense membranes to efficiently acquire the oxygen necessary to support life. A forming process used industrially in the production of multilayer capacitors and electronic substrates was adapted to form the thin membranes required. The process, known as the tape casting, involves the suspension consisting of solvents and binders. The suspension is passed under a blade, resulting in the production of ceramic membranes between 0.1 and 0.5 mm thick. Once fired, the stabilized zirconia membranes were assembled into the cell design by employing a zirconium phosphate solution as the sealing agent. The resulting ceramic-to-ceramic seals were found to be structurally sound and gas-tight. Furthermore, by using a zirconia-based solution to assemble the cell, the problem of a thermal expansion mismatch was alleviated. By adopting an industrial forming process to produce thin membranes, an electrochemical cell for obtaining oxygen from carbon dioxide was produced. The proposed cell design is unique in that it does not require a complicated manifold system for separating the various gases present in this process, nor does it require a series of complex electrical connections. Thus, the device can reliably obtain the vital oxygen supply from the toxic carbon dioxide atmosphere.

Suspension state increases reattachment of breast cancer cells by up-regulating lamin A/C.

PubMed

Zhang, Xiaomei; Lv, Yonggang

2017-12-01

Extravasation is a rate-limiting step of tumor metastasis, for which adhesion to endothelium of circulating tumor cells (CTCs) is the prerequisite. The suspension state of CTCs undergoing detachment from primary tumor is a persistent biomechanical cue, which potentially regulates the biophysical characteristics and cellular behaviors of tumor cells. In this study, breast tumor cells MDA-MB-231 in suspension culture condition were used to investigate the effect of suspension state on reattachment of CTCs. Our study demonstrated that suspension state significantly increased the adhesion ability of breast tumor cells. In addition, suspension state markedly promoted the formation of stress fibers and focal adhesions and reduced the motility in reattached breast cancer cells. Moreover, lamin A/C was reversibly accumulated at posttranscriptional level under suspension state, improving the cell stiffness of reattached breast cancer cells. Disruption of actin cytoskeleton by cytochalasin D caused lamin A/C accumulation. Conversely, decreasing actomyosin contraction by ROCK inhibitor Y27632 reduced lamin A/C level. Knocking down lamin A/C weakened the suspension-induced increase of adhesion, and also abolished the suspension-induced decrease of motility and increase of stress fibers and focal adhesion in reattaching tumor cells, suggesting a crucial role of lamin A/C. In conclusion, it was demonstrated that suspension state promoted the reattachment of breast tumor cells by up-regulating lamin A/C via cytoskeleton disruption. These findings highlight the important role of suspension state for tumor cells in tumor metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

TC-tuned biocompatible suspension of La0.73Sr0.27MnO3 for magnetic hyperthermia.

PubMed

Prasad, N K; Rathinasamy, K; Panda, D; Bahadur, D

2008-05-01

La(1-x)Sr(x)MnO(3), a ferromagnet with high magnetization and Curie temperature T(C) below 70 degrees C, enables its use for magnetic hyperthermia treatment of cancer with a possibility of in vivo temperature control. We found that La(0.73)Sr(0.27)MnO(3) particles of size range 20-100 nm showed saturation magnetization around 38 emu/g at 20 kOe and a T(C) value of 45 degrees C. Aqueous suspension of these nanoparticles was prepared using a polymer, acrypol 934, and the biocompatibility of the suspension was examined using HeLa cells. A good heating ability of the magnetic suspension was obtained in the presence of AC magnetic field, and it was found to increase with the amplitude of field. The suspension having concentration of 0.66 mg/mL (e.g., 0.66 mg of nanoparticles with acropyl per milliliter of culture media) was observed to be biocompatible even after 96 h of treatment, as estimated by sulforhodamine B and trypan blue dye exclusion assays. Further, the treatment with the aforementioned concentration did not alter the microtubule cytoskeleton or the nucleus of the cells. However, the bare particles (concentration of 0.66 mg of nanoparticles per milliliter of culture media, but without acropyl) decreased the viability of cell significantly. Our in vitro studies suggest that the suspension (concentration of 0.66 mg/mL) may further be analyzed for in vivo studies. Copyright 2007 Wiley Periodicals, Inc.

Biocompatible Colloidal Suspensions Based on Magnetic Iron Oxide Nanoparticles: Synthesis, Characterization and Toxicological Profile

PubMed Central

Coricovac, Dorina-Elena; Moacă, Elena-Alina; Pinzaru, Iulia; Cîtu, Cosmin; Soica, Codruta; Mihali, Ciprian-Valentin; Păcurariu, Cornelia; Tutelyan, Victor A.; Tsatsakis, Aristidis; Dehelean, Cristina-Adriana

2017-01-01

The use of magnetic iron oxide nanoparticles in biomedicine has evolved intensely in the recent years due to the multiple applications of these nanomaterials, mainly in domains like cancer. The aim of the present study was: (i) to develop biocompatible colloidal suspensions based on magnetic iron oxide nanoparticles as future theranostic tools for skin pathology and (ii) to test their effects in vitro on human keratinocytes (HaCat cells) and in vivo by employing an animal model of acute dermal toxicity. Biocompatible colloidal suspensions were obtained by coating the magnetic iron oxide nanoparticles resulted during the solution combustion synthesis with a double layer of oleic acid, as innovative procedure in increasing bioavailability. The colloidal suspensions were characterized in terms of dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro effects of these suspensions were tested by means of Alamar blue assay and the noxious effects at skin level were measured using non-invasive methods. The in vitro results indicated a lack of toxicity on normal human cells induced by the iron oxide nanoparticles colloidal suspensions after an exposure of 24 h to different concentrations (5, 10, and 25 μg·mL−1). The dermal acute toxicity test showed that the topical applications of the colloidal suspensions on female and male SKH-1 hairless mice were not associated with significant changes in the quality of barrier skin function. PMID:28400730

Biostimulation effects of low-energy laser radiation on yeast cell suspensions

NASA Astrophysics Data System (ADS)

Anghel, Sorin; Stanescu, Constantin S.; Giosanu, Dana; Neagu, Ionica; Savulescu, Geta; Iorga-Siman, Ion

2000-02-01

This paper presents work to determine the effects produced by low energy laser radiation on the metabolism and growth of a yeast cell suspension. As experimental material, we used young yeast culture in liquid medium, then distributed on a solid medium, to obtain isolated colonies. As laser source, we used a He-Ne laser, and the irradiation was made with different exposure times. Form each irradiated material, a sample of white grape sterile must was sowed, that has fermented at 18 divided by 20 degrees C for 10 divided by 15 days, after that some properties was tested. Some microscopic studies were also made. The results prove some influence of low energy laser irradiation, which can induce mutations, with new properties of the irradiated material. These mutations can be obtained in a positive sense, with new and important perspectives in wine industry. Also, we observed an inhibitory effect of the laser radiation on the yeast cell growth, due, probably to the too high values of the exposure.

Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

PubMed

Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

2017-08-01

Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or pharmacological screening. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

PubMed

Tibbe, Arjan G J; de Grooth, Bart G; Greve, Jan; Dolan, Gerald J; Rao, Chandra; Terstappen, Leon W M M

2002-03-01

Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension. Copyright 2002 Wiley-Liss, Inc.

Xanthones from roots, hairy roots and cell suspension cultures of selected Hypericum species and their antifungal activity against Candida albicans.

PubMed

Zubrická, Daniela; Mišianiková, Anna; Henzelyová, Jana; Valletta, Alessio; De Angelis, Giulia; D'Auria, Felicia Diodata; Simonetti, Giovanna; Pasqua, Gabriella; Čellárová, Eva

2015-11-01

Highest xanthone contents were found in Hypericum pulchrum and H. annulatum untransformed roots. The best anti- Candida activity was obtained for hairy roots extracts of H. tetrapterum clone 2 ATCC 15834. Extracts of root cultures, hairy roots and cell suspensions of selected Hypericum spp. were screened for the presence of xanthones and tested for their antifungal activity against Candida albicans strain ATCC 10231. At least one of the following xanthones, 5-methoxy-2-deprenylrheediaxanthone; 1,3,6,7-tetrahydroxyxanthone; 1,3,5,6-tetrahydroxyxanthone; paxanthone; kielcorin or mangiferin was identified in methanolic extracts of the untransformed root cultures. The highest total xanthone content, with five xanthones, was found in untransformed H. pulchrum and H. annulatum root cultures. Hairy roots and the controls of H. tetrapterum contained 1,7-dihydroxyxanthone, while hairy root cultures and the corresponding controls of H. tomentosum contained toxyloxanthone B, 1,3,6,7- and 1,3,5,6-tetrahydroxyxanthone. Two xanthones, cadensin G and paxanthone, were identified in cell suspension cultures of H. perforatum. Their content increased about two-fold following elicitation with salicylic acid. The anti-Candida activity of the obtained extracts ranged from MIC 64 to >256 µg ml(-1). Among the extracts of Hypericum untransformed roots, the best antifungal activity was obtained for extracts of H. annulatum grown under CD conditions. Extracts of hairy roots clones A4 and 7 ATCC15834 of H. tomentosum and clone 2 ATCC15834 of H. tetrapterum displayed inhibition of 90% of Candida growth with 256 μg ml(-1). Extracts from chitosan-elicitated cells did not show antifungal activity.

Proteomic Analysis of Grape Berry Cell Cultures Reveals that Developmentally Regulated Ripening Related Processes Can Be Studied Using Cultured Cells

PubMed Central

Sharathchandra, Ramaschandra G.; Stander, Charmaine; Jacobson, Dan; Ndimba, Bongani; Vivier, Melané A.

2011-01-01

Background This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first. Methodology/Principal Findings In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions. Conclusions The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks responsible for berry development and ripening. PMID:21379583

Effect of spaceflight on natural killer cell activity

NASA Technical Reports Server (NTRS)

Rykova, Marina P.; Sonnenfeld, Gerald; Lesniak, A. T.; Taylor, Gerald R.; Meshkov, Dimitrii O.; Mandel, Adrian D.; Medvedev, Andrei E.; Berry, Wallace D.; Fuchs, Boris B.; Konstantinova, Irina V.

1992-01-01

The effects of spaceflight on immune cell function were determined in rats flown on Cosmos 2044. Control groups included vivarium, synchronous, and antiorthostatically suspended rats. The ability of natural killer cells to lyse two different target cell lines was determined. Spleen and bone marrow cells obtained from flight rats showed significantly inhibited cytotoxicity for YAC-1 target cells compared with cells from synchronous control rats. This could have been due to exposure of the rats to microgravity. Antiorthostatic suspension did not affect the level of cytotoxicity from spleen cells of suspended rats for YAC-1 cells. On the other hand, cells from rats flown in space showed no significant differences from vivarium and synchronous control rats in cytotoxicity for K-562 target cells. Binding of natural killer cells to K-562 target cells was unaffected by spaceflight. Antiorthostatic suspension resulted in higher levels of cytotoxicity from spleen cells for Cr-51-labeled K-562 cells. The results indicate differential effects of spaceflight on function of natural killer cells. This shows that spaceflight has selective effects on the immune response.

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium.

PubMed

Peng, Lin; Yu, Xiao; Li, Chengyuan; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

2016-04-01

Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-gamma production in suspension and inside microcarriers in protein-free media.

PubMed

Ballez, J S; Mols, J; Burteau, C; Agathos, S N; Schneider, Y J

2004-03-01

We have recently developed a protein-free medium (PFS) able to support the growth of Chinese hamster ovary (CHO) cells in suspension. Upon further supplementation with some plant protein hydrolysates, medium performances reached what could be observed in serum-containing media [Burteau et al. In Vitro Cell. Dev. Biol.-Anim. 39 (2003) 291]. Now, we describe the use of rice and wheat protein hydrolysates, as non-nutritional additives to the culture medium to support productivity and cell growth in suspension or in microcarriers. When CHO-320 cells secreting recombinant interferon-gamma (IFN-gamma) were cultivated in suspension in a bioreactor with our PFS supplemented with wheat hydrolysates, the maximum cell density increased by 25% and the IFN-gamma secretion by 60% compared to the control PFS. A small-scale perfusion system consisting of CHO-320 cells growing on and inside fibrous microcarriers under discontinuous operation was first developed. Under these conditions, rice protein hydrolysates stimulated recombinant IFN-gamma secretion by 30% compared to the control PFS. At the bioreactorscale, similar results were obtained but when compared to shake-flasks studies, nutrients, oxygen or toxic by-products gradients inside the microcarriers seemed to be the main limitation of the system. An increase of the perfusion rate to maintain glucose concentration over 5.5 mM and dissolved oxygen (DO) at 60% was able to stimulate the production of IFN-gamma to a level of 6.6 mug h(-1) g(-1) of microcarriers after 160 h when a cellular density of about 4 x 10(8) cell g(-1) of carriers was reached.

Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor.

PubMed

Tapia, Felipe; Vogel, Thomas; Genzel, Yvonne; Behrendt, Ilona; Hirschel, Mark; Gangemi, J David; Reichl, Udo

2014-02-12

Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀(HA units/100 μL) and 1.8 × 10(10)virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus. Copyright © 2013 Elsevier Ltd. All rights reserved.

Prechlorination of algae-laden water: The effects of transportation time on cell integrity, algal organic matter release, and chlorinated disinfection byproduct formation.

PubMed

Qi, Jing; Lan, Huachun; Liu, Ruiping; Miao, Shiyu; Liu, Huijuan; Qu, Jiuhui

2016-10-01

The prechlorination-induced algal organic matter (AOM) released from Microcystis aeruginosa (M. aeruginosa) cells has been reported to serve as a source of precursors for chlorinated disinfection byproducts (DBPs). However, previous studies have mainly focused on the precursors either extracted directly from the cell suspension or derived immediately after algal suspension prechlorination. This study aims to investigate the impacts of water transportation time after algal suspension prechlorination on cell integrity, AOM release, and DBP formation during the dissolved phase chlorination. The damage to cell integrity after prechlorination was indicated to depend not only on chlorine dose but also on transportation time. The highest dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) values were observed at 2 mg/L chlorine preoxidation before transportation, but were obtained at 0.4 mg/L chlorine after 480-min simulated transportation. The variation of DON with transportation time was indicated to be mainly influenced by the small molecular weight nitrogenous organic compounds, such as amino acids. Additionally, formation of the corresponding chlorinated carbonaceous disinfection byproducts (C-DBPs) and nitrogenous disinfection byproducts (N-DBPs) during the dissolved phase chlorination showed the same variation tendency as DOC and DON respectively. The highest C-DBP (98.4 μg/L) and N-DBP (5.5 μg/L) values were obtained at 0.4 mg/L chlorine preoxidation after 480-min simulated transportation. Therefore, when prechlorination is applied for algae-laden water pretreatment, not only chlorine dose but also transportation time needs to be considered with regard to their effects on cell integrity, AOM release, and chlorinated DBP formation. Copyright © 2016. Published by Elsevier Ltd.

Optimization of multicomponent aqueous suspensions of lithium iron phosphate (LiFePO4) nanoparticles and carbon black for lithium-ion battery cathodes.

PubMed

Li, Jianlin; Armstrong, Beth L; Daniel, Claus; Kiggans, Jim; Wood, David L

2013-09-01

Addition of polyethyleneimine (PEI) to aqueous LiFePO4 nanoparticle suspensions improves stability and reduces agglomerate size, which is beneficial to lithium-ion battery cathode manufacturing. This research examines the effect of both PEI concentration and molecular weight (MW) on dispersing LiFePO4 and Super P C45 in multicomponent aqueous suspensions. It is demonstrated that the optimal conditions for obtaining stable suspensions with minimal agglomerate size are 1.5 wt% PEI with MW=2000 g mol(-1) and 5.0 wt% PEI with MW=10,000 g mol(-1) for LiFePO4 and Super P C45, respectively. The mixing sequence also affects rheological properties of these suspensions. It is found that dispersing the LiFePO4 and Super P C45 separately yielded suspensions with superior properties (Newtonian rheological behavior, smaller agglomerate size, improved settling, etc.). In particular, dispersing the LiFePO4 prior to the Super P C45 when making the final multicomponent suspension is found to be beneficial, which was evidenced by higher half-cell discharge capacity. Copyright © 2013 Elsevier Inc. All rights reserved.

Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion*

PubMed Central

Li, Dan; Peng, Shi-yun; Zhang, Zhen-wu; Feng, Rui-cheng; Li, Lu; Liang, Jie; Tai, Sheng; Teng, Chun-bo

2013-01-01

The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is prerequisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid (EDTA) digestion method to disassociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRP1-positive cells in cell suspensions prepared through cold digestion was consistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells. PMID:23825145

A reliable method for spectrophotometric determination of glycine betaine in cell suspension and other systems.

PubMed

Valadez-Bustos, Ma Guadalupe; Aguado-Santacruz, Gerardo Armando; Tiessen-Favier, Axel; Robledo-Paz, Alejandrina; Muñoz-Orozco, Abel; Rascón-Cruz, Quintin; Santacruz-Varela, Amalio

2016-04-01

Glycine betaine is a quaternary ammonium compound that accumulates in a large variety of species in response to different types of stress. Glycine betaine counteracts adverse effects caused by abiotic factors, preventing the denaturation and inactivation of proteins. Thus, its determination is important, particularly for scientists focused on relating structural, biochemical, physiological, and/or molecular responses to plant water status. In the current work, we optimized the periodide technique for the determination of glycine betaine levels. This modification permitted large numbers of samples taken from a chlorophyllic cell line of the grass Bouteloua gracilis to be analyzed. Growth kinetics were assessed using the chlorophyllic suspension to determine glycine betaine levels in control (no stress) cells and cells osmotically stressed with 14 or 21% polyethylene glycol 8000. After glycine extraction, different wavelengths and reading times were evaluated in a spectrophotometer to determine the optimal quantification conditions for this osmolyte. Optimal results were obtained when readings were taken at a wavelength of 290 nm at 48 h after dissolving glycine betaine crystals in dichloroethane. We expect this modification to provide a simple, rapid, reliable, and cheap method for glycine betaine determination in plant samples and cell suspension cultures. Copyright © 2016 Elsevier Inc. All rights reserved.

Plant regeneration from protoplasts of embryogenic cell suspensions of Coffea arabica L. cv. caturra.

PubMed

Acuna, J R; de Pena, M

1991-09-01

Coffee plants were regenerated from protoplasts isolated from embryogenic cell suspension cultures derived from somatic embryos of Coffea arabica L. cv. caturra. Yields of viable protoplasts ranged from 1×10(5) to 6×10(5) protoplast/g fresh weight. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the total. Plating efficiencies of protoplast ranged from 1 to 10%. Embryogenic protocolonies obtained after several subcultures in a medium supplemented with 0.5 mg/l each of benzylaminopurine, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid, were transferred to a medium lacking plant growth regulators. Well differentiated embryos were formed in selected protocolonies that contained many embryos-like structures. Approximately 70% of the somatic embryos developed into green rooted plantlets which were succesfully transferred to vessels containing sterilized scoria. Plants grown for two months in scoria were finally transferred to greenhouse.

[Isolation, purification and primary culture of rat pancreatic beta-cells].

PubMed

Liu, Yu-Pu; Lü, Qing-Guo; Tong, Nan-Wei

2009-01-01

To isolate and purify rat pancreatic beta-cells and to explore the best conditions for the primary culture of the pancreatic beta-cells in vitro. The pancreas of Norman Wistar rats were digested by collagenase V. The islets were purified by mesh sieve. The activity of the islets was stimulated by different concentrations of glucose and detected by dithizone dye. The purified islets were put into RPMI-1640 nutritive medium for culture overnight. The cultured islets were digested again with trypsin and DNAase to obtain the suspension containing single pancreatic cells. The beta-cells were separated and purified in a fluorescence-activated cell sorter (FACS) in the medium containing 2.8 mmol/L glucose. The purified beta-cells were identified by immunohistochemistry and glucose stimulating test. Ham's F-10 with different concentrations of glucose and 3-Isobutyl-1-methylxanthine (IBMX) were used as nutritive medium for the primary cell culture for 24 hours. The best conditions for the culture were identified. An average of 550 +/- 90 islets with fine activities were obtained per rat. The purification with FACS obtained about 5688 beta-cells per rat, with a recovery rate of (93.69 +/- 1.26)% and a purity of (85.5 +/- 1.24)%. A concentration of 10.0 mmol/L and 16.0 mmol/L glucose in primary culture for 24 hours produced the highest survival rates of beta-cells, but IBMX did not increase the survival rates of beta-cells. FACS is effective in purifying pancreatic beta-cells from the suspension with a medium containing 2.8 mmol/L glucose. Pancreatic beta-cells maintain relatively high activities in Ham's F-10 medium containing 10.0-16.0 mmol/L glucose in primary culture.

CELLS INVOLVED IN THE IMMUNE RESPONSE

PubMed Central

Singhal, Sharwan K.; Richter, Maxwell

1968-01-01

Cell suspensions of immune rabbit lymph nodes and spleen were capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine when maintained in culture with the specific antigen in vitro. They did not respond to other, non-cross-reacting antigens. The blastogenic response obtained with immune lymph node cells could be correlated with the antibody synthesizing capacity of fragment cultures prepared from the same lymph nodes. Cell suspensions of immune bone marrow responded to non-cross-reacting antigens only whereas cell suspensions of immune thymus, sacculus rotundus, and appendix did not respond when exposed to any of the antigens tested. On the other hand, neither fragments nor cell suspensions prepared from lymph nodes, spleen, and thymus of normal, unimmunized rabbits responded with antibody formation and blastogenesis when exposed to any of the antigens. However, normal bone marrow cells responded with marked blastogenesis and tritiated thymidine uptake. The specificity of this in vitro bone marrow response was demonstrated by the fact that the injection of a protein antigen in vivo resulted in the loss of reactivity by the marrow cell to that particular antigen but not to the other, non-cross-reacting antigens. Furthermore, bone marrow cells of tolerant rabbits failed to respond to the specific antigen in vitro. It was also demonstrated that normal bone marrow cells incubated with antigen are capable of forming antibody which could be detected by the fluorescent antibody technique. This response of the bone marrow cells has been localized to the lymphocyte-rich fraction of the bone marrow. It is concluded that the bone marrow lymphocyte, by virtue of its capacity to react with blastogenesis and mitosis and with antibody formation upon initial exposure to the antigen, a capacity not possessed by lymphocytes of the other lymphoid organs, has a preeminent role in the sequence of cellular events culminating in antibody formation. PMID:4176224

Three sesquiterpene compounds biosynthesised from artemisinic acid using suspension-cultured cells of Averrhoa carambola (Oxalidaceae).

PubMed

Yang, Li; Zhu, Jianhua; Song, Liyan; Shi, Xiaojian; Li, Xingyi; Yu, Rongmin

2012-01-01

A new sesquiterpene glycoside, artemisinic acid 3-β-O-β-D-glucopyranoside (3, 31.24%) and other two biotransformation products, 3-β-hydroxyartemisinic acid (2, 36.69%) and 3-β-hydroxyartemisinic acid β-D-glucopyranosyl ester (4, 7.03%), were biosynthesised after artemisinic acid (1) was administered to the cultured cells of Averrhoa carambola. The three biotransformation products were obtained for the first time by using the suspension-cultured cells of A. carambola as a new biocatalyst system, and their structures were identified on the basis of the physico-chemical properties, NMR and mass spectral analyses. The results indicate that the cultured cells of A. carambola have the abilities to hydroxylate and glycosylate sesquiterpene compounds in a regio- and stereoselective manner. Furthermore, the anti-tumour activity of compounds 3 and 4 was evaluated against K562 and HeLa cell lines. Compound 4 showed strong activity against HeLa cell line, with the IC₅₀ value of 0.56 µmol mL⁻¹.

Do rice suspension-cultured cells treated with abscisic acid mimic developing seeds?

PubMed

Matsuno, Koya; Fujimura, Tatsuhito

2015-08-01

Starch synthesis is activated in the endosperm during seed development and also in rice suspension cells cultured with abscisic acid. In the anticipation that the mechanisms of starch synthesis are similar between the endosperm and the suspension cells cultured with abscisic acid, expression of genes involved in starch synthesis was evaluated in the suspension cells after abscisic acid treatment. However, it was found that the regulatory mechanism of starch synthesis in the suspension cells cultured with abscisic acid was different from that in developing seeds. Expression analyses of genes involved in oil bodies, which accumulate in the embryo and aleurone layer, and seed storage proteins, which accumulate mainly in the endosperm, showed that the former were activated in the suspension cells cultured with abscisic acid, but the latter were not. Master regulators for embryogenesis, OsVP1 (homologue of AtABI3) and OsLFL1 (homologue of AtFUS3 or AtLFL2), were expressed in the suspension cells at levels comparable to those in the embryo. From these results, it is suggested that interactions between regulators and abscisic acid control the synthesis of phytic acid and oil bodies in the cultured cells and embryo. We suggest that the system of suspension cells cultured with abscisic acid helps to reveal the mechanisms of phytic acid and oil body synthesis in embryo.

Antitumour, antimicrobial and catalytic activity of gold nanoparticles synthesized by different pH propolis extracts

NASA Astrophysics Data System (ADS)

Gatea, Florentina; Teodor, Eugenia Dumitra; Seciu, Ana-Maria; Covaci, Ovidiu Ilie; Mănoiu, Sorin; Lazăr, Veronica; Radu, Gabriel Lucian

2015-07-01

The Romanian propolis was extracted in five different media, respectively, in water (pH 6.8), glycine buffer (pH 2.5), acetate buffer (pH 5), phosphate buffer (pH 7.4) and carbonate buffer (pH 9.2). The extracts presented different amounts of flavonoids and phenolic acids, increasing pH leading to higher concentrations of active compounds. Five variants of gold nanoparticles suspensions based on different pH Romanian propolis aqueous extracts were successfully synthesized. The obtained nanoparticles presented dimensions between 20 and 60 nm in dispersion form and around 18 nm in dried form, and different morphologies (spherical, hexagonal, triangular). Fourier transform infrared spectroscopy proved the attachment of organic compounds from propolis extracts to the colloidal gold suspensions and X-ray diffraction certified that the suspensions contain metallic gold. The obtained propolis gold nanoparticles do not exhibit any antibacterial or antifungal activity, but presented different catalytic activities and toxicity on tumour cells.

The happy destiny of frozen haematopoietic stem cells: from immature stem cells to mature applications.

PubMed

de Vries, E G E; Vellenga, E; Kluin-Nelemans, J C; Mulder, N H

2004-09-01

Forty years ago, van Putten described in the European Journal of Cancer (see this issue) quantitative studies on the optimal storage techniques of mouse and monkey bone marrow suspensions. Survival of the animals after irradiation following injection with stored bone marrow cell suspensions was the endpoint. He observed some species differences, but based on the data obtained considered a careful trial of the glycerol-polyvinylpyrrolide (PVP) combination for storage of marrow in man was indicated. In spite of this, dimethyl sulphoxide has become the 'standard' cryopreservant for human marrow stem cells. Over the last 40 years, there has been a tremendous increase in knowledge about haematopoietic stem cells and their use in the clinic. Haematopoietic stem cells are now known to travel between the bone marrow and peripheral blood and are the best-characterised adult stem cells. These cells are currently widely used for transplantations in the clinic and are obtained from a wide variety of sources. These include the bone marrow, peripheral blood, cord blood, autologous as well as allogeneic stem cells from related or unrelated donors. Increasingly, data has become available that adult haematopoietic stem cells can generate differentiated cells belonging to other cell types, a process called "developmental plasticity". Thus, they may contribute to non-haematopoietic tissue repair in multiple organ systems. This has created a whole new potential therapeutic armamentarium for the application of haematopoietic stem cells outside of the area of malignancies and haematopoietic disorders.

THE PRIMARY CELL WALL. Progress Report, February 1, 1963 to October 31, 1963

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

Varner, J.E.

1964-10-31

Progress is reported in studies of hydroxyproline synthesis in sycamore cells grown in suspensions with and without the addition of D/sub 2/O. The relation of hydroxyproline to cellulose in cell walls was investigated in suspensions of cells from sycamore and ginkgo cell suspensions. (C.H.)

Epoxidation of Short-Chain Alkenes by Resting-Cell Suspensions of Propane-Grown Bacteria

PubMed Central

Hou, Ching T.; Patel, Ramesh; Laskin, Allen I.; Barnabe, Nancy; Barist, Irene

1983-01-01

Sixteen new cultures of propane-utilizing bacteria were isolated from lake water from Warinanco Park, Linden, N.J. and from lake and soil samples from Bayway Refinery, Linden, N.J. In addition, 19 known cultures obtained from culture collections were also found to be able to grow on propane as the sole carbon and energy source. In addition to their ability to oxidize n-alkanes, resting-cell suspensions of both new cultures and known cultures grown on propane oxidize short-chain alkenes to their corresponding 1,2-epoxides. Among the substrate alkenes, propylene was oxidized at the highest rate. In contrast to the case with methylotrophic bacteria, the product epoxides are further metabolized. Propane and other gaseous n-alkanes inhibit the epoxidation of propylene. The optimum conditions for in vivo epoxidation are described. Results from inhibition studies indicate that a propane monooxygenase system catalyzes both the epoxidation and hydroxylation reactions. Experiments with cell-free extracts show that both hydroxylation and epoxidation activities are located in the soluble fraction obtained after 80,000 × g centrifugation. PMID:16346338

Adherent culture conditions enrich the side population obtained from the cochlear modiolus-derived stem/progenitor cells.

PubMed

Chao, Ting-Ting; Wang, Chih-Hung; Chen, Hsin-Chien; Shih, Cheng-Ping; Sytwu, Huey-Kang; Huang, Kun-Lun; Chen, Shao-Yuan

2013-05-01

Previously, our group reported that sphere-forming cells derived from the organ of Corti represent the stem/progenitor cells (SPCs) of the cochlea due to their properties of self-renewal and multipotency. However, long-term propagation of sphere-forming cells under suspension culture conditions may fail to maintain the characteristic stemness of these cells. Therefore, this study investigated whether an adherent culture system would be beneficial in terms of preserving more stem-like cells for long-term manipulations in vitro. Isolated modiolus-derived SPCs were placed on poly-d-lysine-coated petri dishes to form the so-called "adherent" culture system. Modiolus SPCs cultured under adherent conditions exhibited a significantly increased percentage of cells with the side population (SP) phenotype (18.6%) compared with cells cultured under conventional suspension culture conditions (0.8%). Even after repeated passages, modiolus SPCs cultured under adherent culture conditions preserved more SP phenotype cells. In comparison with the non-SP phenotype cells, the sorted SP cells exhibited more stem-like but less differentiated properties, with an upregulated expression of the ATP-binding cassette subfamily G member 2 (ABCG2), Nestin, Sox2, and Nanog proteins. Furthermore, Retinoic acid (RA) treatment confirmed the expression of the multipotent differentiation markers in the SP cells, including TUJ1, pancytokeratin, glial fibrillary acidic protein (GFAP), and p27(Kip1). Employment of an adherent culture system, instead of a suspension culture system, resulted in the enrichment of the SP cells from SPCs while retaining their stemness and multipotency. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Semi-synthetic preparation of 1-O-(1'-/sup 14/C)hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

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

Weber, N.; Mangold, H.K.

1985-04-01

Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-(1'-/sup 14/C)hexadecyl-sn-glycerol or rac-1-O-(1'-/sup 14/C)hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-(1'-/sup 14/C)hexadecyl-sn-glycero-3-phosphocholine. 1-O-(1'-14C)Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity.

Macrolide Antibiotics Improve Phagocytic Capacity and Reduce Inflammation In Sulfur Mustard-Exposed Monocytes

DTIC Science & Technology

2008-12-01

phagocytotic function and on inflammatory cytokines/mediators production in vitro using SM-exposed monocyte THP - 1 cells. Using flow cytometry we found...in vitro using SM-exposed monocyte THP - 1 cells. 2. MATERIALS AND METHODS 2.1 Reagents Sulfur mustard (2,2’-dichlorodiethyl sulfide; 4 mM) was...monocyte THP - 1 cells were obtained from ATCC (Manassas, VA). Cells were grown as suspension in the optimized media as formulated by the manufacturer and

Dielectric properties of isolated adrenal chromaffin cells determined by microfluidic impedance spectroscopy.

PubMed

Sabuncu, A C; Stacey, M; Craviso, G L; Semenova, N; Vernier, P T; Leblanc, N; Chatterjee, I; Zaklit, J

2018-02-01

Knowledge of the dielectric properties of biological cells plays an important role in numerical models aimed at understanding how high intensity ultrashort nanosecond electric pulses affect the plasma membrane and the membranes of intracellular organelles. To this end, using electrical impedance spectroscopy, the dielectric properties of isolated, neuroendocrine adrenal chromaffin cells were obtained. Measured impedance data of the cell suspension, acquired between 1kHz and 20MHz, were fit into a combination of constant phase element and Cole-Cole models from which the effect of electrode polarization was extracted. The dielectric spectrum of each cell suspension was fit into a Maxwell-Wagner mixture model and the Clausius-Mossotti factor was obtained. Lastly, to extract the cellular dielectric parameters, the cell dielectric data were fit into a granular cell model representative of a chromaffin cell, which was based on the inclusion of secretory granules in the cytoplasm. Chromaffin cell parameters determined from this study were the cell and secretory granule membrane specific capacitance (1.22 and 7.10μF/cm 2 , respectively), the cytoplasmic conductivity, which excludes and includes the effect of intracellular membranous structures (1.14 and 0.49S/m, respectively), and the secretory granule milieu conductivity (0.35S/m). These measurements will be crucial for incorporating into numerical models aimed at understanding the differential poration effect of nanosecond electric pulses on chromaffin cell membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

PubMed Central

Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

2016-01-01

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

Elicitation of Jerusalem artichoke (Helianthus tuberosus L.) cell suspension culture for enhancement of inulin production and altered degree of polymerisation.

PubMed

Ma, Chunquan; Zhou, Dong; Wang, Haitao; Han, Dongming; Wang, Yang; Yan, Xiufeng

2017-01-01

Plant cell suspension cultures have emerged as a potential source of secondary metabolites for food additives and pharmaceuticals. In this study inulin accumulation and its degree of polymerisation (DP) in the treated cells in the same medium were investigated after treatment with six types of elicitors. An in vitro cell suspension culture of Jerusalem artichoke (Helianthus tuberosus L.) was optimised by adding an extra nitrogen source. According to the growth kinetics, a maximum biomass of 5.48 g L -1 was obtained from the optimal cell suspension medium consisted of Murashige and Skoog basic medium (MS) + 1.0 mg L -1 α-naphthalene acetic acid (NAA) + 1.0 mg L -1 6-benzylaminopurine (6-BA) + 0.5 mg L -1 proline + 1.0 mg L -1 glutamine. Methyl jasmonate (MeJA, 250 µmol L -1 ) treatment for 15 days led to the highest levels of inulin (2955.27 ± 9.81 mg L -1 compared to control of 1217.46 ± 0.26 mg L -1 ). The elicited effect of five elicitors to the suspension cells of Jerusalem artichoke is as follows: AgNO 3 (Ag, 10 µmol L -1 ), salicylic acid (SA, 75 µmol L -1 ), chitosan (KJT, 40 mg L -1 ), Trichoderma viride (Tv, 90 mg L -1 ), yeast extract (YE, 0.25 mg L -1 ), and the corresponding content of inulin is increased by 2.05-, 1.93-, 1.76-, 1.44- and 1.18-fold compared to control, respectively. The obvious effect on the percentage of lower DP in inulin was observed in cells treated with 40 mg L -1 KJT, 0.25 mg L -1 YE and 10 µmol L -1 Ag. Among the six types of elicitors, the descending order of inulin content is MeJA > Ag > SA > KJT > Tv > YE. For the purpose inulin with lower DP and its application to prebiotic food, three elicitors, including KJT, YE and Ag, can be used for the elicitation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis

USDA-ARS?s Scientific Manuscript database

Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transfe...

Inflorescence proliferation for somatic embryogenesis induction and suspension-derived plant regeneration from banana (Musa AAA, cv. 'Dwarf Cavendish') male flowers.

PubMed

Pérez-Hernández, Juan Bernardo; Rosell-García, Purificación

2008-06-01

Availability of explants with adequate embryogenic competence is one of the most important limitations for the development of regenerable cell suspensions in banana. To increase the number and ease of accessibility to potentially embryogenic explants, a novel methodology is described by which young male flower clusters isolated from adult plants are induced to form new flower buds and proliferate in vitro. Different concentrations of the plant growth regulator thidiazuron (TDZ) induced inflorescence proliferation, which could be maintained over time as a continuous source of young flower buds. Intensity of proliferation was evaluated during successive subcultures. At the third cycle of proliferation, the highest multiplication rate (2.89) was obtained on the medium containing 5 microM TDZ. Newly generated floral tissues were assessed for embryogenic competence, resulting in an average embryogenic frequency of 12.5%. The observed embryogenic capacity, together with the recurrent availability of immature flowers, allowed for the direct initiation of cell suspensions from bulked explant cultures. Regular observation and regeneration tests during the development of suspended cell cultures confirmed their embryogenic condition. Produced embryos successfully matured and germinated to regenerate hundreds of somatic in vitro plants.

Application of flow cytometry with a fluorescent dye to measurement of intracellular nitric oxide in plant cells.

PubMed

Kępczyński, Jan; Cembrowska-Lech, Danuta

2018-04-27

A simple and rapid method involving flow cytometry and NO-specific probe (DAF-FM DA) proved useful for detection and determination of intracellular NO production in Medicago truncatula suspension cells and leaves as well as in cells of Avena fatua, Amaranthus retroflexus embryos and leaves. The measurement of nitric oxide (NO) in plant material is important for examining the regulatory roles of endogenous NO in various physiological processes. The possibility of detecting and determining intracellular NO production by flow cytometry (FCM) with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA), an NO-specific probe in Medicago truncatula cells in suspension and leaves as well as in cells of embryos and leaves of Avena fatua L. or Amaranthus retroflexus L. was explored. To detect and measure NO production by cell suspension or embryos and leaves, the recommended DAF-FM DA concentration is 5 or 10 µM, respectively, applied for 30 min. Exogenous NO increased the intensity of the fluorescent signal in embryos and leaves of both plants, while carboxy-PTIO (cPTIO), an NO scavenger, decreased it. Thus, these results demonstrate that NO can be detected and an increase and a decrease of its intracellular level can be estimated. Wounding was observed to increase the fluorescence signal, indicating an increase in the intracellular NO level. In addition, the levels of exogenous and endogenous ascorbic acid were demonstrated to have no effect on the NO-related fluorescence signal, indicating the signal's specificity only in relation with NO. The applicability of the proposed method for detection and determination of NO was confirmed (1) by in situ NO imaging in cell suspensions and (2) by determining the NO concentration in embryos and leaves using the Griess reagent. In view of the data obtained, FCM is recommended as a rapid and simple method with which to detect and determine intracellular NO production in plant cells.

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII.

PubMed

Swiech, Kamilla; Kamen, Amine; Ansorge, Sven; Durocher, Yves; Picanço-Castro, Virgínia; Russo-Carbolante, Elisa M S; Neto, Mário S A; Covas, Dimas T

2011-11-24

Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37 °C. We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34 °C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

Laboratory production of human prolactin from CHO cells adapted to serum-free suspension culture.

PubMed

Arthuso, Fernanda Santos; Bartolini, Paolo; Soares, Carlos Roberto Jorge

2012-08-01

Human prolactin (hPRL) is a polypeptide with 199 amino acids and a molecular mass of 23 kDa. Previously, a eukaryotic hPRL expression vector was used to transfect Chinese hamster ovary (CHO) cells: this work describes a fast and practical laboratory adaptation of these transfected cells, in ~40 days, to grow in suspension in serum-free medium. High cell densities of up to 4.0 × 10(6) cell/ml were obtained from spinner flask cultures and a stable and continuous production process was developed for at least 30 days. Two harvesting strategies were set up, 50 or 100 % of the total conditioned medium being collected daily and replaced by fresh culture medium. The volumetric productivity was 5-7 μg hPRL/ml, as determined directly in the collected medium via reversed-phase HPLC (RP-HPLC). A two-step process based on a cationic exchanger followed by size exclusion chromatography was applied to obtain purified hPRL from conditioned medium. Two hPRL isoforms, non-glycosylated and glycosylated, could also be separated by high-performance size-exclusion chromatography (HPSEC) and, when analyzed by RP-HPLC, HPSEC, Western blotting, and bioassay, were found to be comparable to the World Health Organization International Reference Reagent of hPRL. These results are useful for the practical scale-up to the pilot and industrial scale of a bioprocess based on CHO cell culture.

Anti-Cancer Activity of Resveratrol and Derivatives Produced by Grapevine Cell Suspensions in a 14 L Stirred Bioreactor.

PubMed

Nivelle, Laetitia; Hubert, Jane; Courot, Eric; Jeandet, Philippe; Aziz, Aziz; Nuzillard, Jean-Marc; Renault, Jean-Hugues; Clément, Christophe; Martiny, Laurent; Delmas, Dominique; Tarpin, Michel

2017-03-16

In the present study, resveratrol and various oligomeric derivatives were obtained from a 14 L bioreactor culture of elicited grapevine cell suspensions (Vitis labrusca L.). The crude ethyl acetate stilbene extract obtained from the culture medium was fractionated by centrifugal partition chromatography (CPC) using a gradient elution method and the major stilbenes contained in the fractions were subsequently identified by using a 13 C-NMR-based dereplication procedure and further 2D NMR analyses including HSQC, HMBC, and COSY. Beside δ-viniferin (2), leachianol F (4) and G (4'), four stilbenes (resveratrol (1), ε-viniferin (5), pallidol (3) and a newly characterized dimer (6)) were recovered as pure compounds in sufficient amounts to allow assessment of their biological activity on the cell growth of three different cell lines, including two human skin malignant melanoma cancer cell lines (HT-144 and SKMEL-28) and a healthy human dermal fibroblast HDF line. Among the dimers obtained in this study, the newly characterized resveratrol dimer (6) has never been described in nature and its biological potential was evaluated here for the first time. ε-viniferin as well as dimer (6) showed IC 50 values on the three tested cell lines lower than the ones exerted by resveratrol and pallidol. However, activities of the first two compounds were significantly decreased in the presence of fetal bovine serum although that of resveratrol and pallidol was not. The differential tumor activity exerted by resveratrol on healthy and cancer lines was also discussed.

Lowering of the cavitation threshold in aqueous suspensions of porous silicon nanoparticles for sonodynamic therapy applications

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

Sviridov, A. P., E-mail: asagittarius89@gmail.com; Osminkina, L. A.; Nikolaev, A. L.

2015-09-21

A significant decrease of the cavitation threshold in aqueous suspensions of porous silicon nanoparticles (PSi NPs) with sizes about 100 nm as compared with pure water was observed for ultrasound irradiation (USI) with therapeutic frequency (0.88 MHz) and intensities (about 1 W/cm{sup 2}). This effect is explained by porous morphology of PSi NPs, which promotes the nucleation of cavitation bubbles. In vitro experiments revealed a suppression of the proliferation of cancer cells with the introduced PSi NPs after exposure to USI related to the enhanced cavitation processes, which led to the cell destruction. The obtained results demonstrate that PSi NPs are prospectivemore » for applications as sonosensitizers in mild cancer therapy.« less

Purification and cultivation of human pituitary growth hormone secreting cells

NASA Technical Reports Server (NTRS)

Hymer, W. C.

1978-01-01

The maintainance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro was studied. The primary approach was the testing of agents which may be expected to increase the release of the human growth hormone (hGH). A procedure for tissue procurement is described along with the methodologies used to dissociate human pituitary tissue (obtained either at autopsy or surgery) into single cell suspensions. The validity of the Biogel cell column perfusion system for studying the dynamics of GH release was developed and documented using a rat pituitary cell system.

Opposite extremes in ethylene/nitric oxide ratio induce cell death in suspension culture and root apices of tomato exposed to salt stress.

PubMed

Poór, P; Borbély, P; Kovács, Judit; Papp, Anita; Szepesi, Ágnes; Takács, Z; Tari, Irma

2014-12-01

The plant hormone ethylene or the gaseous signalling molecule nitric oxide (NO) may enhance salt stress tolerance by maintaining ion homeostasis, first of all K+/Na+ ratio of tissues. Ethylene and NO accumulation increased in the root apices and suspension culture cells of tomato at sublethal salt stress caused by 100 mM NaCl, however, the induction phase of programmed cell death (PCD) was different at lethal salt concentration. The production of ethylene by root apices and the accumulation of NO in the cells of suspension culture did not increase during the initiation of PCD after 250 mM NaCl treatment. Moreover, cells in suspension culture accumulated higher amount of reactive oxygen species which, along with NO deficiency contributed to cell death induction. The absence of ethylene in the apical root segments and the absence of NO accumulation in the cell suspension resulted in similar ion disequilibrium, namely K+/Na+ ratio of 1.41 ± 0.1 and 1.68 ± 0.3 in intact plant tissues and suspension culture cells, respectively that was not tolerated by tomato.

A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids

PubMed Central

Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A.; Falvo D’Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

2016-01-01

A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future. PMID:27144306

A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

PubMed

Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A; Falvo D'Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

2016-01-01

A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

PubMed

Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

2014-01-01

Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

Reflection coefficients of permeant molecules in human red cell suspensions.

PubMed

Owen, J D; Eyring, E M

1975-08-01

The Staverman reflection coefficient, sigma for several permeant molecules was determined in human red cell suspensions with a Durrum stopped-flow spectrophotometer. This procedure was first used with dog, cat, and beef red cells and with human red cells. The stopped-flow technique used was similar to the rapid-flow method used by those who originally reported sigma measurements in human red cells for molecules which rapidly penetrate the red cell membrane. The sigma values we obtained agreed with those previously reported for most of the slow penetrants, except malonamide, but disagreed with all the sigma values previously reported for the rapid penetrants. We were unable to calculate an "equivalent pore radius" with our sigma data. The advantages of our equipment and our experimental procedure are discussed. Our sigma data suggest that sigma is indirectly proportional to the log of the nonelectrolyte permeability coefficient, omega. Since a similar trend has been previously shown for log omega and molar volume of the permeant molecules, a correlatioo was shown between sigma and molar volume suggesting the membrane acts as a sieve.

Rheological behaviour of a suspension of microswimmers varying in motor characteristics

NASA Astrophysics Data System (ADS)

Tirumkudulu, Mahesh; Karmakar, Richa; Gulvady, Ranjit; Venkatesh, K. V.

2013-11-01

A suspension of motile cells exhibits complex rheological properties due to their collective motion. We measure the shear viscosity of suspensions of Escherichia coli strains varying in motor characteristics such as duration of run and tumble. At low cell densities, all strains irrespective of their motor characteristics exhibiting a linear increase in viscosity with cell density suggesting that the cells behave as a suspension of rods with an effective aspect ratio set by the motor characteristics of the bacteria. As the cell density is increased beyond a critical value, the viscosity drops sharply signaling the presence of strongly coordinated motion among bacteria. The critical density depends not only on the magnitude of shear but also the motor characteristics of individual cells. High shear rate disrupts the coordinated motion reducing its behavior, once again, to a suspension of inactive particles. The authors acknowldege financial support from Department of Science and Technology, India.

Motor characteristics determine the rheological behavior of a suspension of microswimmers

NASA Astrophysics Data System (ADS)

Karmakar, Richa; Gulvady, Ranjit; Tirumkudulu, Mahesh S.; Venkatesh, K. V.

2014-07-01

A suspension of motile cells exhibits complex rheological properties due to their collective motion. We measure the shear viscosity of a suspension of Escherichia coli strains varying in motor characteristics such as duration of run and tumble. At low cell densities, all strains irrespective of their motor characteristics exhibit a linear increase in viscosity with cell density suggesting that the cells behave as a suspension of passive rods with an effective aspect ratio set by the motor characteristics of the bacteria. As the cell density is increased beyond a critical value, the viscosity drops sharply signaling the presence of strongly coordinated motion among bacteria. The critical density depends not only on the magnitude of shear but also the motor characteristics of individual cells. High shear rate disrupts the coordinated motion reducing its behavior, once again, to a suspension of inactive particles.

Effect of Magnetic Nanoparticles on Tobacco BY-2 Cell Suspension Culture

PubMed Central

Krystofova, Olga; Sochor, Jiri; Zitka, Ondrej; Babula, Petr; Kudrle, Vit; Adam, Vojtech; Kizek, Rene

2012-01-01

Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles), electronics (high-resolution imaging, logical circuits on the molecular level) and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases) or imaging (contrast agents). Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs) and modified magnetic nanoparticles (MNPs) on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis—total protein content, thiols—reduced (GSH) and oxidized (GSSG) glutathione, phytochelatins PC2-5, glutathione S-transferase (GST) activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension culture. The obtained results are discussed, especially in connection with already published data. Possible mechanisms of NPs’ and MNPs’ toxicity are introduced. PMID:23343980

Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation

PubMed Central

Yango, Pamela; Altman, Eran; Smith, James F.; Klatsky, Peter C.; Tran, Nam D.

2015-01-01

Objective To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. Design In vitro human testicular tissues. Setting Academic research unit. Patients Adult testicular tissues (n = 4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n = 3). Intervention(s) Testicular tissue vs. single cell suspension cryopreservation. Main Outcome Measures Cell viability, total cell recovery per milligram of tissue, as well as, viable and SSEA-4+ cell recovery. Results Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Conclusions Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient age, type of samples cryopreserved, and end points of therapeutic applications. PMID:25241367

A Solvent-Free Surface Suspension Melt Technique for Making Biodegradable PCL Membrane Scaffolds for Tissue Engineering Applications.

PubMed

Suntornnond, Ratima; An, Jia; Tijore, Ajay; Leong, Kah Fai; Chua, Chee Kai; Tan, Lay Poh

2016-03-21

In tissue engineering, there is limited availability of a simple, fast and solvent-free process for fabricating micro-porous thin membrane scaffolds. This paper presents the first report of a novel surface suspension melt technique to fabricate a micro-porous thin membrane scaffolds without using any organic solvent. Briefly, a layer of polycaprolactone (PCL) particles is directly spread on top of water in the form of a suspension. After that, with the use of heat, the powder layer is transformed into a melted layer, and following cooling, a thin membrane is obtained. Two different sizes of PCL powder particles (100 µm and 500 µm) are used. Results show that membranes made from 100 µm powders have lower thickness, smaller pore size, smoother surface, higher value of stiffness but lower ultimate tensile load compared to membranes made from 500 µm powder. C2C12 cell culture results indicate that the membrane supports cell growth and differentiation. Thus, this novel membrane generation method holds great promise for tissue engineering.

Abrogation of E-cadherin-mediated cellular aggregation allows proliferation of pluripotent mouse embryonic stem cells in shake flask bioreactors.

PubMed

Mohamet, Lisa; Lea, Michelle L; Ward, Christopher M

2010-09-23

A fundamental requirement for the exploitation of embryonic stem (ES) cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times. Here we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/-) or the neutralising antibody DECMA-1 (EcadAb), allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers. This is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are necessary to increase cell viability of hES cells in suspension.

Induction of betacyanin formation in Chenopodium album cell cultures by co-cultivation with the duckweed Wolffia arrhiza.

PubMed

Rudat, A; Ehwald, R

1994-02-01

Cells of Chenopodium album and whole plants of the duckweed Wolffia arrhiza were cocultivated. In the presence of Wolffia arrhiza the synthesis of a red-violet pigment (betacyanin) was induced in several cells or cell clusters of Chenopodium album in the light. The exchange of solutes through the liquid phase was necessary for the induction of pigment formation. The red-violet cells could be selected and subcultivated resulting in a red callus. A reddish cell suspension was obtained in liquid culture in the presence of the duckweeds.

Biomek Cell Workstation: A Variable System for Automated Cell Cultivation.

PubMed

Lehmann, R; Severitt, J C; Roddelkopf, T; Junginger, S; Thurow, K

2016-06-01

Automated cell cultivation is an important tool for simplifying routine laboratory work. Automated methods are independent of skill levels and daily constitution of laboratory staff in combination with a constant quality and performance of the methods. The Biomek Cell Workstation was configured as a flexible and compatible system. The modified Biomek Cell Workstation enables the cultivation of adherent and suspension cells. Until now, no commercially available systems enabled the automated handling of both types of cells in one system. In particular, the automated cultivation of suspension cells in this form has not been published. The cell counts and viabilities were nonsignificantly decreased for cells cultivated in AutoFlasks in automated handling. The proliferation of manual and automated bioscreening by the WST-1 assay showed a nonsignificant lower proliferation of automatically disseminated cells associated with a mostly lower standard error. The disseminated suspension cell lines showed different pronounced proliferations in descending order, starting with Jurkat cells followed by SEM, Molt4, and RS4 cells having the lowest proliferation. In this respect, we successfully disseminated and screened suspension cells in an automated way. The automated cultivation and dissemination of a variety of suspension cells can replace the manual method. © 2015 Society for Laboratory Automation and Screening.

Three-dimensional behavior of ice crystals and biological cells during freezing of cell suspensions.

PubMed

Ishiguro, H; Koike, K

1998-09-11

Behavior of ice crystals and human red blood cells during extracellular-freezing was investigated in three-dimensions using a confocal laser scanning microscope(CLSM), which noninvasively produces tomograms of biological materials. Physiological saline and physiological saline with 2.4 M glycerol were used for suspension. Various cooling rates for directional solidification were used for distinctive morphology of the ice crystals. Addition of acridine orange as a fluorescent dye into the cell suspension enabled ice crystal, cells and unfrozen solution to be distinguished by different colors. The results indicate that the microscopic structure is three-dimensional for flat, cellular, and dendritic solid-liquid interfaces and that a CLSM is very effective in studying three-dimensional structure during the freezing of cell suspensions.

Equivalent Air Spring Suspension Model for Quarter-Passive Model of Passenger Vehicles.

PubMed

Abid, Haider J; Chen, Jie; Nassar, Ameen A

2015-01-01

This paper investigates the GENSIS air spring suspension system equivalence to a passive suspension system. The SIMULINK simulation together with the OptiY optimization is used to obtain the air spring suspension model equivalent to passive suspension system, where the car body response difference from both systems with the same road profile inputs is used as the objective function for optimization (OptiY program). The parameters of air spring system such as initial pressure, volume of bag, length of surge pipe, diameter of surge pipe, and volume of reservoir are obtained from optimization. The simulation results show that the air spring suspension equivalent system can produce responses very close to the passive suspension system.

43 CFR 3212.11 - How do I obtain a suspension of operations or a suspension of operations and production on my lease?

Code of Federal Regulations, 2011 CFR

2011-10-01

... INTERIOR MINERALS MANAGEMENT (3000) GEOTHERMAL RESOURCE LEASING Lease Suspensions, Cessation of Production, Royalty Rate Reductions, and Energy Policy Act Royalty Rate Conversions § 3212.11 How do I obtain a...

Comparison of the oxygen exchange between photosynthetic cell suspensions and detached leaves of Euphorbia characias L

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

Carrier, P.; Chagvardieff, P.; Tapie, P.

1989-11-01

Using a mass-spectrometric {sup 16}O{sub 2}/{sup 18}O{sub 2}-isotope technique, we compared the nature and the relative importance of oxygen exchange in photomixotrophic (PM) and photoautotrophic (PA) suspensions of Euphorbia characias L. with those in intact leaves of the same species. Young and mature leaves, dividing and nondividing cell suspensions were characterized in short-term experiments. On chlorophyll basis, the gross photosynthetic activities at CO{sub 2} saturating concentration of PA and PM suspensions varied little from those of leaves. On dry weight basis, gross photosynthesis of PA suspensions was equal to that of leaves because of their similar chlorophyll content. This wasmore » not the case in PM suspensions where gross photosynthesis was lower and largely varied during the growth cycle. The CO{sub 2} compensation point of PA cells was much higher than that of leaves. Oxygen uptakes were analyzed in terms of mitochondrial respiration, photorespiration and light stimulation of oxygen uptake (LSOU), often identified to Mehler-type reactions. In Pa and PM suspensions, mitochondrial respiration rates were higher than in leaves by a factor of 1.5 to 4.5. In PM suspensions, photorespiration and LSOU were observed only in nondividing cells. Photorespiration and LSOU rates were comparable in PA suspensions and leaves. Our results demonstrate that photorespiration of PA suspensions has not been affected by the 2% CO{sub 2} concentration imposed during 2 years of culture.« less

Serotonin Signal Transduction in Two Groups of Autistic Patients

DTIC Science & Technology

2013-12-01

modification as learned from the initial studies. Treat cells with escitalopram or r-citalopram and test for increased coupling between G proteins and...improved medications. 6 Figure 1 (above). Escitalopram translocates Gs in cells obtained from High 5HT subjects. Lymphoblasts from High...5HT/High IS or Normal 5HT/moderate IS (two lines each) were grown in suspension and treated with escitalopram or control (r-citalopram, @10 µM) for

A Low-Cost Thermistor Device for Measurements of Metabolic Heat in Yeast Cells in Suspension.

ERIC Educational Resources Information Center

Keeling, Richard P.

1980-01-01

Provides illustrated directions for the construction and use of a low-cost thermistor device. Attached to a servo-type millivolt chart recorder, the device will record minute temperature changes and will simulate data obtained from an oxygen polarograph. Includes results of experiments with baker's yeast. (Author/CS)

Electrical resistivity of the liquid phase of vesicular suspensions prepared by different methods

NASA Astrophysics Data System (ADS)

Vitkova, V.; Antonova, K.; Popkirov, G.; Mitov, M. D.; Ermakov, Yu A.; Bivas, I.

2010-11-01

Giant lipid vesicles are obtained mainly by two methods of formation: (i) electroformation and (ii) gentle hydration (spontaneous swelling). Very often the electoformation is carried out in experimental cells consisting of indium-tin oxide (ITO) coated plates as electrodes and various polymer spacers. In the present work, the influence of the ITO coatings and the polymer spacers on the electrical resistivity of the liquid medium of electroformed vesicle suspensions is examined by means of electrochemical impedance spectroscopy (EIS). Our study is intended to point out possible implications of the electroformation method, especially in cases when phenomena, related to electric properties of the vesicle membranes, are investigated.

Bioactive Glass-Ceramic Scaffolds from Novel ‘Inorganic Gel Casting’ and Sinter-Crystallization

PubMed Central

Elsayed, Hamada; Rincón Romero, Acacio; Ferroni, Letizia; Gardin, Chiara; Zavan, Barbara; Bernardo, Enrico

2017-01-01

Highly porous wollastonite-diopside glass-ceramics have been successfully obtained by a new gel-casting technique. The gelation of an aqueous slurry of glass powders was not achieved according to the polymerization of an organic monomer, but as the result of alkali activation. The alkali activation of a Ca-Mg silicate glass (with a composition close to 50 mol % wollastonite—50 mol % diopside, with minor amounts of Na2O and P2O5) allowed for the obtainment of well-dispersed concentrated suspensions, undergoing progressive hardening by curing at low temperature (40 °C), owing to the formation of a C–S–H (calcium silicate hydrate) gel. An extensive direct foaming was achieved by vigorous mechanical stirring of partially gelified suspensions, comprising also a surfactant. The open-celled structure resulting from mechanical foaming could be ‘frozen’ by the subsequent sintering treatment, at 900–1000 °C, causing substantial crystallization. A total porosity exceeding 80%, comprising both well-interconnected macro-pores and micro-pores on cell walls, was accompanied by an excellent compressive strength, even above 5 MPa. PMID:28772531

Photolabeling of Tonoplast from Sugar Beet Cell Suspensions by [3H]5-(N-Methyl-N-Isobutyl)-Amiloride, an Inhibitor of the Vacuolar Na+/H+ Antiport 1

PubMed Central

Barkla, Bronwyn J.; Charuk, Jeffrey H. M.; Cragoe, Edward J.; Blumwald, Eduardo

1990-01-01

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na+/H+ antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na+/H+ exchange in a competitive manner with a Ki of 2.5 and 5.9 micromolar for ΔpH-dependent 22Na+ influx in tonoplast vesicles and Na+-dependent H+ efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [3H]MIA to tonoplast membranes revealed a high affinity binding component with a Kd of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na+/H+ antiport. Photolabeling of the tonoplast with [3H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog. Images Figure 7 PMID:16667602

Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport.

PubMed

Barkla, B J; Charuk, J H; Cragoe, E J; Blumwald, E

1990-07-01

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na(+)/H(+) antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na(+)/H(+) exchange in a competitive manner with a K(i) of 2.5 and 5.9 micromolar for DeltapH-dependent (22)Na(+) influx in tonoplast vesicles and Na(+)-dependent H(+) efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [(3)H]MIA to tonoplast membranes revealed a high affinity binding component with a K(d) of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na(+)/H(+) antiport. Photolabeling of the tonoplast with [(3)H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.

Stable expression of recombinant human coagulation factor XIII in protein-free suspension culture of Chinese hamster ovary cells.

PubMed

Chun, B H; Bang, W G; Park, Y K; Woo, S K

2001-11-01

The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture system with a spin filter is employed.

Equivalent Air Spring Suspension Model for Quarter-Passive Model of Passenger Vehicles

PubMed Central

Abid, Haider J.; Chen, Jie; Nassar, Ameen A.

2015-01-01

This paper investigates the GENSIS air spring suspension system equivalence to a passive suspension system. The SIMULINK simulation together with the OptiY optimization is used to obtain the air spring suspension model equivalent to passive suspension system, where the car body response difference from both systems with the same road profile inputs is used as the objective function for optimization (OptiY program). The parameters of air spring system such as initial pressure, volume of bag, length of surge pipe, diameter of surge pipe, and volume of reservoir are obtained from optimization. The simulation results show that the air spring suspension equivalent system can produce responses very close to the passive suspension system. PMID:27351020

Optimized suspension culture: the rotating-wall vessel

NASA Technical Reports Server (NTRS)

Hammond, T. G.; Hammond, J. M.

2001-01-01

Suspension culture remains a popular modality, which manipulates mechanical culture conditions to maintain the specialized features of cultured cells. The rotating-wall vessel is a suspension culture vessel optimized to produce laminar flow and minimize the mechanical stresses on cell aggregates in culture. This review summarizes the engineering principles, which allow optimal suspension culture conditions to be established, and the boundary conditions, which limit this process. We suggest that to minimize mechanical damage and optimize differentiation of cultured cells, suspension culture should be performed in a solid-body rotation Couette-flow, zero-headspace culture vessel such as the rotating-wall vessel. This provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis, characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. Optimization of suspension culture is achieved by applying three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Next, rotation in the rotating-wall vessel induces both Coriolis and centrifugal forces, directly dependent on terminal velocity and minimized as terminal velocity is minimized. Last, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. In the transduction of mechanical culture conditions into cellular effects, several lines of evidence support a role for multiple molecular mechanisms. These include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as protein kinase C. The discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and biological effects, emphasizing cellular processes important for the industrial production of biological pharmaceuticals and devices.

Extraction and Estimation of Secondary Metabolites from Date Palm Cell Suspension Cultures.

PubMed

Naik, Poornananda M; Al-Khayri, Jameel M

2017-01-01

The health benefits of dates arise from their content of phytochemicals, known for having pharmacological properties, including flavonoids, carotenoids, phenolic acids, sterols, procyanidins, and anthocyanins. In vitro cell culture technology has become an attractive means for the production of biomass and bioactive compounds. This chapter describes step-by-step procedures for the induction and proliferation of callus from date palm offshoots on Murashige and Skoog (MS) medium supplemented with plant growth regulators. Subsequently cell suspension cultures are established for optimum biomass accumulation, based on the growth curve developed by packed cell volume as well as fresh and dry weights. The highest production of biomass occurs at the 11th week after culturing. Moreover, this chapter describes methodologies for the extraction and analysis of secondary metabolites of date palm cell suspension cultures using high-performance liquid chromatography (HPLC). The optimum level of catechin, caffeic acid, apigenin, and kaempferol from the cell suspension cultures establishes after the 11th and 12th weeks of culture. This protocol is useful for scale-up production of secondary metabolites from date palm cell suspension cultures.

Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.

PubMed

Reuter, Lauri J; Bailey, Michael J; Joensuu, Jussi J; Ritala, Anneli

2014-05-01

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Dynamic and rheological properties of soft biological cell suspensions

PubMed Central

Yazdani, Alireza; Li, Xuejin

2016-01-01

Quantifying dynamic and rheological properties of suspensions of soft biological particles such as vesicles, capsules, and red blood cells (RBCs) is fundamentally important in computational biology and biomedical engineering. In this review, recent studies on dynamic and rheological behavior of soft biological cell suspensions by computer simulations are presented, considering both unbounded and confined shear flow. Furthermore, the hemodynamic and hemorheological characteristics of RBCs in diseases such as malaria and sickle cell anemia are highlighted. PMID:27540271

Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions

PubMed Central

Pless-Petig, Gesine; Walter, Björn; Bienholz, Anja

2018-01-01

Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/106 cells after cold storage, 5 ± 3 nmol/106 cells after rewarming vs. control 29 ± 6 nmol/106 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec−1 per 106 cells after rewarming vs. control 232 ± 83 pmol sec−1 per 106 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/106 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production—as elicited by an inhibitor of the respiratory chain, antimycin A—can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment with subsequent energy deficiency is a key factor for the lack of attachment of cold-stored hepatocyte suspensions. PMID:29390882

Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions.

PubMed

Pless-Petig, Gesine; Walter, Björn; Bienholz, Anja; Rauen, Ursula

2017-12-01

Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/10 6 cells after cold storage, 5 ± 3 nmol/10 6 cells after rewarming vs. control 29 ± 6 nmol/10 6 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec -1 per 10 6 cells after rewarming vs. control 232 ± 83 pmol sec -1 per 10 6 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/10 6 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production-as elicited by an inhibitor of the respiratory chain, antimycin A-can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment with subsequent energy deficiency is a key factor for the lack of attachment of cold-stored hepatocyte suspensions.

[Neuron differential attachment purification and its influence factor].

PubMed

Li, Jun; Feng, Daxiong; Huang, Yize; Ye, Fei

2010-02-01

Neuron purification is essential to procedure of various nerve cell experimental research, however, at present there is few reports on the effect of various factors on neural axons during purification. To find out a simple method of neuron purification, and to investigate the influence factors of corresponding purification culture in dorsal root ganglion (DRG) tissue culture on beta3-tubulin positive axon. The DRGs were obtained from the 3 days neonatal SD rat microscopically and were made into cell suspension. Then, the amount of attached DRG neurons and nonneuronal cells in poly-D-lysine (PDL) group, PDL/Laminin (PDL/LN) group and collagen-I (Col I) group was observed from 10 to 100 minutes. Then, the extension and arborization of beta3-tubulin positive axons were observed after 72 hours completely randomised DRG tissue culture for the research of the influences among culture substrates (PDL, PDL/LN, and Col I), FBS (0, 5%, and 10%), 5 fluorouracil (5-Fu, 0, 20, and 40 micromol/L), and cytarabine (Ara-C, 0, 10, and 20 micromol/L). Adherent cells were observed instantly after inoculation by inverted phase contrast microscope and inverted fluorescence microscope; after cell suspension was removed, adherent growth of DRGn cells and non-DRGn cells were still seen. In PDL group, the amount of NSE negative cells was significantly higher than that of NSE positive cells at 10 and 30 minutes (P < 0.05); the amount of NSE positive cells was significantly higher than that of NSE negative cells at 80, 90 and 100 minutes (P < 0.05). In PDL/LN group, there was no significant difference (P > 0.05) in the amount of NSE negative cells and NSE positive cells at 10, 20, 30, 40, and 50 minutes; the amount of NSE positive cells was significantly higher (P < 0.05) than that of NSE negative cells at 60, 70, 80, 90, 100 minutes. In Col I group, the amount of NSE negative cells was higher than that of NSE positive cells at 10-40 minutes, but showing no significant difference (P > 0.05); the amount of NSE positive cells was significantly higher (P < 0.05) than that of NSE negative cells at 70-100 minutes. At 72 hours after DRG tissue culture, the best result of beta3-tubulin positive axon extension and arborization was obtained when the substrate level was PDL/LN, and the average length of PDL/LN level was significantly larger than that of other two substrates (P < 0.05). The highest number of beta3-tubulin positive axon distal end was obtained at 5% concentration level of FBS (P < 0.05), but showing no significant differences in beta3-tubulin positive axon length among three levels (P > 0.05). Both the most of beta3-tubulin positive axon distal ends and the longest beta3-tubulin positive axon average length were obtained at 0 micromol/L concentration level of 5-Fu, showing significant differences between 0 micromol/L level and 20, 40 micromol/L levels (P < 0.05). A similar result of 33-tubulin positive axon distal end was got at the 0 micromol/L level and 10 micromol/L level of Ara-C, which was significantly higher than that of 20 micromol/L level (P < 0.05). A purified DRG neuron suspension for neuron culture could be obtained via PDL differential attachment for 30 minutes. When DRG neuron culture, neuron special medium, PDL/LN substrate and 10 micromol/L Ara-C are recommended in beta3-tubulin positive axon research.

Growing Bacteriophage M13 in Liquid Culture.

PubMed

Green, Michael R; Sambrook, Joseph

2017-11-01

Stocks of bacteriophage M13 are usually grown in liquid culture. The infected bacteria do not lyse but, instead, grow at a slower than normal rate to form a dilute suspension. The inoculum of bacteriophage is almost always a freshly picked plaque or a suspension of bacteriophage particles obtained from a single plaque, as described here. Infected cells contain up to 200 copies of double-stranded, replicative-form DNA and extrude several hundred bacteriophage particles per generation. Thus, a 1-mL culture of infected cells can produce enough double-stranded viral DNA (1-2 mg) for restriction mapping and recovery of cloned DNA inserts and sufficient single-stranded DNA (∼5-10 mg) for site-directed mutagenesis, DNA sequencing, or synthesis of radiolabeled probes. The titer of bacteriophages in the supernatant from infected cells is so high (∼10 12 pfu/mL) that a small aliquot serves as a permanent stock of the starting plaque. © 2017 Cold Spring Harbor Laboratory Press.

Impact of Motile Bacterial Suspensions on Viscous Fingering and Mixing

NASA Astrophysics Data System (ADS)

Chui, Jane; Auradou, Harold; de Anna, Pietro; Fahrner, Karen; Berg, Howard; Juanes, Ruben

2017-11-01

Viscous fingering is a hydrodynamic instability that occurs when a less viscous fluid displaces a more viscous one. Instead of progressing as a uniform front, the less viscous fluid forms fingers to create complex patterns. Understanding how these patterns and their associated gradients evolve over time is of critical importance in characterizing the mixing of two fluids, which in turn is important to applications such as enhanced oil recovery, bioremediation, and microfluidics. Here, we investigate the impact of replacing the less viscous fluid with an active suspension of motile bacteria. In this series of experiments, a suspension of motile Escherichia coli capable of collective swimming is injected into a microfluidic Hele-Shaw cell under viscous fingering conditions. Through videomicroscopy, we obtain high-resolution concentration fields to determine the evolution of the mixing zone (region with concentration gradients). We quantify the impact that active suspensions have on the formation of viscous fingering patterns and mixing efficiency between the two fluids, and-conversely-report details of the collective swimming behavior in the presence of a viscous-gradient front.

Cultivation of cottontail rabbit epidermal (Sf1Ep) cells on microcarrier beads and their use for suspension cultivation of Treponema pallidum subsp. pallidum.

PubMed Central

Riley, B S; Cox, D L

1988-01-01

In vitro propagation of Treponema pallidum can be achieved by cocultivation with Sf1Ep cells. This study had two objectives: (i) to achieve suspension cultivation of Sf1Ep cells and (ii) to develop procedures for achieving the replication of T. pallidum in those cell cultures. Seven suspension cultures of Sf1Ep cells yielded an average of 7.2 x 10(8) T. pallidum (36-fold increase) after 12 days. Images PMID:3063209

Photoacoustic detection of induced melanoma in vitro using a mouse model

NASA Astrophysics Data System (ADS)

Gupta, Sagar; Bhattacharya, Kiran; Newton, Jessica R.; Quinn, Thomas P.; Viator, John A.

2012-03-01

Metastasis is a life threatening complex physiological phenomenon that involves the movement of cancer cells from one organ to another by means of blood and lymph. An understanding about metastasis is extremely important to device diagnostic systems to detect and monitor its spread within the body. For the first time we report rapid photoacoustic detection of the induced metastatic melanoma in mice in vitro using photoacoustic flowmetry. A new photoacoustic flow system is developed, that employs photoacoustic excitation coupled with an ultrasound transducer capable of determining the presence of individual, induced mouse melanoma cells (B16/F10) within the circulating system in vitro. Tumor was induced in mice by injecting mouse melanoma cells through tail vein into the C57BL/6 mice. A luciferase based in vivo bioluminescence imaging is performed to confirm the tumor load and multiple metastases in the tumor-induced mice. 1ml of blood obtained through cardiac puncture of the induced metastasized mice was treated to lyse the red blood cells (RBC) and enriched, leaving the induced melanoma in the peripheral blood mononuclear suspension (PBMC). A photoacoustic flowsystem coupled with an ultrasound transducer is used to detect the individual circulating metastatic melanoma cells from the enriched cell suspension.

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures.

PubMed

Cetin, Emine Sema; Babalik, Zehra; Hallac-Turk, Filiz; Gokturk-Baydar, Nilgun

2014-09-23

Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells. Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl2 and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl2 gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6. As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.

Regio-selective deglycosylation of icariin by cell suspension cultures of Glycyrrhiza uralensis and Morus alba.

PubMed

Zhang, De-Wu; Tao, Xiao-Yu; Chen, Ri-Dao; Yu, Li-Yan; Dai, Jun-Gui

2015-01-01

Biotransformations of icariin (1) by cell suspension cultures of Glycyrrhiza uralensis and Morus alba yielded two new metabolites, icaruralins A and B (2 and 3), and one known metabolite, baohuoside I (4). Their structures were determined on the basis of extensive spectroscopic analysis. This is the first report that the cell suspension cultures of G. uralensis and M. alba possess deglycosylation functionality.

Optical filter based on Fabry-Perot structure using a suspension of goethite nanoparticles as electro-optic material

NASA Astrophysics Data System (ADS)

Abbas, Samir; Dupont, Laurent; Dozov, Ivan; Davidson, Patrick; Chanéac, Corinne

2018-02-01

We have investigated the feasibility of optical tunable filters based on a Fabry-Perot etalon that uses a suspension of goethite (α-FeOOH) nanorods as electro-optic material for application in optical telecommunications in the near IR range. These synthetic nanoparticles have a high optical anisotropy that give rise to a very strong Kerr effect in their colloidal suspensions. Currently, these particles are dispersed in aqueous solvent, with pH2 to ensure the colloidal electrostatic stability. However, the high conductivity of these suspensions requires using high-frequency electric fields (f > 1 MHz), which brings about a high power consumption of the driver. To decrease the field frequency, we have changed the solvent to ethylene glycol which has a lower electrical conductivity than the aqueous solvent. We have built a Fabry-Perot cell, filled with this colloidal suspension in the isotropic phase, and showed that a phase shift of 14 nm can be obtained in a field of 3V/μm. Therefore, the device can operate as a tunable filter. A key advantage of this filter is that it is, by principle, completely insensitive to the polarization of the input light. However, several technological issues still need to be solved, such as ionic contamination of the suspension from the blocking layers, and dielectrophoretic and thermal effects.

Serotonin Signal Transduction in Two Groups of Autistic Patients

DTIC Science & Technology

2012-10-01

as learned from the initial studies. Treat cells with escitalopram or r-citalopram and test for increased coupling between G proteins and effector...Figure 1 (top). Escitalopram translocates Gs in cells obtained from High 5HT subjects. Lymphoblasts from High 5HT/High IS or Normal 5HT/moderate IS...two lines each) were grown in suspension and treated with escitalopram or control (r-citalopram, @10 µM) for 3days, harvested and membranes were

Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

NASA Astrophysics Data System (ADS)

Chen, Kun; Qin, Yejun; Zheng, Feng; Sun, Menghong; Shi, Daren

2006-07-01

A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis.

Enhancement of viniferin production in Vitis vinifera L. cv. Alphonse Lavallée Cell suspensions by low-energy ultrasound alone and in combination with methyl jasmonate.

PubMed

Santamaria, Anna Rita; Innocenti, Marzia; Mulinacci, Nadia; Melani, Fabrizio; Valletta, Alessio; Sciandra, Ilaria; Pasqua, Gabriella

2012-11-07

This study examined for the first time the effect of low-energy ultrasound (US), used alone or in combination with methyl jasmonate (MeJA), on viniferin production in cell cultures of Vitis vinifera L. cv Alphonse Lavallée. Cell suspensions were exposed for 2 min to US (power 30, 60, and 90 mW cm(-3)). The highest viniferin production was obtained at 30 mW cm(-3). When sonication was performed twice, the effect on viniferin production was negligible, whereas triple sonication slightly increased production. US treatment at 30 mW cm(-3) for 5 min decreased viniferin production and induced cellular death. The combined use of MeJA and US (2 min) increased the production of δ-viniferin, the dominant stilbene, more than each elicitor used alone. These results suggest that low-energy US, alone and in combination with MeJA, can act as a physical elicitor to stimulate viniferin production in V. vinifera cell cultures.

20 CFR 416.1330 - Suspension due to failure to apply for and obtain other benefits.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false Suspension due to failure to apply for and obtain other benefits. 416.1330 Section 416.1330 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Suspensions and Terminations § 416.1330...

Histamine release, formation of prostaglandin-like activity (SRS-C) and mast cell degranulation by the direct lytic factor (DLF) and phospholipase A of cobra venom.

PubMed

Damerau, B; Lege, L; Oldigs, H D; Vogt, W

1975-01-01

Cobra venom, alone and in combination, on mast cell degranulation, histamine release and formation of prostaglandin-like activity (SRS-C) was studied in perfused guinea-pig lungs and in mast cell-containing rat peritoneal cell suspensions. For comparison, the effect of equivalent doses of whole cobra venom was investigated. 1. Cobra venom caused mast cell degranulation, histamine release and SRS-C formation in both systems. For comparable effects much higher doses had to be used in guine-pig lungs than in rat peritoneal cell suspensions. 2. Phase A showed little degranulation of mast cells in both systems, a limited histamine release in rat peritoneal cell suspensions and none in perfused guinea-pig lungs. It caused a considerable SRS-C formation in both, lung tissue and peritoneal cell suspensions. 3. DLF caused histamine release, SRS-C formation and mast cell degranulation in both systems; in rat peritoneal cell suspensions it acted almost as strong as equivalent doses of cobra venom, in guinea pig lungs it was much less active. 4. In rat peritoneal cell suspensions the effects of DLF and phase A in combination did not exceed the sum of their single effects. In guinea-pig lungs these two substances interacted in a potentiating synergism. It is concluded that DLF is the main cytotoxic principle of cobra venom, whereas ph-ase A alone is not cytotoxic. The difference in the synergism of DLF and ph-ase A between rat peritoneal cells and guinea-pig lungs may be due to two different actions of DLF and species differences as regards sensitivity against these actions.

Protein specific fluorescent microspheres for labelling a protein

NASA Technical Reports Server (NTRS)

Rembaum, Alan (Inventor)

1982-01-01

Highly fluorescent, stable and biocompatible microspheres are obtained by copolymerizing an acrylic monomer containing a covalent bonding group such as hydroxyl, amine or carboxyl, for example, hydroxyethylmethacrylate, with an addition polymerizable fluorescent comonomer such as dansyl allyl amine. A lectin or antibody is bound to the covalent site to provide cell specificity. When the microspheres are added to a cell suspension the marked microspheres will specifically label a cell membrane by binding to a specific receptor site thereon. The labeled membrane can then be detected by fluorescence of the fluorescent monomer.

Suspension culture process for H9N2 avian influenza virus (strain Re-2).

PubMed

Wang, Honglin; Guo, Suying; Li, Zhenguang; Xu, Xiaoqin; Shao, Zexiang; Song, Guicai

2017-10-01

H9N2 avian influenza virus has caused huge economic loss for the Chinese poultry industry since it was first identified. Vaccination is frequently used as a control method for the disease. Meanwhile suspension culture has become an important tool for the development of influenza vaccines. To optimize the suspension culture conditions for the avian influenza H9N2 virus (Re-2 strain) in Madin-Darby Canine Kidney (MDCK) cells, we studied the culture conditions for cell growth and proliferation parameters for H9N2 virus replication. MDCK cells were successfully cultured in suspension, from a small scale to industrial levels of production, with passage time and initial cell density being optimized. The influence of pH on the culture process in the reactor has been discussed and the process parameters for industrial production were explored via amplification of the 650L reactor. Subsequently, we cultivated cells at high cell density and harvested high amounts of virus, reaching 10log2 (1:1024). Furthermore an animal experiment was conducted to detect antibody. Compared to the chicken embryo virus vaccine, virus cultured from MDCK suspension cells can produce a higher amount of antibodies. The suspension culture process is simple and cost efficient, thus providing a solid foundation for the realization of large-scale avian influenza vaccine production.

Enhanced Production of Anthraquinones and Phenolic Compounds and Biological Activities in the Cell Suspension Cultures of Polygonum multiflorum

PubMed Central

Thiruvengadam, Muthu; Rekha, Kaliyaperumal; Rajakumar, Govindasamy; Lee, Taek-Jun; Kim, Seung-Hyun; Chung, Ill-Min

2016-01-01

Anthraquinones (AQs) and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of Polygonum multiflorum. We wanted to optimize the effects of plant growth regulators (PGRs), media, sucrose, l-glutamine, jasmonic acid (JA), and salicylic acid (SA) for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum. The medium containing Murashige and Skoog (MS) salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM); 3 and 2.93 g dry mass (DM)) and AQ production (emodin 295.20 and 282 mg/g DM; physcion 421.55 and 410.25 mg/g DM) were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA) induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds) production as well as biological activities in the cell suspension cultures of P. multiflorum. This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds) from cell suspension cultures, and the phytochemicals can be used for various biological activities. PMID:27854330

Extraction of pectins and hemicelluloses from cell walls of suspension culture cells of cotton by endopolygalacturonase treatment

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

An, Jinhua; Maness, N.O.; Mort, A.J.

1989-04-01

Walls of cotton suspension cultures were treated with a highly purified endopolygalacturonase coded for by a gene from Erwinia carotovora expressed in E. coli. About 80 percent of the walls galacturonic acid was solubilized and could be fractionated by ion exchange chromatography into three classes: (1) tri- and tetra-galacturonides from digestion of homogalacturonans, (2) high molecular weight fragments rich in galacturonic acid, rhamnose and other neutral sugars indicative of rhamnogalacturonan I, and (3) a well defined low molecular weight polymer resembling rhamnogalacturonan II. Treatment of the insoluble wall residue with alkali allowed extraction of the remainder of the wall galacturonicmore » acid as high molecular weight polymers resembling rhamnogalacturonan I but in association with what is probably xyloglucan. The purified polymers will be compared to those obtained by others from different species, especially those of sycamore.« less

Suppression of humoral response during the course of Candida albicans infection in mice.

PubMed

Valdez, J C; Meson, O E; de Valdez, G A; Sirena, A

1984-10-30

This paper aims at demonstrating the non-specific immunosuppression as regards thyme-dependent antigens sheep erythrocytes (SRBC) during the course of Candida albicans systemic infection. Three lots of syngeneic/BALB/c mice, 8-12 weeks of age, were used. The first normal lot was inoculated via the intraperitoneal route with a (SRBC) suspension (4 X 10(8) cells ml) in a Hank's balanced saline solution. The primary response of antibodies formed by splenic cells was measured from 4 to 8 days after inoculation using the direct plaque forming cells technique. The second lot was infected by the same route with a suspension of Candida albicans (1 X 10(7) cells). Positive retrocultures from the blood and kidneys of these infected mice were obtained. These yeasts cultivated in a Sabouraud medium were harvested after 20 h at 37 degrees C. Following the same methodology the immune response to SRBC was determined. The serum obtained from infected mice was transferred to a third lot of mice at different intervals during the course of the infection. The immune response to SRBC was done by the direct plaque-forming cells technique. Controls were carried out using normal donors and recipients. A suppression of the immune response was obtained as from the 2nd day of inoculation up to the 28th day. It was not possible to transfer such suppression passively by means of the serum. These results suggest that the systemic infection by Candida albicans induce a non-specific immunosuppression in the organism, already demonstrated in viral infections, bacteria, protozoaria and metazoaria in mammals. In some way, this will contribute to explain the mechanisms of immune response to Candida albicans.

Spectral analysis of fundus autofluorescence pattern as a tool to detect early stages of degeneration in the retina and retinal pigment epithelium.

PubMed

Feldman, Tatiana B; Yakovleva, Marina A; Larichev, Andrey V; Arbukhanova, Patimat M; Radchenko, Alexandra Sh; Borzenok, Sergey A; Kuzmin, Vladimir A; Ostrovsky, Mikhail A

2018-05-22

The aim of this work is the determination of quantitative diagnostic criteria based on the spectral characteristics of fundus autofluorescence to detect early stages of degeneration in the retina and retinal pigment epithelium (RPE). RPE cell suspension samples were obtained from the cadaver eyes with and without signs of age-related macular degeneration (AMD). Fluorescence analysis at an excitation wavelength of 488 nm was performed. The fluorescence lifetimes of lipofuscin-granule fluorophores were measured by counting time-correlated photon method. Comparative analysis of fluorescence spectra of RPE cell suspensions from the cadaver eyes with and without signs of AMD showed a significant difference in fluorescence intensity at 530-580 nm in response to fluorescence excitation at 488 nm. It was notably higher in eyes with visual pathology than in normal eyes regardless of the age of the eye donor. Measurements of fluorescence lifetimes of lipofuscin fluorophores showed that the contribution of photooxidation and photodegradation products of bisretinoids to the total fluorescence at 530-580 nm of RPE cell suspensions was greater in eyes with visual pathology than in normal eyes. Because photooxidation and photodegradation products of bisretinoids are markers of photodestructive processes, which can cause RPE cell death and initiate degenerative processes in the retina, quantitative determination of increases in these bisretinoid products in lipofuscin granules may be used to establish quantitative diagnostic criteria for degenerative processes in the retina and RPE.

Mesenchymal stem cell sheets exert anti-stenotic effects in a rat arterial injury model.

PubMed

Homma, Jun; Sekine, Hidekazu; Matsuura, Katsuhisa; Kobayashi, Eiji; Shimizu, Tatsuya

2018-05-04

Restenosis after catheter or surgical intervention substantially affects the prognosis of arterial occlusive disease. Mesenchymal stem cells (MSCs) may have anti-stenotic effects on injured arteries. MSC transplantation from the adventitial side of an artery is safer than endovascular transplantation but has not been extensively examined. In this study, a rat model of femoral artery injury was used to compare the anti-stenotic effects of transplanted cell sheets and transplanted cell suspensions. Rat adipose-derived stem cells (ASCs) were used as the source of MSCs. For both cell sheets and suspensions, 6×106 MSCs were transplanted on the day of arterial injury. MSC sheets attenuated neointimal hyperplasia more than MSC suspensions (intima-to-media ratio in haematoxylin/eosin-stained sections: 0.55±0.13 vs. 1.14±0.12; P<0.05). Cell engraftment (assessed by immunohistochemistry or bioluminescence imaging of luciferase-expressing cells), arterial re-endothelialisation (evaluated by immunohistochemical staining for rat endothelial cell antigen-1) and restriction of vascular smooth muscle cell proliferation in the neointima (double-staining of alpha-smooth muscle actin and phospho-histone H3) were greater when MSC sheets were applied than when MSC suspensions were used. In conclusion, MSC sheets exhibited better anti-stenotic and cell engraftment properties than MSC suspensions. MSC sheet transplantation from the adventitial side is a promising therapy for prevention of arterial restenosis.

Study of dust re-suspension at low pressure in a dedicated wind-tunnel

NASA Astrophysics Data System (ADS)

Rondeau, Anthony; Sabroux, Jean-Christophe; Chassefière, Eric

2015-04-01

The atmosphere of several telluric planets or satellites are dusty. Such is the case of Earth, Venus, Mars and Titan, each bearing different aeolian processes linked principally to the kinematic viscosity of the near-surface atmosphere. Studies of the Martian atmosphere are particularly relevant for the understanding of the dust re-suspension phenomena at low pressure (7 mbar). It turns out that operation of fusion reactors of the tokamak design produces significant amount of dust through the erosion of plasma-facing components. Such dust is a key issue, both regarding the performance and the safety of a fusion reactor such as ITER, under construction in Cadarache, France. Indeed, to evaluate the explosion risk in the ITER fusion reactor, it is essential to quantify the re-suspended dust fraction as a function of the dust inventory that can be potentially mobilized during a loss of vacuum accident (LOVA), with air or water vapour ingress. A complete accident sequence will encompass dust re-suspension from near-vacuum up to atmospheric pressure. Here, we present experimental results of particles re-suspension fractions measured at 1000, 600 and 300 mbar in the IRSN BISE (BlowIng facility for airborne releaSE) wind tunnel. Both dust monolayer deposits and multilayer deposits were investigated. In order to obtain experimental re-suspension data of dust monolayer deposits, we used an optical microscope allowing to measure the re-suspended particles fraction by size intervals of 1 µm. The deposits were made up of tungsten particles on a tungsten surface (an ubiquitous plasma facing component) and alumina particles on a glass plate, as a surrogate. A comparison of the results with the so-called Rock'nRoll dust re-suspension model (Reeks and Hall, 2001) is presented and discussed. The multilayer deposits were made in a vacuum sedimentation chamber allowing to obtain uniform deposits in terms of thickness. The re-suspension experimental data of such deposits were obtained thanks to a photovoltaic cell on which the alumina dust was deposited, taking advantage of the well-known loss of efficiency of dusty solar panels (e.g., Spirit Mars Rover before and after a "cleaning event"). The short circuit intensity of the cell being a function of the amount (mg/cm2) of dust deposited, it is possible to determine directly on-line the fraction of dust re-suspended during an experiment in the wind tunnel. Thus, we highlighted two kinds of mechanisms, with a transition region. Above a threshold and at each step increase of airflow velocity, a fraction of the deposit is quickly mobilised (in a few seconds). After a transition gap (approximately two minutes), a low linear increase of the re-suspension fraction is observed. This phenomenon is likely due to random bursts characterizing turbulent airflows. In addition, we brought to light a dust mobilisation mechanism by particle clustering, likely due to the adhesion forces between particles outmatching the adhesion forces between particles and the panel surface. These experimental data will allow to validate a re-suspension model for multi-layer deposits taking into account the effect of low pressure and of dust mobilisation by particles clustering. Reeks, M.W. and Hall, D., 2001. Kinetic models for particle resuspension in turbulent flows: theory and measurement. J. Aerosol Sci., 32: 1-31.

Cell specific, variable density, polymer microspheres

NASA Technical Reports Server (NTRS)

Yen, Shiao-Ping S. (Inventor); Rembaum, Alan (Inventor); Molday, Robert S. (Inventor)

1977-01-01

Biocompatible polymeric microspheres having an average diameter below about 3 microns and having density at least 15% greater or lesser than organic cells and having covalent binding sites are provided in accordance with this invention. The microspheres are obtained by copolymerizing a hydroxy or amine substituted acrylic monomer such as hydroxyethylmethacrylate with a light or dense comonomer such as a fluoromonomer. A lectin or antibody is bound to the hydroxy or amine site of the bead to provide cell specificity. When added to a cell suspension the marked bead will specifically label the cell membrane by binding to specific receptor sites thereon. The labelled membrane can then be separated by density gradient centrifugation.

An easy “SteamDrop” method for high quality plant chromosome preparation

PubMed Central

2014-01-01

Background The chromosome preparation is a crucial step for obtaining satisfactory results in molecular cytogenetic researches. The preparation of plant chromosomes for molecular cytogenetic purposes remains a challenge for some species. In contrast to human chromosome preparation, the processes occurring during plant chromosome preparation and causing chromosome spreading are still poorly understood. Results We studied the dynamics of plant chromosome spreading after dropping cell suspension on slides. We showed that steam stimulates cytoplasm hydrolysis and rapid chromosome spreading and that chromosomes stretch during this chromosome spreading. Based on these observations, we developed a novel method, named “SteamDrop”, for the preparation of well-spread mitotic and pachytene chromosomes and successfully used it for 28 plant species with large and small chromosomes. We applied cell suspensions in ethanol instead of the commonly used ethanol/acetic acid fixative. Mitotic and meiotic chromosomes prepared via “SteamDrop” were used in fluorescent in situ hybridization (FISH) experiments with repetitive and unique DNA probes. Long storage of cell suspensions in ethanol did not impair the quality of chromosome preparations. Conclusion The SteamDrop procedure provides a robust and routine method for high quality plant chromosome preparations. The method can be applied for metaphase as well as pachytene chromosome preparation in wide range of species. The chromosomes prepared by SteamDrop are well suitable for repetitive and unique DNA visualization. PMID:24602284

Intracerebral Cell Implantation: Preparation and Characterization of Cell Suspensions.

PubMed

Rossetti, Tiziana; Nicholls, Francesca; Modo, Michel

2016-01-01

Intracerebral cell transplantation is increasingly finding a clinical translation. However, the number of cells surviving after implantation is low (5-10%) compared to the number of cells injected. Although significant efforts have been made with regard to the investigation of apoptosis of cells after implantation, very little optimization of cell preparation and administration has been undertaken. Moreover, there is a general neglect of the biophysical aspects of cell injection. Cell transplantation can only be an efficient therapeutic approach if an optimal transfer of cells from the dish to the brain can be ensured. We therefore focused on the in vitro aspects of cell preparation of a clinical-grade human neural stem cell (NSC) line for intracerebral cell implantation. NSCs were suspended in five different vehicles: phosphate-buffered saline (PBS), Dulbecco's modified Eagle medium (DMEM), artificial cerebral spinal fluid (aCSF), HypoThermosol, and Pluronic. Suspension accuracy, consistency, and cell settling were determined for different cell volume fractions in addition to cell viability, cell membrane damage, and clumping. Maintenance of cells in suspension was evaluated while being stored for 8 h on ice, at room temperature, or physiological normothermia. Significant differences between suspension vehicles and cellular volume fractions were evident. HypoThermosol and Pluronic performed best, with PBS, aCSF, and DMEM exhibiting less consistency, especially in maintaining a suspension and preserving viability under different storage conditions. These results provide the basis to further investigate these preparation parameters during the intracerebral delivery of NSCs to provide an optimized delivery process that can ensure an efficient clinical translation.

Rapid diagnosis of primary ciliary dyskinesia: cell culture and soft computing analysis.

PubMed

Pifferi, Massimo; Bush, Andrew; Montemurro, Francesca; Pioggia, Giovanni; Piras, Martina; Tartarisco, Gennaro; Di Cicco, Maria; Chinellato, Iolanda; Cangiotti, Angela M; Boner, Attilio L

2013-04-01

Diagnosis of primary ciliary dyskinesia (PCD) sometimes requires repeated nasal brushing to exclude secondary ciliary alterations. Our aim was to evaluate whether the use of a new method of nasal epithelial cell culture can speed PCD diagnosis in doubtful cases and to identify which are the most informative parameters by means of a multilayer artificial neural network (ANN). A cross-sectional study was performed in patients with suspected PCD. All patients underwent nasal brushing for ciliary motion analysis, ultrastructural assessment and evaluation of ciliary function after ciliogenesis in culture by ANN. 151 subjects were studied. A diagnostic suspension cell culture was obtained in 117 nasal brushings. A diagnosis of PCD was made in 36 subjects (29 of whom were children). In nine out of the 36 patients the diagnosis was made only after a second brushing, because of equivocal results of both tests at first examination. In each of these subjects diagnosis of PCD was confirmed by cell culture results. Cell culture in suspension evaluated by means of ANN allows the separation of PCD from secondary ciliary dyskinesia patients after only 5 days of culture and allows diagnosis to be reached in doubtful cases, thus avoiding the necessity of a second sample.

Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

PubMed

Mélida, Hugo; Largo-Gosens, Asier; Novo-Uzal, Esther; Santiago, Rogelio; Pomar, Federico; García, Pedro; García-Angulo, Penélope; Acebes, José Luis; Álvarez, Jesús; Encina, Antonio

2015-04-01

Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment. © 2015 Institute of Botany, Chinese Academy of Sciences.

Optimization of Native and Formaldehyde iPOND Techniques for Use in Suspension Cells.

PubMed

Wiest, Nathaniel E; Tomkinson, Alan E

2017-01-01

The isolation of proteins on nascent DNA (iPOND) technique developed by the Cortez laboratory allows a previously unparalleled ability to examine proteins associated with replicating and newly synthesized DNA in mammalian cells. Both the original, formaldehyde-based iPOND technique and a more recent derivative, accelerated native iPOND (aniPOND), have mostly been performed in adherent cell lines. Here, we describe modifications to both protocols for use with suspension cell lines. These include cell culture, pulse, and chase conditions that optimize sample recovery in both protocols using suspension cells and several key improvements to the published aniPOND technique that reduce sample loss, increase signal to noise, and maximize sample recovery. Additionally, we directly and quantitatively compare the iPOND and aniPOND protocols to test the strengths and limitations of both. Finally, we present a detailed protocol to perform the optimized aniPOND protocol in suspension cell lines. © 2017 Elsevier Inc. All rights reserved.

PATHWAYS OF GLUCOSE CATABOLISM IN BACILLUS CEREUS1

PubMed Central

Goldman, Manuel; Blumenthal, Harold J.

1964-01-01

Goldman, Manuel (The University of Michigan, Ann Arbor), and Harold J. Blumenthal. Pathways of glucose catabolism in Bacillus cereus. J. Bacteriol. 87:377–386. 1964.—Estimates by a radiorespirometric method of the pathways of glucose catabolism of resting-cell suspensions of Bacillus cereus strain terminalis indicate that the Embden-Meyerhof pathway predominates at every stage of development, including the sporogenic and germinative phases. At the filamentous, granular, forespore, and transitional stages, 98% of the glucose was catabolized by the Embden-Meyerhof pathway, and the remainder by the hexose monophosphate oxidative pathway. Estimates of the pathways in resting spore-suspensions arrested at defined stages of development indicate that 20% of the glucose was catabolized through the hexose monophosphate pathway in germinated spores, and 10% in the swollen and elongated stages of postgermination. In cells which had completed the first cell division, the figure fell to about 2%, a level similar to that found for vegetative cells at later stages of development. The key Embden-Meyerhof enzymes, hexokinase, phosphohexoisomerase, phosphofructokinase, and aldolase, as well as several other enzymes, were present at all stages of germination and postgerminative development, supporting the radioisotopic data obtained with whole cells. As indicated by the release of C14O2 from glucose-6-C14, terminal respiration of resting-cell suspensions operates maximally in vegetative cells at the granular, fore-spore, and transitional stages. There was marked inhibition of terminal respiration during the development of spores into vegetative cells. Only slight activity occurred in the earliest vegetative stages, and maximal operation developed after about ten cell divisions. Fumarase was absent in spores until sometime late in the elongation stage. At this point, a weak but definite activity appeared which increased during later stages of development so that, by the end of about the sixth cell division, fumarase had a specific activity about 80 times that observed at elongation. PMID:14151060

Isolation of Nuclei and Nucleoli.

PubMed

Pendle, Alison F; Shaw, Peter J

2017-01-01

Here we describe methods for producing nuclei from Arabidopsis suspension cultures or root tips of Arabidopsis, wheat, or pea. These methods could be adapted for other species and cell types. The resulting nuclei can be further purified for use in biochemical or proteomic studies, or can be used for microscopy. We also describe how the nuclei can be used to obtain a preparation of nucleoli.

Nanometer-scale sizing accuracy of particle suspensions on an unmodified cell phone using elastic light scattering.

PubMed

Smith, Zachary J; Chu, Kaiqin; Wachsmann-Hogiu, Sebastian

2012-01-01

We report on the construction of a Fourier plane imaging system attached to a cell phone. By illuminating particle suspensions with a collimated beam from an inexpensive diode laser, angularly resolved scattering patterns are imaged by the phone's camera. Analyzing these patterns with Mie theory results in predictions of size distributions of the particles in suspension. Despite using consumer grade electronics, we extracted size distributions of sphere suspensions with better than 20 nm accuracy in determining the mean size. We also show results from milk, yeast, and blood cells. Performing these measurements on a portable device presents opportunities for field-testing of food quality, process monitoring, and medical diagnosis.

Growing Arabidopsis in vitro: cell suspensions, in vitro culture, and regeneration.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

2014-01-01

An understanding of basic methods in Arabidopsis tissue culture is beneficial for any laboratory working on this model plant. Tissue culture refers to the aseptic growth of cells, organs, or plants in a controlled environment, in which physical, nutrient, and hormonal conditions can all be easily manipulated and monitored. The methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Techniques, including callus production, cell suspension cultures, and plant regeneration, are all indispensable tools for the study of cellular biochemical and molecular processes. Plant regeneration is a key technology for successful stable plant transformation, while cell suspension cultures can be exploited for metabolite profiling and mining. In this chapter we report methods for the successful and highly efficient in vitro regeneration of plants and production of stable cell suspension lines from leaf explants of both Arabidopsis thaliana and Arabidopsis halleri.

The effects of tissue processing on markers for T and B cells from solid tissues.

PubMed

Millard, P R; Rabin, B S; Whiteside, T L; Hubbard, J D

1977-03-01

Suspensions of lymphoid cells from tissues have been used for the determination of the quantitative relationship between the T and B cell populations. The distribution of the lymphocytes within a given tissue, however, cannot be demonstrated once such a suspension has been prepared. Various methods of characterizing lymphocytes within tissues were evaluated. The method of tissue preparation can alter the capability of detecting the lymphocyte markers. Fluorescein-labeled anti-immunoglobulin sera reacted equally well with lymphocytes in tissue regardless of the method of tissue preparation. Complement-coated sheep erythrocytes were less effective in detecting lymphocyte markers in tissue sections than in cell suspensions. Quantitative assays of lymphocytes could be done in suspensions only. Unaltered sheep erythrocytes did not bind to T lymphocytes in tissue. T lymphocytes could be identified in tissue sections, however, by the use of anti-human T cell serum.

Jasmonic and salicylic acids enhanced phytochemical production and biological activities in cell suspension cultures of spine gourd (Momordica dioica Roxb).

PubMed

Chung, Ill-Min; Rekha, Kaliyaperumal; Rajakumar, Govindasamy; Thiruvengadam, Muthu

2017-03-01

In vitro cell suspension culture was established for the production of commercially valuable phytochemicals in Momordica dioica. The influence of elicitors in jasmonic acid (JA) and salicylic acid (SA) increased their effect on phytochemical production and biomass accumulation in M. dioica. The results indicate that compared with non-elicited cultures, JA- and SA-elicited cell suspension cultures had significantly enhanced phenolic, flavonoid, and carotenoid production, as well as antioxidant, antimicrobial, and antiproliferative activities. Furthermore, elicited cultures produced 22 phenolic compounds, such as flavonols, hydroxycinnamic acids, and hydroxybenzoic acids. Greater biomass production, phytochemical accumulation, and biological activity occurred in JA- than in SA-elicited cell cultures. This study is the first to successfully establish M. dioica cell suspension cultures for the production of phenolic compounds and carotenoids, as well as for biomass accumulation.

Investigation of the hydrodynamic response of cells in drop on demand piezoelectric inkjet nozzles.

PubMed

Cheng, Eric; Yu, Haoran; Ahmadi, Ali; Cheung, Karen C

2016-01-29

Cell motion within a liquid suspension inside a piezoelectrically actuated, cylindrical inkjet printhead was studied using high speed imaging and a low depth of field setup. For each ejected droplet, a cell within the inkjet nozzle was observed to exhibit one of three possible behaviors which are termed: cell travel, cell ejection and cell reflection. Cell reflection is an undesirable phenomenon which may adversely affect an inkjet's capability in dispensing cells and a possible reason why it was previously reported that the rate of cells dispensed did not follow the expected Poisson distribution. Through the study of the cells motions, it was hypothesized that the rheological properties of the media in the cell suspension play an important role in influencing the cell behaviors exhibited. This was experimentally studied with the tracking of cells within the inkjet nozzle in a 10% w/v Ficoll PM400 cell suspension. The effect of cell reflection was eliminated using the higher density and viscosity Ficoll PM400 suspension. The presented work is the first in-depth study of the cell behaviors occurring within a piezoelectric inkjet nozzle during the printing process. The understanding of the hydrodynamics during a droplet ejection and its effect on the suspended cells are imperative towards achieving reliable cell dispensing for biofabrication applications.

Study of immobilized and extracellular saccharase of watermelon.

PubMed

Stano, Ján; Siekel, Peter; Micieta, Karol; Barth, Alfred

2006-01-01

A simple, rapid and reproducible procedure for the identification and determination of extracellular saccharase from culture medium of watermelon cell suspension cultures is described. The culture medium (without cells) was used for the identification and determination of extracellular enzyme activity. Intracellular activity was estimated from the cell suspension. Watermelon cell suspension was permeabilized by Tween 80 and immobilized by glutaraldehyde. The highest saccharase activity was at pH 4.6 at a temperature of 50 degrees C. The hydrolysis of substrate was linear 5h after reaching 60% conversion. The cells had high saccharase activity and good stability, and in long-term storage they showed convenient physico-mechanical properties.

Strategies to Suspension Serum-Free Adaptation of Mammalian Cell Lines for Recombinant Glycoprotein Production.

PubMed

Caron, Angelo Luis; Biaggio, Rafael Tagé; Swiech, Kamilla

2018-01-01

Serum-free suspension cultures are preferably required for recombinant protein production due to its readiness in upstream/downstream processing and scale-up, therefore increasing process productivity and competitiveness. This type of culture replaces traditional cell culturing as the presence of animal-derived components may introduce lot-a-lot variability and adventitious pathogens to the process. However, adapting cells to serum-free conditions is challenging, time-consuming, and cell line and medium dependent. In this chapter, we present different approaches that can be used to adapt mammalian cell lines from an anchorage-dependent serum supplemented culture to a suspension serum-free culture.

A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

PubMed

Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

2015-09-01

A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 μI of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed.

Regulation of callus status and cell-suspending culture in naked seed oat (Avena nuda).

PubMed

Cui, L; Fan, Y

1998-01-01

The original calli were obtained by inducing culture of mature embryos of naked seed oat on N6 medium. The original calli were white-colored tumor forms, soft outside and hard inside. These kinds of calli are easy to differentiate into plantlets, and they are not the friable type. Friable embryogenic calli could be obtained by cycled regulated culture on IM1-IM4 medium for 7-8 months from the original calli. They became vigorous, lightish yellow in color, with small grainy forms. Well-separated and fast-growing suspending cell lines have been obtained from the above-mentioned embryogenic calli in the liquid medium. Regenerated plants have been obtained for this kind of suspension line by culturing on the medium for differentiation. The surviving percentage for such plantlets was over 95% after planting in the soil.

Collective hydrodynamics of swimming micro-organisms

NASA Astrophysics Data System (ADS)

Pedley, Timothy

2007-11-01

Since the work of Kessler in the 1980s, and before, there has been considerable interest among fluid dynamicists and physicists in the collective behaviour of swimming micro-organisms in suspension. Since all such cells are denser than the water in which they swim, bioconvection patterns result from upswimming of cells in a chamber of finite depth and from gyrotaxis of bottom-heavy cells in a uniform fluid. Bioconvection has been analysed for dilute suspensions; the theory will be briefly re-examined with emphasis on the additional stress induced by the cells' swimming motions (each cell can be regarded as a force-dipole, or stresslet), because of the new instabilities revealed by Simha & Ramaswamy (2002) for uniform suspensions in the absence of gravity. Even more fascinating coherent structures arise in concentrated suspensions, of bacteria for example, in which cell-cell interactions cannot be ignored. The hypothesis is that such structures emerge from purely hydrodynamic interactions between cells. A variety of models have been developed, which are outlined briefly, but particular attention will be paid to our own model in which cells are represented as inertia-free ``spherical squirmers,'' whose behaviour is dominated by near-field hydrodynamics. Pairwise interactions are computed precisely, and Stokesian dynamics in a periodic box is used to simulate an infinite suspension. Trajectories are computed deterministically, but the long-time spreading of a 3D suspension, from random initial conditions, is diffusive; scaling arguments can be used to estimate the effective diffusivity. However, in 2D there is a strong tendency towards aggregation into clumps or bands. [Recent work reported here has been performed in collaboration with T Ishikawa and J T Locsei.

Some potential blood flow experiments for space

NASA Technical Reports Server (NTRS)

Cokelet, G. R.; Meiselman, H. J.; Goldsmith, H. L.

1979-01-01

Blood is a colloidal suspension of cells, predominantly erythrocytes, (red cells) in an aqueous solution called plasma. Because the red cells are more dense than the plasma, and because they tend to aggregate, erythrocyte sedimentation can be significant when the shear stresses in flowing blood are small. This behavior, coupled with equipment restrictions, has prevented certain definitive fluid mechanical studies from being performed with blood in ground-based experiments. Among such experiments, which could be satisfactorily performed in a microgravity environment, are the following: (1) studies of blood flow in small tubes, to obtain pressure-flow rate relationships, to determine if increased red cell aggregation can be an aid to blood circulation, and to determine vessel entrance lengths, and (2) studies of blood flow through vessel junctions (bifurcations), to obtain information on cell distribution in downstream vessels of (arterial) bifurcations, and to test flow models of stratified convergent blood flows downstream from (venous) bifurcations.

Establishment and characterization of American elm cell suspension cultures

Treesearch

Steven M. Eshita; Joseph C. Kamalay; Vicki M. Gingas; Daniel A. Yaussy

2000-01-01

Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A700 and media conductivity. Combined cell growth data for all experiments within a...

Studies on Rapidly Frozen Suspensions of Yeast Cells by Differential Thermal Analysis and Conductometry

PubMed Central

Mazur, Peter

1963-01-01

Few, if any, yeast cells survived rapid cooling to -196°C and subsequent slow warming. After rapid freezing, the suspensions absorbed latent heat of fusion between -15° and 0°C during warming, and the relation between the amount of heat absorbed and the concentration of cells was the same as that in equivalent KCl solutions, indicating that frozen suspensions behave thermally like frozen solutions. The amount of heat absorbed was such that more than 80 per cent of the intracellular solution had to be frozen. The conductometric behavior of frozen suspensions showed that cell solutes were still inside the cells and surrounded by an intact cell membrane at the time heat was being absorbed. Two models are consistent with these findings. The first assumes that intracellular freezing has taken place; the second that all freezable water has left the cells and frozen externally. The latter model is ruled out because rapidly cooled cells do not shrink by an amount equal to the volume of water that would have to be withdrawn to prevent internal freezing. PMID:13934216

Simulation and analysis of vertical displacement characteristics of three wheels reverse trike vehicle with PID controller application

NASA Astrophysics Data System (ADS)

Wibowo, Lambang, Lullus; Erick Chandra, N.; Muhayat, Nurul; Jaka S., B.

2017-08-01

The purpose of this research is to obtain a mathematical model (Full Vehicle Model) and compare the performance of passive and active suspension systems of a Three-Wheels Reverse Trike vehicle. Vehicle suspension system should able to provide good steering handling and passenger comfort. Vehicle suspension system generally only uses passive suspension components with fix spring and damper coefficients. An active suspension developed from the traditional (passive) suspension design can directly control the actuator force in the suspension system. In this paper, modeling and simulation of passive and active suspension system for a Full Vehicle Model is performed using Simulink-MATLAB software. Ziegler & Nichols tuning method is used to obtain controller parameters of Proportional Integral Derivative (PID) controller. Comparison between passive and active suspension with PID controller is conducted for disturbances input of single bump road surface profile 0.1 meters. The results are the displacement and acceleration of the vehicle body in the vertical direction of active suspension system with PID control is better in providing handling capabilities and comfort for the driver than of passive suspension system. The acceleration of 1,8G with the down time of 2.5 seconds is smaller than the acceleration of 2.5G with down time of 5.5 seconds.

Lipid profile of in vitro oil produced through cell culture of Jatropha curcas.

PubMed

Correa, Sandra M; Atehortúa, Lucía

2012-01-01

Recent increases in energy demands as a consequence of population growth and industrialization, and pollution caused during the extraction and combustion of fossil fuel sources have driven the development of new energy sources that do not cause pollution and are inexpensive and renewable. Consequently, it is necessary to develop alternative ways of generating biofuels that put less pressure on agricultural lands and water supplies, and ensure ecosystems conservation. In order to achieve the proposed goals related to energetic coverage and independence, several approaches have been developed, including biodiesel production using vegetal oils as feedstock. The aim of the current research project was to apply a nonconventional bioprocess for in vitro biomass and oil production of Jatropha curcas, for assessing different J. curcas varieties, where seed tissue was isolated and used for callus induction. Once friable callus was obtained, cell suspension cultures were established. The cell viability, fatty acid content, and characteristics were used to select the most promising cell line according to its fatty acid profile and ability to grow and develop under in vitro conditions. Oil produced by cell suspension culture of the Jatropha varieties studied was extracted and characterized by GC/MS. Differences encountered among Jatropha varieties were related to their fatty acid profiles, oil content (% on dry basis), and cell viability measurements (%).

Unilateral lower limb suspension does not mimic bed rest or spaceflight effects on human muscle fiber function

NASA Technical Reports Server (NTRS)

Widrick, J. J.; Trappe, S. W.; Romatowski, J. G.; Riley, D. A.; Costill, D. L.; Fitts, R. H.

2002-01-01

We used Ca2+-activated skinned muscle fibers to test the hypothesis that unilateral lower leg suspension (ULLS) alters cross-bridge mechanisms of muscle contraction. Soleus and gastrocnemius biopsies were obtained from eight subjects before ULLS, immediately after 12 days of ULLS (post-0 h), and after 6 h of reambulation (post-6 h). Post-0 h soleus fibers expressing type I myosin heavy chain (MHC) showed significant reductions in diameter, absolute and specific peak Ca2+-activated force, unloaded shortening velocity, and absolute and normalized peak power. Fibers obtained from the gastrocnemius were less affected by ULLS, particularly fibers expressing fast MHC isoforms. Post-6 h soleus fibers produced less absolute and specific peak force than did post-0 h fibers, suggesting that reambulation after ULLS induced cell damage. Like bed rest and spaceflight, ULLS primarily affects soleus over gastrocnemius fibers. However, in contrast to these other models, slow soleus fibers obtained after ULLS showed a decrease in unloaded shortening velocity and a greater reduction in specific force.

Preparation of an Ultrafine Rebamipide Ophthalmic Suspension with High Transparency.

PubMed

Matsuda, Takakuni; Hiraoka, Shogo; Urashima, Hiroki; Ogura, Ako; Ishida, Tatsuhiro

2017-01-01

A 2% commercially available, milky-white, rebamipide micro-particle suspension is used to treat dry eyes, and it causes short-term blurring of the patient's vision. In the current study, to improve the transparency of a rebamipide suspension, we attempted to obtain a clear rebamipide suspension by transforming the rebamipide particles to an ultrafine state. In the initial few efforts, various rebamipide suspensions were prepared using a neutralizing crystallization method with additives, but the suspensions retained their opaque quality. However, as a consequence of several critical improvements in the neutralizing crystallization methods such as selection of additives for crystallization, process parameters during crystallization, the dispersion method, and dialysis, we obtained an ultrafine rebamipide suspension (2%) that was highly transparent (transmittance at 640 nm: 59%). The particle size and transparency demonstrated the fewest level of changes at 25°C after 3 years, compared to initial levels. During that period, no obvious particle sedimentation was observed. The administration of this ultrafine rebamipide suspension (2%) increased the conjunctival mucin, which was comparable to the commercially available micro-particle suspension (2%). The corneal and conjunctival concentration of rebamipide following ocular administration of the ultrafine suspension was slightly higher than that of the micro-particle suspension. The ultrafine rebamipide suspension (eye-drop formulation) with a highly transparent ophthalmic clearness should improve a patient's QOL by preventing even a shortened period of blurred vision.

The biological acoustic sensor to record the interactions of the microbial cells with the phage antibodies in conducting suspensions.

PubMed

Guliy, О I; Zaitsev, B D; Borodina, I A; Shikhabudinov, А М; Teplykh, A A; Staroverov, S A; Fomin, A S

2018-02-01

The acoustic biological sensor for the analysis of the bacterial cells in conducting suspension was developed. The sensor represented the two channel delay line based on the piezoelectric plate of Y-X lithium niobate thick of 0.2mm. Two pairs of the interdigital transducers (IDT) for the excitation and reception of shear horizontal acoustic wave of zero order (SH 0 ) in each channel were deposited by the method of photolithography. One channel of the delay line was electrically shorted by the deposition of thin aluminum film between IDTs. The second channel remained as electrically open. The liquid container with the volume of 5ml was fixed on the plate surface between IDTs by the glue, which did not cause the additional insertion loss. For the first time the influence of the conductivity of the cell suspension on the registration of the specific and nonspecific interactions of the bacterial cells with phage-antibodies (phage-Abs) was studied by means of the developed sensor. The dependencies of the change in insertion loss and phase of the output signal on the conductivity of the buffer solution at specific/nonspecific interactions for the electrically open and shorted channels of the delay line were obtained. It was shown that the sensor successfully registered the interactions of microbial cells with phage-Abs in the range of the conductivity of 2-20 μS/cm on the model samples A. brasilense Sp245 - specific phage-Abs. The sensor in the time regime of the operation fast reacted on the specific/nonspecific interaction and the time of the stabilization of the output parameters did not exceed 10min. Copyright © 2017 Elsevier B.V. All rights reserved.

High-level production of human interleukin-10 fusions in tobacco cell suspension cultures

PubMed Central

Kaldis, Angelo; Ahmad, Adil; Reid, Alexandra; McGarvey, Brian; Brandle, Jim; Ma, Shengwu; Jevnikar, Anthony; Kohalmi, Susanne E; Menassa, Rima

2013-01-01

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER. PMID:23297698

Biotransformation of perfumery terpenoids, (−)-ambrox® by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala

PubMed Central

2012-01-01

Background Biotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm) with fungal and plant cell culture. Results Biotransformation of (−)-ambrox (1) with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmala yielded oxygenated products, 3β-hydroxyambrox (2), 6β-hydroxyambrox (3), 1α-hydroxy-3oxoambrox (4), 1α,3β-dihydroxyambrox (5), 13,14,15,16-tetranorlabdane-3-oxo-8,12-diol (6), 3-oxoambrox (7), 2α-hydroxyambrox (8), 3β-hydroxysclareolide (9), and 2α,3β-dihydroxyambrox (10). Metabolite 4 was found to be new compound. These metabolites were structurally characterized on the basis of spectroscopic studies. Conclusion Nine oxygenated metabolites of (−)-ambrox (1) were obtained from Macrophomina phaseolina and Peganum harmala. Enzymatic system of screened organisms introduced hydroxyl and keto functionalities at various positions of compound 1 in a stereo- and regio-controlled manner. PMID:22863186

Enhanced Biosynthesis of Withanolides by Elicitation and Precursor Feeding in Cell Suspension Culture of Withania somnifera (L.) Dunal in Shake-Flask Culture and Bioreactor

PubMed Central

Sivanandhan, Ganeshan; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

2014-01-01

The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period. PMID:25089711

Cell specific, variable density, polymer microspheres

NASA Technical Reports Server (NTRS)

Yen, Shiao-Ping S. (Inventor); Rembaum, Alan (Inventor); Molday, Robert S. (Inventor)

1978-01-01

Biocompatible polymeric microspheres having an average diameter below about 3 microns and having a density at least 15% greater or lesser than organic cells and having covalent binding sites are provided in accordance with this invention. The microspheres are obtained by copolymerizing a hydroxy or amine substituted acrylic monomer such as hydroxyethylmethacrylate with a light or dense comonomer such as a fluoromonomer. A lectin or antibody is bound to the hydroxy or amine site of the bead to provide cell specificity. When added to a cell suspension the marked bead will specifically label the cell membrane by binding to specific receptor sites thereon. The labelled membrane can then be separated by density gradient centrifugation.

Method of electrode fabrication for solid oxide electrochemical cells

DOEpatents

Jensen, R.R.

1990-11-20

A process for fabricating cermet electrodes for solid oxide electrochemical cells by sintering is disclosed. First, a porous metal electrode is fabricated on a solid oxide cell, such as a fuel cell by, for example, sintering, and is then infiltrated with a high volume fraction stabilized zirconia suspension. A second sintering step is used to sinter the infiltrated zirconia to a high density in order to more securely attach the electrode to the solid oxide electrolyte of the cell. High performance fuel electrodes can be obtained with this process. Further electrode performance enhancement may be achieved if stabilized zirconia doped with cerium oxide, chromium oxide, titanium oxide, and/or praseodymium oxide for electronic conduction is used. 5 figs.

Method of electrode fabrication for solid oxide electrochemical cells

DOEpatents

Jensen, Russell R.

1990-01-01

A process for fabricating cermet electrodes for solid oxide electrochemical cells by sintering is disclosed. First, a porous metal electrode is fabricated on a solid oxide cell, such as a fuel cell by, for example, sintering, and is then infiltrated with a high volume fraction stabilized zirconia suspension. A second sintering step is used to sinter the infiltrated zirconia to a high density in order to more securely attach the electrode to the solid oxide electrolyte of the cell. High performance fuel electrodes can be obtained with this process. Further electrode performance enhancement may be achieved if stabilized zirconia doped with cerium oxide, chromium oxide, titanium oxide, and/or praseodymium oxide for electronic conduction is used.

CELL SEPARATION ON ANTIGEN-COATED COLUMNS

PubMed Central

Wigzell, Hans; Andersson, Birger

1969-01-01

Glass and plastic bead columns coated with antigenic protein molecules were used as an immunological filter for cell populations containing immune cells of relevant specificity. A selective elimination of these immune cells from the passing cell suspension was regularly noted and it approached, in some experiments, complete abolition of the specific immune reactivity of the filtered cell population. This specific retention of immune cells by antigenic columns could be selectively blocked by the presence of free antigen molecules in the medium during filtration. The results obtained support the concept of a cell-associated antigen-specific receptor being present on the outer surface of immune cells, displaying the same antigen-binding specificity as the potential product of the cell, the humoral antibody. Using the present bead column system, results were obtained indicating that this receptor was an active product of the immune cells and not any passively adsorbed, cytophilic antibody. Antigenic bead columns may very well constitute a tool for the production in vitro of cell populations being specifically deprived of immune reactivity and allow detailed analysis of the characteristics of the cell-associated antibody of immune cells. PMID:5782770

Uniform Embryoid Body Production and Enhanced Mesendoderm Differentiation with Murine Embryonic Stem Cells in a Rotary Suspension Bioreactor.

PubMed

Lei, Xiaohua; Deng, Zhili; Duan, Enkui

2016-01-01

Embryonic stem cells (ESCs) are capable of differentiating into almost all cell types in vitro and hold great promise for drug screening, developmental studies and have a huge potential in many therapeutic areas. ESCs can aggregate to form embryoid body (EB) in static suspension culture by spontaneous differentiation, which resembles an intact embryo; while static suspension culture cannot prevent agglomeration of cells and offers little control over the size and shape of EBs, it results in aggregation of EBs into large, irregular masses, which prejudice the efficiency of differentiation of cells. Recently, bioreactor-based platforms have been shown to not only offer a beneficial effect on increasing diffusion of nutrients and oxygen which promotes cell viability and proliferation but also display local biomechanical properties (e.g., low fluid shear stresses and hydrodynamic force) in tissue development and organogenesis. This chapter describes a protocol for using a rotary suspension bioreactor to produce embryoid bodies and process the differentiation of mouse embryonic stem cells (mESCs), and to assess the efficiency of EB differentiation in the bioreactor by real-time PCR and immunostaining.

A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

PubMed

Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

2016-01-01

We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Regio- and stereoselectivities in plant cell biotransformation

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

Hamada, H.

1995-12-01

The ability of plant cultured cells to convert foreign substrates into more useful substances is of considerable interest. Therefore I have studied biotransformation of foreign substrate by plant cell suspension cultures. In this presentation, I report regio- and stereoselectivities in biotransformation of steroids and indole alkaloids and taxol by plant (tobacco, periwinkle, moss, orchid) cell suspension cultures.

Semi-solid electrode cell having a porous current collector and methods of manufacture

DOEpatents

Chiang, Yet-Ming; Carter, William Craig; Cross, III, James C.; Bazzarella, Ricardo; Ota, Naoki

2017-11-21

An electrochemical cell includes an anode, a semi-solid cathode, and a separator disposed therebetween. The semi-solid cathode includes a porous current collector and a suspension of an active material and a conductive material disposed in a non-aqueous liquid electrolyte. The porous current collector is at least partially disposed within the suspension such that the suspension substantially encapsulates the porous current collector.

Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

PubMed

Ayaydin, Ferhan; Kotogány, Edit; Ábrahám, Edit; Horváth, Gábor V

2017-01-01

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.

Predicting human blood viscosity in silico

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

Fedosov, Dmitry A.; Pan, Wenxiao; Caswell, Bruce

2011-07-05

Cellular suspensions such as blood are a part of living organisms and their rheological and flow characteristics determine and affect majority of vital functions. The rheological and flow properties of cell suspensions are determined by collective dynamics of cells, their structure or arrangement, cell properties and interactions. We study these relations for blood in silico using a mesoscopic particle-based method and two different models (multi-scale/low-dimensional) of red blood cells. The models yield accurate quantitative predictions of the dependence of blood viscosity on shear rate and hematocrit. We explicitly model cell aggregation interactions and demonstrate the formation of reversible rouleaux structuresmore » resulting in a tremendous increase of blood viscosity at low shear rates and yield stress, in agreement with experiments. The non-Newtonian behavior of such cell suspensions (e.g., shear thinning, yield stress) is analyzed and related to the suspension’s microstructure, deformation and dynamics of single cells. We provide the flrst quantitative estimates of normal stress differences and magnitude of aggregation forces in blood. Finally, the flexibility of the cell models allows them to be employed for quantitative analysis of a much wider class of complex fluids including cell, capsule, and vesicle suspensions.« less

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

Contribution of flow cytometry to the diagnosis of gastric lymphomas in endoscopic biopsy specimens.

PubMed

Almasri, N M; Zaer, F S; Iturraspe, J A; Braylan, R C

1997-07-01

Gastric lymphomas seem to have unique clinical, pathologic, and immunophenotypic features that set them apart from nodal lymphomas. Microscopic examination of endoscopic biopsy specimens is the most frequent procedure used to diagnose gastric tumors, but it is very difficult, and sometimes impossible, to recognize lymphomas in endoscopic samples by histologic or even immunohistologic methods. Because most gastric lymphomas are of B-cell origin, we used flow cytometry to assess B-cell clonality in gastric biopsy specimens containing dense lymphocytic infiltrates thought to represent lymphoma. We prepared viable cell suspensions from unfixed specimens obtained from 29 consecutive patients who had a previous microscopic diagnosis of suspicious gastric lymphoid infiltrates. We performed immunophenotypic studies with multicolor flow cytometry, and we assessed clonality by examination of immunoglobulin (Ig) light-chain expression analyzed exclusively on B cells identified by anti-CD20 or CD19 antibodies. The mean number of cells recovered was 1.04 x 10(6), from an average of 5.5 gastric biopsy fragments per patient. In 26 of the 29 patients, the number of cells was adequate for analysis. We detected B-cell monoclonality in 16 cases, including 5 in which the percentage of clonal B cells was less than 5%. Of the 16 cases, only 8 could be diagnosed as lymphomas on morphologic grounds alone; the remaining 8 patients had either suspicious lymphoid infiltrates or chronic gastritis. The three cases with an insufficient number of cells were considered non-neoplastic either on histologic grounds alone or in conjunction with Southern analysis of Ig genes. We conclude that flow cytometric immunophenotypic analysis of freshly prepared cell suspensions obtained from endoscopic biopsy specimens can be used to evaluate gastric lymphocytic infiltrates. Specifically, the analysis of surface Ig light-chain expression on B cells distinguishes between monoclonal (lymphoma) and polyclonal (nonlymphoma) infiltrates. The rapidity, ease, quantitative properties, and sensitivity of this technique make it a supplement to the morphologic assessment of gastric lymphoid infiltrates.

Studies of uranium-sodium suspensions. Part I. Construction and operation of experimental loop

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

Bett, F L; Hilditch, R J; Mepham, R G

1961-08-01

An experimnental uranium- sodium suspension loop was operated for 4320 hr. The design, construction, commissioning, and operation of the loop to the point where a comnplete stable suspension was obtained is described.

Fast Micromethod: Determination of DNA Integrity in Cell Suspensions and in Solid Tissues.

PubMed

Bihari, Nevenka

2017-01-01

The Fast Micromethod is a rapid and convenient microplate procedure for the determination of DNA integrity in cell suspensions and in solid tissues. The procedure is based on the ability of fluorochromes to preferentially interact with double-stranded DNA in alkaline conditions. Rapid sample lysis is followed by denaturation at high pH during 15 min. Only 30 ng of DNA from cell suspensions or tissue homogenates per single well are required for the analyses. The whole analysis is performed within 3 h or less (for one 96-well microplate).The Fast Micromethod is broadly used in biology and medicine. Its applications range from environmental pollution tests in marine invertebrates to the analysis of biopsy samples in cancer patients to detect DNA alterations caused by irradiation or chemotherapy.The procedure presented here describes the Fast Micromethod applied for the determination of DNA integrity in cell suspensions (HeLa cells) and solid tissues (mussel gills).

Gamma-irradiated bacterial preparation having anti-tumor activity

DOEpatents

Vass, Arpad A.; Tyndall, Richard L.; Terzaghi-Howe, Peggy

1999-01-01

A bacterial preparation from Pseudomonas species isolated #15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

In vitro cytotoxic effects of benzalkonium chloride in corticosteroid injection suspension.

PubMed

Davis, Daniel; Cyriac, Mathew; Ge, Dongxia; You, Zongbing; Savoie, Felix H

2010-01-01

Some deleterious effects on cartilage and even severe arthropathy have been reported after intra-articular corticosteroid injections. The objective of the present in vitro study was to determine if an injectable corticosteroid suspension is toxic to articular chondrocytes and synovial cells. Human and bovine articular chondrocytes, bovine synovial cells, mouse C3H10T1/2 cells, and human osteosarcoma MG-63 cells were treated for thirty minutes in monolayer or suspension culture with an injectable corticosteroid suspension or its chemical components, including betamethasone sodium phosphate, betamethasone acetate, and benzalkonium chloride (as preservative). Cell viability was determined by means of microscopy or flow cytometry analysis. In monolayer culture, the betamethasone corticosteroids per se did not cause cell death, whereas benzalkonium chloride caused death of articular chondrocytes. In suspension culture, betamethasone sodium phosphate at dosages of as high as 6 mg/mL did not cause significant death of human or bovine articular chondrocytes (p > 0.05). In contrast, benzalkonium chloride caused a death rate of 10.6% in human articular chondrocytes at a dosage of 10 microg/mL (p < 0.01), 21.0% at a dosage of 13.3 microg/mL (p < 0.01), and 99.3% and 99.4% at dosages of 20 and 200 microg/mL, respectively (p < 0.001 for both). Similarly, benzalkonium chloride caused death of bovine articular chondrocytes, bovine synovial cells, C3H10T1/2 cells, and MG-63 cells in a dose-dependent manner. When treated with a combination of betamethasone sodium phosphate and 200 microg/mL benzalkonium chloride, >99% of human or bovine articular chondrocytes were dead (p < 0.001). The injectable corticosteroid suspension caused death in in vitro culture of human and bovine articular chondrocytes as well as bovine synovial cells because of its preservative benzalkonium chloride. The betamethasone corticosteroids per se did not cause significant chondrocyte death under the conditions tested.

Cellulose nanocrystals from grape pomace: Production, properties and cytotoxicity assessment.

PubMed

Coelho, Caroline C S; Michelin, Michele; Cerqueira, Miguel A; Gonçalves, Catarina; Tonon, Renata V; Pastrana, Lorenzo M; Freitas-Silva, Otniel; Vicente, António A; Cabral, Lourdes M C; Teixeira, José A

2018-07-15

Cellulose nanocrystals (CNCs) were obtained from grape pomace through chemical and physical pretreatments. Bleached cellulose pulp was subjected to acid hydrolysis (AH) for 30 or 60 min and an ultrasound treatment to obtain CNCs (AH 30S and AH 60S ). Compositional analyses of untreated (UGP) and pretreated (PGP) grape pomace showed the effectiveness of pretreatment in removing non-cellulosic components, recovering 80.1% cellulose in PGP (compared to 19.3% of UGP). Scanning and transmission electron microscopies were used to evaluate the CNCs morphology. AH in combination with ultrasound treatment led to needle-shaped structures and apparently more dispersed suspensions. Crystallinity index and thermal stability were studied by X-ray diffraction and thermogravimetric analysis, respectively. The AH 60S sample presented high aspect ratio, crystallinity and thermal stability. CNCs toxicity was evaluated by exposing Caco-2 cells to CNCs suspension and evaluating their viability. Results showed that CNCs are non-toxic, opening the opportunity for their use on food and pharmaceutical applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Impact of noncovalent interactions between apple condensed tannins and cell walls on their transfer from fruit to juice: studies in model suspensions and application.

PubMed

Le Bourvellec, Carine; Le Quere, Jean-Michel; Renard, Catherine M G C

2007-09-19

The adsorption of procyanidins (condensed tannins) on cell-wall material was quantified by bringing into contact solutions of procyanidins and suspensions of cell-wall material. A model was developed on the basis of the Langmuir isotherm formulation and a factorial experimental design. The parameters that influenced the adsorption were the concentration and molecular weight of the procyanidins, the ionic strength of the solution, the temperature, and the apple cell-wall concentration. The model was applied to partitioning of procyanidins from apple between juice and mash. The parameters to be taken into account are the composition of the apples and, specifically, (i) the concentration and molecular weight of the procyanidins, (ii) their acidity and pH as a determinant of the ionic strength, and (iii) their cell-wall content and the temperature at pressing. To estimate the ability of the model to relate procyanidin concentrations in the juice to their concentration in the apple, apples of three varieties of widely different procyanidin compositions were pressed in conditions that prevent oxidation. In these conditions, yields in the juice were >80% for phenolic acids or catechin monomers but <50% for procyanidins, with the lowest rates obtained for the higher polymers in accordance with the model.

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a Technicon H-1.

PubMed

Ross, D W; Bishop, C; Henderson, A; Kaplow, L

1990-01-01

We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.

Targeted antitumoral dehydrocrotonin nanoparticles with L-ascorbic acid 6-stearate.

PubMed

Frungillo, Lucas; Martins, Dorival; Teixeira, Sérgio; Anazetti, Maristela Conti; Melo, Patrícia da Silva; Durán, Nelson

2009-12-01

Tumoral cells are known to have a higher ascorbic acid uptake than normal cells. Therefore, the aim of this study was to obtain polymeric nanoparticles containing the antitumoral compound trans-dehydrocrotonin (DHC) functionalized with L-ascorbic acid 6-stearate (AAS) to specifically target this system tumoral cells. Nanoparticle suspensions (NP-AAS-DHC) were prepared by the nanoprecipitation method. The systems were characterized for AAS presence by thin-layer chromatography and for drug loading (81-88%) by UV-Vis spectroscopy. To further characterize these systems, in vitro release kinetics, size distribution (100-140 nm) and Zeta potential by photon-correlation spectroscopic method were used. In vitro toxicity against HL60 cells was evaluated by tetrazolium reduction and Trypan blue exclusion assays. Cell death by apoptosis was quantified and characterized by flow cytometry and caspase activity. Zeta potential analyses showed that the system has a negatively charged outer surface and also indicate that AAS is incorporated on the external surface of the nanoparticles. In vitro release kinetics assay showed that DHC loaded in nanoparticles had sustained release behavior. In vitro toxicity assays showed that NP-AAS-DHC suspension was more effective as an antitumoral than free DHC or NP-DHC and increased apoptosis induction by receptor-mediated pathway. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Fluid flows created by swimming bacteria drive self-organization in confined suspensions

PubMed Central

Lushi, Enkeleida; Wioland, Hugo; Goldstein, Raymond E.

2014-01-01

Concentrated suspensions of swimming microorganisms and other forms of active matter are known to display complex, self-organized spatiotemporal patterns on scales that are large compared with those of the individual motile units. Despite intensive experimental and theoretical study, it has remained unclear the extent to which the hydrodynamic flows generated by swimming cells, rather than purely steric interactions between them, drive the self-organization. Here we use the recent discovery of a spiral-vortex state in confined suspensions of Bacillus subtilis to study this issue in detail. Those experiments showed that if the radius of confinement in a thin cylindrical chamber is below a critical value, the suspension will spontaneously form a steady single-vortex state encircled by a counter-rotating cell boundary layer, with spiral cell orientation within the vortex. Left unclear, however, was the flagellar orientation, and hence the cell swimming direction, within the spiral vortex. Here, using a fast simulation method that captures oriented cell–cell and cell–fluid interactions in a minimal model of discrete particle systems, we predict the striking, counterintuitive result that in the presence of collectively generated fluid motion, the cells within the spiral vortex actually swim upstream against those flows. This prediction is then confirmed by the experiments reported here, which include measurements of flagella bundle orientation and cell tracking in the self-organized state. These results highlight the complex interplay between cell orientation and hydrodynamic flows in concentrated suspensions of microorganisms. PMID:24958878

Design analysis of formula student race car suspension system

NASA Astrophysics Data System (ADS)

Wirawan, Julian Wisnu; Ubaidillah, Aditra, Rama; Alnursyah, Rafli; Rahman, Rizki Abdul; Cahyono, Sukmaji Indro

2018-02-01

Design analysis of suspension especially for racecar suspension is very crucial to achieve maximum performance and handling. Suspension design may vary depending on the road terrain and the vehicle purpose itself, such as high speed or off-road vehicle. This paper focused on the suspension which used for racecar vehicle. The suspension type used was unequal double wishbone. This model is used because of its stability for high-speed usage compared to another kind of suspension. The suspension parameter was calculated to achieve desired performance. The result is the motion ratio of the designed suspension geometry. The obtained value of motion ratio was 1:2 for front suspension and 1:1 for the rear suspension. These calculation result the front suspension is still too soft, which the optimal motion ratio should be kept around 1:1 for better handling. This problem caused by the lack of space for suspension linkage.

Accurate label-free 3-part leukocyte recognition with single cell lens-free imaging flow cytometry.

PubMed

Li, Yuqian; Cornelis, Bruno; Dusa, Alexandra; Vanmeerbeeck, Geert; Vercruysse, Dries; Sohn, Erik; Blaszkiewicz, Kamil; Prodanov, Dimiter; Schelkens, Peter; Lagae, Liesbet

2018-05-01

Three-part white blood cell differentials which are key to routine blood workups are typically performed in centralized laboratories on conventional hematology analyzers operated by highly trained staff. With the trend of developing miniaturized blood analysis tool for point-of-need in order to accelerate turnaround times and move routine blood testing away from centralized facilities on the rise, our group has developed a highly miniaturized holographic imaging system for generating lens-free images of white blood cells in suspension. Analysis and classification of its output data, constitutes the final crucial step ensuring appropriate accuracy of the system. In this work, we implement reference holographic images of single white blood cells in suspension, in order to establish an accurate ground truth to increase classification accuracy. We also automate the entire workflow for analyzing the output and demonstrate clear improvement in the accuracy of the 3-part classification. High-dimensional optical and morphological features are extracted from reconstructed digital holograms of single cells using the ground-truth images and advanced machine learning algorithms are investigated and implemented to obtain 99% classification accuracy. Representative features of the three white blood cell subtypes are selected and give comparable results, with a focus on rapid cell recognition and decreased computational cost. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Single-Beam Acoustic Trapping of Red Blood Cells and Polystyrene Microspheres in Flowing Red Blood Cell Saline and Plasma Suspensions.

PubMed

Liu, Hsiao-Chuan; Li, Ying; Chen, Ruimin; Jung, Hayong; Shung, K Kirk

2017-04-01

Single-beam acoustic tweezers (SBATs) represent a new technology for particle and cell trapping. The advantages of SBATs are their deep penetration into tissues, reduction of tissue damage and ease of application to in vivo studies. The use of these tools for applications in drug delivery in vivo must meet the following conditions: large penetration depth, strong trapping force and tissue safety. A reasonable penetration depth for SBATs in the development of in vivo applications was established in a previous study conducted in water with zero velocity. However, capturing objects in flowing fluid can provide more meaningful results. In this study, we investigated the capability of SBATs to trap red blood cells (RBCs) and polystyrene microspheres in flowing RBC suspensions. Two different types of RBC suspension were prepared in this work: an RBC phosphate-buffered saline (PBS) suspension and an RBC plasma suspension. The results indicated that SBATs successfully trapped RBCs and polystyrene microspheres in a flowing RBC PBS suspension with an average steady velocity of 1.6 cm/s in a 2-mm-diameter polyimide. Furthermore, SBATs were found able to trap RBCs in a flowing RBC PBS suspension at speeds as high as 7.9 cm/s in a polyimide tube, which is higher than the velocity in capillaries (0.03 cm/s) and approaches the velocity in arterioles and venules. Moreover, the results also indicated that polystyrene microspheres can be trapped in an RBC plasma suspension, where aggregation is observed. This work represents a step forward in using this tool in actual in vivo experimentation. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Inactivation and sublethal injury of Escherichia coli and Listeria innocua by high hydrostatic pressure in model suspensions and beetroot juice

NASA Astrophysics Data System (ADS)

Sokołowska, Barbara; Skąpska, Sylwia; Niezgoda, Jolanta; Rutkowska, Małgorzata; Dekowska, Agnieszka; Rzoska, Sylwester J.

2014-01-01

Cells exposed to different physical and chemical treatments, including high hydrostatic pressure (HHP), suffer from injuries that could be reversible in food materials when stored. Escherichia coli and Listeria innocua cells suspended in phosphate-buffered saline (PBS) (model suspensions), and acidified beetroot juice were subjected to a pressure of 400 MPa at a temperature of 20°C for up to 10 min. The difference between the viable and non-injured cells was used to estimate the number of injured survivors. The reduction in E. coli cell number was 3.4-4.1 log after 10 min pressurization in model suspensions and 6.2 log in beetroot juice. Sublethally injured cells in PBS accounted for up to 2.7 log after 10 min HHP treatment and 0.8 log in beetroot juice. The reduction in L. innocua cell number after 10 min pressure treatment reached from 3.8 to 4.8 log, depending on the initial concentration in model suspensions. Among the surviving L. innocua cells, even up to 100% were injured. L. innocua cells were completely inactivated after 1 min HHP treatment in beetroot juice.

Putting the Spotlight Back on Plant Suspension Cultures

PubMed Central

Santos, Rita B.; Abranches, Rita; Fischer, Rainer; Sack, Markus; Holland, Tanja

2016-01-01

Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso®)—the first licensed recombinant pharmaceutical protein derived from plants—is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plant cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models, and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon. PMID:27014320

Separation of cancer cells from a red blood cell suspension using inertial force.

PubMed

Tanaka, Tatsuya; Ishikawa, Takuji; Numayama-Tsuruta, Keiko; Imai, Yohsuke; Ueno, Hironori; Matsuki, Noriaki; Yamaguchi, Takami

2012-11-07

The circulating tumor cell (CTC) test has recently become popular for evaluating prognosis and treatment efficacy in cancer patients. The accuracy of the test is strongly dependent on the precision of the cancer cell separation. In this study, we developed a multistage microfluidic device to separate cancer cells from a red blood cell (RBC) suspension using inertial migration forces. The device was able to effectively remove RBCs up to the 1% hematocrit (Hct) condition with a throughput of 565 μL min(-1). The collection efficiency of cancer cells from a RBC suspension was about 85%, and the enrichment of cancer cells was about 120-fold. Further improvements can be easily achieved by parallelizing the device. These results illustrate that the separation of cancer cells from RBCs is possible using only inertial migration forces, thus paving the way for the development of a novel microfluidic device for future CTC tests.

Bioprocess Forces and Their Impact on Cell Behavior: Implications for Bone Regeneration Therapy

PubMed Central

Brindley, David; Moorthy, Kishaani; Lee, Jae-Ho; Mason, Chris; Kim, Hae-Won; Wall, Ivan

2011-01-01

Bioprocess forces such as shear stress experienced during routine cell culture are considered to be harmful to cells. However, the impact of physical forces on cell behavior is an area of growing interest within the tissue engineering community, and it is widely acknowledged that mechanical stimulation including shear stress can enhance osteogenic differentiation. This paper considers the effects of bioprocess shear stress on cell responses such as survival and proliferation in several contexts, including suspension-adapted cells used for recombinant protein and monoclonal antibody manufacture, adherent cells for therapy in suspension, and adherent cells attached to their growth substrates. The enhanced osteogenic differentiation that fluid flow shear stress is widely found to induce is discussed, along with the tissue engineering of mineralized tissue using perfusion bioreactors. Recent evidence that bioprocess forces produced during capillary transfer or pipetting of cell suspensions can enhance osteogenic responses is also discussed. PMID:21904661

Isolation, Identification, and Culture of Human Lymphatic Endothelial Cells.

PubMed

Lokmic, Zerina

2016-01-01

A protocol describing the isolation of foreskin lymphatic endothelial cells (LECs) and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented herein. To isolate LECs and LM LECs, tissues are mechanically disrupted to make a single-cell suspension, which is then enzymatically digested in dispase and collagenase type II. LECs and LM LECs, in the resulting single-cell suspension, are then sequentially labeled with antibodies recognizing fibroblast and endothelial cell surface antigens CD34 and CD31 and separated from the remaining components in the cell suspension by capture with magnetic beads. Viable LECs and LM LECs are then seeded and expanded on fibronectin-coated flasks. LEC and LM LEC purity is determined immunohistochemically using cell surface markers CD31, CD34, podoplanin, VEGFR-3 and nuclear marker PROX-1. Cells whose purity is >98 % are used for experiments between passage 4 and 6.

9 CFR 113.46 - Detection of cytopathogenic and/or hemadsorbing agents.

Code of Federal Regulations, 2012 CFR

2012-01-01

... volume of a 0.2 percent red blood cell suspension to uniformly cover the surface of the monolayer of cultured cells. Suspensions of washed guinea pig and chicken red blood cells shall be used. These... examine for hemadsorption. (4) If no hemadsorption is apparent, repeat step (b)(2) of this section and...

9 CFR 113.46 - Detection of cytopathogenic and/or hemadsorbing agents.

Code of Federal Regulations, 2011 CFR

2011-01-01

... volume of a 0.2 percent red blood cell suspension to uniformly cover the surface of the monolayer of cultured cells. Suspensions of washed guinea pig and chicken red blood cells shall be used. These... examine for hemadsorption. (4) If no hemadsorption is apparent, repeat step (b)(2) of this section and...

9 CFR 113.46 - Detection of cytopathogenic and/or hemadsorbing agents.

Code of Federal Regulations, 2013 CFR

2013-01-01

... volume of a 0.2 percent red blood cell suspension to uniformly cover the surface of the monolayer of cultured cells. Suspensions of washed guinea pig and chicken red blood cells shall be used. These... examine for hemadsorption. (4) If no hemadsorption is apparent, repeat step (b)(2) of this section and...

9 CFR 113.46 - Detection of cytopathogenic and/or hemadsorbing agents.

Code of Federal Regulations, 2014 CFR

2014-01-01

... volume of a 0.2 percent red blood cell suspension to uniformly cover the surface of the monolayer of cultured cells. Suspensions of washed guinea pig and chicken red blood cells shall be used. These... examine for hemadsorption. (4) If no hemadsorption is apparent, repeat step (b)(2) of this section and...

Bio-inactivation of human malignant cells through highly responsive diluted colloidal suspension of functionalized magnetic iron oxide nanoparticles

NASA Astrophysics Data System (ADS)

Ferreira, Roberta V.; Silva-Caldeira, Priscila P.; Pereira-Maia, Elene C.; Fabris, José D.; Cavalcante, Luis Carlos D.; Ardisson, José D.; Domingues, Rosana Z.

2016-04-01

Magnetic fluids, more specifically aqueous colloidal suspensions containing certain magnetic nanoparticles (MNPs), have recently been gaining special interest due to their potential use in clinical treatments of cancerous formations in mammalians. The technological application arises mainly from their hyperthermic behavior, which means that the nanoparticles dissipate heat upon being exposed to an alternating magnetic field (AMF). If the temperature is raised to slightly above 43 °C, cancer cells are functionally inactivated or killed; however, normal cells tend to survive under those same conditions, entirely maintaining their bioactivity. Recent in vitro studies have revealed that under simultaneous exposure to an AMF and magnetic nanoparticles, certain lines of cancer cells are bio-inactivated even without experiencing a significant temperature increase. This non-thermal effect is cell specific, indicating that MNPs, under alternating magnetic fields, may effectively kill cancer cells under conditions that were previously thought to be implausible, considering that the temperature does not increase more than 5 °C, which is also true in cases for which the concentration of MNPs is too low. To experimentally test for this effect, this study focused on the feasibility of inducing K562 cell death using an AMF and aqueous suspensions containing very low concentrations of MNPs. The assay was designed for a ferrofluid containing magnetite nanoparticles, which were obtained through the co-precipitation method and were functionalized with citric acid; the particles had an average diameter of 10 ± 2 nm and a mean hydrodynamic diameter of approximately 40 nm. Experiments were first performed to test for the ability of the ferrofluid to release heat under an AMF. The results show that for concentrations ranging from 2.5 to 1.0 × 103 mg L-1, the maximum temperature increase was actually less than 2 °C. However, the in vitro test results from K562 cells and suspensions containing these MNPs at concentrations varying within a narrower range from 2.5 to 10 mg L-1, typically under an AMF of 15 kA m-1 at 356 kHz, indicate efficient cytotoxic activity against malignant cells and inhibition of cell growth, even at very low hyperthermally induced temperature increases. The IC50 value varied with time, reaching 3.5 mg L-1 after 10 min under the AMF. Our results effectively demonstrate new prospective uses for such nanoparticles in advanced medical practices in oncology.

Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors.

PubMed

Lesch, H P; Laitinen, A; Peixoto, C; Vicente, T; Makkonen, K-E; Laitinen, L; Pikkarainen, J T; Samaranayake, H; Alves, P M; Carrondo, M J T; Ylä-Herttuala, S; Airenne, K J

2011-06-01

Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.

A system and methodology for high-content visual screening of individual intact living cells in suspension

NASA Astrophysics Data System (ADS)

Renaud, Olivier; Heintzmann, Rainer; Sáez-Cirión, Asier; Schnelle, Thomas; Mueller, Torsten; Shorte, Spencer

2007-02-01

Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

Effect of ultrasonic irradiation on mammalian cells and chromosomes in vitro

NASA Technical Reports Server (NTRS)

Roseboro, J. A.; Buchanan, P.; Norman, A.; Stern, R.

1978-01-01

Human peripheral blood and HeLa cells were irradiated in vitro at the ultrasonic frequency of 65 kHz. The whole blood and HeLa cell suspensions were exposed to continuous and pulsed ultrasonic power levels of 0.12, 0.16, 0.72, 1.12 and 2.24 W for a period of one minute. The method of ultrasonic irradiation was carried out with the whole blood or HeLa cell suspensions coupled directly to a cylindrical transducer while heating of the cell suspensions in excess of 41 C was avoided. Irradiated and unirradiated peripheral blood lymphocyte chromosome cultures were prepared and scored for selected numerical and morphological aberrations. There was no significant difference in the frequency of chromosomal aberrations between irradiated and unirradiated cells.

Detonation nanodiamond complexes with cancer stem cells inhibitors or paracrine products of mesenchymal stem cells as new potential medications

NASA Astrophysics Data System (ADS)

Konoplyannikov, A. G.; Alekseenskiy, A. E.; Zlotin, S. G.; Smirnov, B. B.; Kalsina, S. Sh.; Lepehina, L. A.; Semenkova, I. V.; Agaeva, E. V.; Baboyan, S. B.; Rjumshina, E. A.; Nosachenko, V. V.; Konoplyannikov, M. A.

2015-09-01

Combined use of complexes of the most active chemotherapeutic drugs and detonation nanodiamonds (DND) is a new trend in cancer therapy, which is probably related to selective chemotherapeutic drug delivery by DND to the zone of so-called cancer stem cells (CSC). Stable DND complexes of 4-5 nm size with salinomycin—a strong CSC inhibitor—have been obtained (as a suspension). It has been demonstrated that a complex administration considerably increases the drug antitumor effect on the transplantable tumor of LLC mice. A similar effect has been observed in CSC models in vivo, obtained by exposure of stem cells of normal mice tissues to a carcinogen 1,2-dimethylhydrazine. It has also been found out, that administration of DND complexes with the conditioned medium from mesenchymal stem cells (MSC) cultures to mice results in a considerable stimulation of stem cell pools in normal mice tissues, which can be used in regenerative medicine.

Two-signal electrochemical method for evaluation suppression and proliferation of MCF-7 cells based on intracellular purine.

PubMed

Li, Jinlian; Lin, Runxian; Wang, Qian; Gao, Guanggang; Cui, Jiwen; Liu, Jiguang; Wu, Dongmei

2014-07-01

Two electrochemical signals ascribed to xanthine/guanine and hypanthine/adenine in MCF-7 cells were detected at 0.726 and 1.053 V, respectively. Based on the intensity of signals, the genistein-induced proliferation and suppression of MCF-7 cells could be evaluated. The results showed that with the increase of genistein dose at the range of 10(-9) to 10(-6)M, the two electrochemical signals of MCF-7 cell suspension increased due to the proliferation, whereas the tendency at the high dosage range of more than 10(-5)M was decreased. The proliferation and cytotoxicity obtained by the electrochemical method were in agreement with those obtained by cell counting and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] method. Thus, the two-signal electrochemical method is an effective way to evaluate the effect of drugs on cell activity based on purine metabolism. Copyright © 2014 Elsevier Inc. All rights reserved.

The BioDyn facility on ISS: Advancing biomaterial production in microgravity for commercial applications

NASA Astrophysics Data System (ADS)

Myers, Niki; Wessling, Francis; Deuser, Mark; Anderson, C. D.; Lewis, Marian

1999-01-01

The primary goals of the BioDyn program are to foster use of the microgravity environment for commercial production of bio-materials from cells, and to develop services and processes for obtaining these materials through space processing. The scope of products includes commercial bio-molecules such as cytokines, other cell growth regulatory proteins, hormones, monoclonal antibodies and enzymes; transplantable cells or tissues which can be improved by low-G processes, or which cannot be obtained through standard processes in earth gravity; agriculture biotechnology products from plant cells; microencapsulation for diabetes treatment; and factors regulating cellular aging. To facilitate BioDyn's commercial science driven goals, hardware designed for ISS incorporates the flexibility for interchange between the different ISS facilities including the glovebox, various thermal units and centrifuges. By providing a permanent research facility, ISS is the critical space-based platform required by scientists for carrying out the long-term experiments necessary for developing bio-molecules and tissues using several cell culture modalities including suspension and anchorage-dependent cell types.

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes.

PubMed

Shafa, Mehdi; Krawetz, Roman; Zhang, Yuan; Rattner, Jerome B; Godollei, Anna; Duff, Henry J; Rancourt, Derrick E

2011-12-14

Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Murine D3-MHC-neo(r) ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells.

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

PubMed Central

2011-01-01

Background Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells. PMID:22168552

Isolation, growth, and metabolism of an obligately anaerobic, selenate- respiring bacterium, strain SES-3

USGS Publications Warehouse

Oremland, R.S.; Blum, J.S.; Culbertson, C.W.; Visscher, P.T.; Miller, L.G.; Dowdle, P.; Strohmaier, F.E.

1994-01-01

A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO2 with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Se0). Hence, SES-3 can carry out a complete reduction of selenate to Se0. Washed cell suspensions of selenate- grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of [14C]lactate to 14CO2 coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide m- chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Se0 by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [75Se]selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Se0. This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Se0 may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions.

Chlorogenic acid in a Nicotiana plumbaginifolia cell suspension.

PubMed

Gillet; Mesnard; Fliniaux; Monti; Fliniaux

1999-11-01

A phenylpropanoid compound has been characterized in a Nicotiana plumbaginifolia cell suspension. This compound has been isolated and purified by semi-preparative reverse phase-high performance liquid chromatography. Its structure has been identified by NMR spectroscopy as 5-O-caffeoylquinic acid, which is chlorogenic acid (CA). The influence of culture conditions on the accumulation of this metabolite by N. plumbaginifolia cell suspensions has been studied. Darkness strongly inhibits the CA accumulation. Moreover, it has been shown that feeding experiments with caffeic acid had a deleterious effect upon the CA content. This one was not influenced by a supplementation with quinic acid.

The development of cat testicular sperm cryopreservation protocols: Effects of tissue fragments or sperm cell suspension.

PubMed

Chatdarong, Kaywalee; Thuwanut, Paweena; Morrell, Jane M

2016-01-15

In endangered animals that have been found dead or sterilized for medical reasons, testis is the ultimate source of haploid DNA or sperm. Thus, preservation of testicular sperm may be performed to rescue their genetics. The aim of this study was to evaluate protocols for testicular sperm freezing: as tissue fragments or cell suspension in domestic cats as a model. A pair of testes from each cat (n = 9) were cut into eight equal pieces. Four randomly selected pieces were cryopreserved as: (1) tissue pieces using two-step freezing; (2) tissue pieces using a slow passive cooling device (CoolCell); (3) sperm suspension after single-layer centrifugation (SLC) through colloids; and (4) sperm suspension without being processed through SLC. A testicular piece from each cat served as fresh control. Testicular sperm membrane and DNA integrity were evaluated before, and after, the cryopreservation process. In addition, spermatogenic cell types (testicular sperm, spermatogonia, spermatocyte, and spermatid) present in the suspension samples were counted before and after SLC. The results found that testicular sperm membrane integrity in the suspension after SLC process was higher than that in the fragment form neither using the two-step nor CoolCell freezing, both before and after freezing (before freezing: 92.3 ± 3.4 vs. 81 ± 4.5 and 80.0 ± 7.0; after freezing: 84.5 ± 4.6 vs. 71.2 ± 12 and 76.2 ± 4.6; P ≤ 0.05). Testicular sperm DNA integrity was, however, not different among groups. Furthermore, the samples processed through the SLC had higher ration of sperm cells: other spermatogenic cells than those were not processed through the SLC (88.9 ± 3.8 vs. 30 ± 7.9; P ≤ 0.05). In summary, testicular sperm cryopreserved as a minced suspension is considered suitable in terms of preventing sperm membrane integrity, and SLC is considered a selection tool for enriching haploid sperm cells from castrated or postmortem cats. Copyright © 2016 Elsevier Inc. All rights reserved.

Feasibility and Safety of Intra-arterial Pericyte Progenitor Cell Delivery Following Mannitol-Induced Transient Blood-Brain Barrier Opening in a Canine Model.

PubMed

Youn, Sung Won; Jung, Keun-Hwa; Chu, Kon; Lee, Jong-Young; Lee, Soon-Tae; Bahn, Jae-jun; Park, Dong-Kyu; Yu, Jung-Suk; Kim, So-Yun; Kim, Manho; Lee, Sang Kun; Han, Moon-Hee; Roh, Jae-Kyu

2015-01-01

Stem cell therapy is currently being studied with a view to rescuing various neurological diseases. Such studies require not only the discovery of potent candidate cells but also the development of methods that allow optimal delivery of those candidates to the brain tissues. Given that the blood-brain barrier (BBB) precludes cells from entering the brain, the present study was designed to test whether hyperosmolar mannitol securely opens the BBB and enhances intra-arterial cell delivery. A noninjured normal canine model in which the BBB was presumed to be closed was used to evaluate the feasibility and safety of the tested protocol. Autologous adipose tissue-derived pericytes with platelet-derived growth factor receptor β positivity were utilized. Cells were administered 5 min after mannitol pretreatment using one of following techniques: (1) bolus injection of a concentrated suspension, (2) continuous infusion of a diluted suspension, or (3) bolus injection of a concentrated suspension that had been shaken by repeated syringe pumping. Animals administered a concentrated cell suspension without mannitol pretreatment served as a control group. Vital signs, blood parameters, neurologic status, and major artery patency were kept stable throughout the experiment and the 1-month posttreatment period. Although ischemic lesions were noted on magnetic resonance imaging in several mongrel dogs with concentrated cell suspension, the injection technique using repeated syringe shaking could avert this complication. The cells were detected in both ipsilateral and contralateral cortices and were more frequent at the ipsilateral and frontal locations, whereas very few cells were observed anywhere in the brain when mannitol was not preinjected. These data suggest that intra-arterial cell infusion with mannitol pretreatment is a feasible and safe therapeutic approach in stable brain diseases such as chronic stroke.

Properties of electrophoretic fractions of human embryonic kidney cells separated on space shuttle flight STS-8

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Lewis, M. L.; Barlow, G. H.; Todd, P. W.; Kunze, M. E.; Sarnoff, B. E.; Li, Z. K.

1985-01-01

Suspensions of cultured primary human embryonic kidney cells were subjected to continuous flow electrophoresis on Space Shuttle flight STS-8. The objectives of the experiments were to obtain electrophoretically separated fractions of the original cell populations and to test these fractions for the amount and kind of urokinase (a kidney plasminogen activator that is used medically for digesting blood clots), the morphologies of cells in the individual fractions, and their cellular electrophoretic mobilities after separation and subsequent proliferation. Individual fractions were successfully cultured after return from orbit, and they were found to differ substantially from one another and from the starting sample with respect to all of these properties.

Screening hypochromism (sieve effect) in red blood cells: a quantitative analysis

PubMed Central

Razi Naqvi, K.

2014-01-01

Multiwavelength UV-visible spectroscopy, Kramers-Kronig analysis, and several other experimental and theoretical tools have been applied over the last several decades to fathom absorption and scattering of light by suspensions of micron-sized pigmented particles, including red blood cells, but a satisfactory quantitative analysis of the difference between the absorption spectra of suspension of intact and lysed red blood cells is still lacking. It is stressed that such a comparison is meaningful only if the pertinent spectra are free from, or have been corrected for, scattering losses, and it is shown that Duysens’ theory can, whereas that of Vekshin cannot, account satisfactorily for the observed hypochromism of suspensions of red blood cells. PMID:24761307

Screening hypochromism (sieve effect) in red blood cells: a quantitative analysis.

PubMed

Razi Naqvi, K

2014-04-01

Multiwavelength UV-visible spectroscopy, Kramers-Kronig analysis, and several other experimental and theoretical tools have been applied over the last several decades to fathom absorption and scattering of light by suspensions of micron-sized pigmented particles, including red blood cells, but a satisfactory quantitative analysis of the difference between the absorption spectra of suspension of intact and lysed red blood cells is still lacking. It is stressed that such a comparison is meaningful only if the pertinent spectra are free from, or have been corrected for, scattering losses, and it is shown that Duysens' theory can, whereas that of Vekshin cannot, account satisfactorily for the observed hypochromism of suspensions of red blood cells.

Acid-growth response and alpha-expansins in suspension cultures of bright yellow 2 tobacco

NASA Technical Reports Server (NTRS)

Link, B. M.; Cosgrove, D. J.

1998-01-01

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of alpha-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple alpha-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

Media Screening for Obtaining Haematococcus pluvialis Red Motile Macrozooids Rich in Astaxanthin and Fatty Acids

PubMed Central

Butler, Thomas O.; McDougall, Gordon J.; Campbell, Raymond; Stanley, Michele S.; Day, John G.

2017-01-01

Astaxanthin from Haematococcus pluvialis is commercially produced in a two-stage process, involving green vegetative (macrozooid) and red aplanospore stages. This approach has been scaled up to an industrial process but constraints limit its commercial success and profitability, including: contamination issues, high pigment extraction costs, requirements for high light levels and photo-bleaching in the red stage. However, in addition to the aplanospore stage, this alga can produce astaxanthin in vegetative palmelloid and motile macrozooid cells. In this study, a two-stage process utilising different media in the green stage, with subsequent re-suspension in medium without nitrate was employed to optimise the formation of red motile macrozooids. Optimal growth in the green phase was obtained on cultivation under mixotrophic conditions in EG:JM media followed by re-suspension in medium without nitrate resulting in red motile macrozooids with an astaxanthin content of 2.74% (78.4% of total carotenoids) and a lipid content of 35.3% (rich in unsaturated fatty acids. It is envisaged that the red motile macrozooids could be harvested and fed as a whole-cell product directly in the animal feed and aquaculture sectors, or used as a blend of carotenoids and polyunsaturated fatty acids (PUFAs) in nutraceutical products. PMID:29278377

Media Screening for Obtaining Haematococcus pluvialis Red Motile Macrozooids Rich in Astaxanthin and Fatty Acids.

PubMed

Butler, Thomas O; McDougall, Gordon J; Campbell, Raymond; Stanley, Michele S; Day, John G

2017-12-26

Astaxanthin from Haematococcus pluvialis is commercially produced in a two-stage process, involving green vegetative (macrozooid) and red aplanospore stages. This approach has been scaled up to an industrial process but constraints limit its commercial success and profitability, including: contamination issues, high pigment extraction costs, requirements for high light levels and photo-bleaching in the red stage. However, in addition to the aplanospore stage, this alga can produce astaxanthin in vegetative palmelloid and motile macrozooid cells. In this study, a two-stage process utilising different media in the green stage, with subsequent re-suspension in medium without nitrate was employed to optimise the formation of red motile macrozooids. Optimal growth in the green phase was obtained on cultivation under mixotrophic conditions in EG:JM media followed by re-suspension in medium without nitrate resulting in red motile macrozooids with an astaxanthin content of 2.74% (78.4% of total carotenoids) and a lipid content of 35.3% (rich in unsaturated fatty acids. It is envisaged that the red motile macrozooids could be harvested and fed as a whole-cell product directly in the animal feed and aquaculture sectors, or used as a blend of carotenoids and polyunsaturated fatty acids (PUFAs) in nutraceutical products.

In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits.

PubMed

Keilhoff, G; Fansa, H; Schneider, W; Wolf, G

1999-07-01

In vivo predegeneration of peripheral nerves is presented as a convenient and effective method to obtain activated Schwann cells and an enhanced cell yield following in vitro cultivation. The experiments conducted in rats were aimed at clinical use in gaining Schwann cell suspensions for filling artificial conduits in order to bridge peripheral nerve gaps. The rat sciatic nerve used as a model was transected distally to the spinal ganglia. Predegeneration in vivo was allowed to take place for 1, 2, 3 and 4 days and up to 1, 2 and 3 weeks. The nerve was then resected and prepared for cell cultivation. Schwann cells cultivated from the contralateral untreated nerve served as control. Immunostaining for S100, nerve growth factor receptor and the adhesion molecules N-cadherin and L1 was used to characterize the general state of the cultures. Viability was assessed by fluorescein fluorescence staining, and the proliferation index was determined by bromodeoxyuridine-DNA incorporation. The Schwann cells from predegenerated nerves revealed an increased proliferation rate compared to the control, whereas fibroblast contamination was decreased. Best results were obtained 1 week after predegeneration.

Effects of granulocyte-macrophage colony-stimulating factor and interleukin 6 on the growth of leukemic blasts in suspension culture.

PubMed

Tsao, C J; Cheng, T Y; Chang, S L; Su, W J; Tseng, J Y

1992-05-01

We examined the stimulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL)-6 on the in vitro proliferation of leukemic blast cells from patients with acute leukemia. Bone marrow or peripheral blood leukemic blast cells were obtained from 21 patients, including 14 cases of acute myeloblastic leukemia (AML), four cases of acute lymphoblastic leukemia (ALL), two cases of acute undifferentiated leukemia, and one case of acute mixed-lineage leukemia. The proliferation of leukemic blast cells was evaluated by measuring the incorporation of 3H-thymidine into cells incubated with various concentrations of cytokines for 3 days. GM-CSF stimulated the DNA synthesis (with greater than 2.0 stimulation index) of blast cells in 9 of 14 (64%) AML cases, two cases of acute undifferentiated leukemia and one case of acute mixed-lineage leukemia. Only two cases of AML blasts responded to IL-6 to grow in the short-term suspension cultures. GM-CSF and IL-6 did not display a synergistic effect on the growth of leukemic cells. Moreover, GM-CSF and IL-6 did not stimulate the proliferation of ALL blast cells. Binding study also revealed the specific binding of GM-CSF on the blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia. Our results indicated that leukemic blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia possessed functional GM-CSF receptors.

Quantification of the fraction poorly deformable red blood cells using ektacytometry.

PubMed

Streekstra, G J; Dobbe, J G G; Hoekstra, A G

2010-06-21

We describe a method to obtain the fraction of poorly deformable red blood cells in a blood sample from the intensity pattern in an ektacytometer. In an ektacytometer red blood cells are transformed into ellipsoids by a shear flow between two transparent cylinders. The intensity pattern, due to a laser beam that is sent through the suspension, is projected on a screen. When measuring a healthy red blood cell population iso-intensity curves are ellipses with an axial ratio equal to that of the average red blood cell. In contrast poorly deformable cells result in circular iso-intensity curves. In this study we show that for mixtures of deformable and poorly deformable red blood cells, iso-intensity curves in the composite intensity pattern are neither elliptical nor circular but obtain cross-like shapes. We propose a method to obtain the fraction of poorly deformable red blood cells from those intensity patterns. Experiments with mixtures of poorly deformable and deformable red blood cells validate the method and demonstrate its accuracy. In a clinical setting our approach is potentially of great value for the detection of the fraction of sickle cells in blood samples of patients with sickle cell disease or to find a measure for the parasitemia in patients infected with malaria.

Simulations of molecular diffusion in lattices of cells: insights for NMR of red blood cells.

PubMed Central

Regan, David G; Kuchel, Philip W

2002-01-01

The pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance (NMR) experiment, conducted on a suspension of red blood cells (RBC) in a strong magnetic field yields a q-space plot consisting of a series of maxima and minima. This is mathematically analogous to a classical optical diffraction pattern. The method provides a noninvasive and novel means of characterizing cell suspensions that is sensitive to changes in cell shape and packing density. The positions of the features in a q-space plot characterize the rate of exchange across the membrane, cell dimensions, and packing density. A diffusion tensor, containing information regarding the diffusion anisotropy of the system, can also be derived from the PGSE NMR data. In this study, we carried out Monte Carlo simulations of diffusion in suspensions of "virtual" cells that had either biconcave disc (as in RBC) or oblate spheroid geometry. The simulations were performed in a PGSE NMR context thus enabling predictions of q-space and diffusion tensor data. The simulated data were compared with those from real PGSE NMR diffusion experiments on RBC suspensions that had a range of hematocrit values. Methods that facilitate the processing of q-space data were also developed. PMID:12080109

Simulations of molecular diffusion in lattices of cells: insights for NMR of red blood cells.

PubMed

Regan, David G; Kuchel, Philip W

2002-07-01

The pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance (NMR) experiment, conducted on a suspension of red blood cells (RBC) in a strong magnetic field yields a q-space plot consisting of a series of maxima and minima. This is mathematically analogous to a classical optical diffraction pattern. The method provides a noninvasive and novel means of characterizing cell suspensions that is sensitive to changes in cell shape and packing density. The positions of the features in a q-space plot characterize the rate of exchange across the membrane, cell dimensions, and packing density. A diffusion tensor, containing information regarding the diffusion anisotropy of the system, can also be derived from the PGSE NMR data. In this study, we carried out Monte Carlo simulations of diffusion in suspensions of "virtual" cells that had either biconcave disc (as in RBC) or oblate spheroid geometry. The simulations were performed in a PGSE NMR context thus enabling predictions of q-space and diffusion tensor data. The simulated data were compared with those from real PGSE NMR diffusion experiments on RBC suspensions that had a range of hematocrit values. Methods that facilitate the processing of q-space data were also developed.

Ex vivo biomechanical characterization of syringe-needle ejections for intracerebral cell delivery.

PubMed

Wahlberg, Brendon; Ghuman, Harmanvir; Liu, Jessie R; Modo, Michel

2018-06-15

Intracerebral implantation of cell suspensions is finding its clinical translation with encouraging results in patients with stroke. However, the survival of cells in the brain remains poor. Although the biological potential of neural stem cells (NSCs) is widely documented, the biomechanical effects of delivering cells through a syringe-needle remain poorly understood. We here detailed the biomechanical forces (pressure, shear stress) that cells are exposed to during ejection through different sized needles (20G, 26G, 32G) and syringes (10, 50, 250 µL) at relevant flow rates (1, 5, 10 µL/min). A comparison of 3 vehicles, Phosphate Buffered Saline (PBS), Hypothermosol (HTS), and Pluronic, indicated that less viscous vehicles are favorable for suspension with a high cell volume fraction to minimize sedimentation. Higher suspension viscosity was associated with greater shear stress. Higher flow rates with viscous vehicle, such as HTS reduced viability by ~10% and also produced more apoptotic cells (28%). At 5 µL/min ejection using a 26G needle increased neuronal differentiation for PBS and HTS suspensions. These results reveal the biological impact of biomechanical forces in the cell delivery process. Appropriate engineering strategies can be considered to mitigate these effects to ensure the efficacious translation of this promising therapy.

Electrochemical performance of solid oxide fuel cells having electrolytes made by suspension and solution precursor plasma spraying

NASA Astrophysics Data System (ADS)

Marr, M.; Kuhn, J.; Metcalfe, C.; Harris, J.; Kesler, O.

2014-01-01

Yttria-stabilized zirconia (YSZ) electrolytes were deposited by suspension plasma spraying (SPS) and solution precursor plasma spraying (SPPS). The electrolytes were evaluated for permeability, microstructure, and electrochemical performance. With SPS, three different suspensions were tested to explore the influence of powder size distribution and liquid properties. Electrolytes made from suspensions of a powder with d50 = 2.6 μm were more gas-tight than those made from suspensions of a powder with d50 = 0.6 μm. A peak open circuit voltage of 1.00 V was measured at 750 °C with a cell with an electrolyte made from a suspension of d50 = 2.6 μm powder. The use of a flammable suspension liquid was beneficial for improving electrolyte conductivity when using lower energy plasmas, but the choice of liquid was less important when using higher energy plasmas. With SPPS, peak electrolyte conductivities were comparable to the peak conductivities of the SPS electrolytes. However, leak rates through the SPPS electrolytes were higher than those through the electrolytes made from suspensions of d50 = 2.6 μm powder. The electrochemical test data on SPPS electrolytes are the first reported in the literature.

SUSPENSION CULTURE AND PLANT REGENERATION OF TYPHA LATIFOLIA

EPA Science Inventory

This study is the first reported attempt to generate a growth curve from Typha latifolia L. (broadleaf cattail) callus cells in suspension culture. Several media and hormone combinations were tested for their capacity to induce callus cell formation from T. latifolia leaf section...

Development, characterization, and optimization of a new suspension chicken-induced pluripotent stem cell line for the production of Newcastle disease vaccine

USDA-ARS?s Scientific Manuscript database

Traditionally, substrates for production of vaccines have been embryonated eggs or adherent cell culture. The daunting challenge of scaling up these technologies in the face of an outbreak has been a limitation for industrial applicability. Suspension cell lines are better suited in many ways to e...

Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspension-cultured cells by abscisic acid.

PubMed

Koike, M; Takezawa, D; Arakawa, K; Yoshida, S

1997-06-01

Suspension-cultured cells derived from immature embryos of winter wheat (Triticum aestivum L. cv. Chihoku) were used in experiments designed to obtain clues to the mechanism of the ABA-induced development of freezing tolerance. Cultured cells treated with 50 microM ABA for 5 d at 23 degrees C acquired the maximum level of freezing tolerance (LT50; -21.6 degrees C). The increased freezing tolerance of ABA-treated cells was closely associated with the remarkable accumulation of 19-kDa polypeptides in the plasma membrane. The 19-kDa polypeptide components were isolated by preparative gel electrophoresis and were further separated into one major (AWPM-19) and other minor polypeptide components by Tricine-SDS-PAGE. N-terminal amino acid sequence of AWPM-19 was determined, and a cDNA clone encoding AWPM-19 was isolated by PCR from the library prepared from the ABA-treated cultured cells. The cDNA clone (WPM-1) encoded a 18.9 kDa hydrophobic polypeptide with four putative membrane spanning domains and with a high pI value (10.2). Expression of WPM-1 mRNA was dramatically induced by 50 microM ABA within a few hours. These results suggest that the AWPM-19 might be closely associated with the ABA-induced increase in freezing tolerance in wheat cultured cells.

Comparison of hindlimb unloading and partial weight suspension models for spaceflight-type condition induced effects on white blood cells

NASA Astrophysics Data System (ADS)

Wilson, Jolaine M.; Krigsfeld, Gabriel S.; Sanzari, Jenine K.; Wagner, Erika B.; Mick, Rosemarie; Kennedy, Ann R.

2012-01-01

Animal models are frequently used to assist in the determination of the long- and short-term effects of space flight. The space environment, including microgravity, can impact many physiological and immunological system parameters. It has been found that ground based models of microgravity produce changes in white blood cell counts, which negatively affects immunologic function. As part of the Center of Acute Radiation Research (CARR), we compared the acute effects on white blood cell parameters induced by the more traditionally used animal model of hindlimb unloading (HU) with a recently developed reduced weightbearing analog known as partial weight suspension (PWS). Female ICR mice were either hindlimb unloaded or placed in the PWS system at 16% quadrupedal weightbearing for 4 h, 1, 2, 7 or 10 days, at which point complete blood counts were obtained. Control animals (jacketed and non-jacketed) were exposed to identical conditions without reduced weightbearing. Results indicate that significant changes in total white blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were observed within the first 2 days of exposure to each system. These differences in blood cell counts normalized by day 7 in both systems. The results of these studies indicate that there are some statistically significant changes observed in the blood cell counts for animals exposed to both the PWS and HU simulated microgravity systems.

Enhancement of tissue engineered bone formation by a low pressure system improving cell seeding and medium perfusion into a porous scaffold.

PubMed

Wang, Juyong; Asou, Yoshinori; Sekiya, Ichiro; Sotome, Shinichi; Orii, Hisaya; Shinomiya, Kenichi

2006-05-01

To obtain more extensive bone formation in composites of porous ceramics and bone marrow stromal cells (BMSCs), we hypothesized that a low-pressure system would serve to facilitate the perfusion of larger number of BMSCs into the porous scaffold, enhancing bone formation within the composites. After culturing BMSCs in osteogenic medium, porous blocks of beta-tricalcium phosphate (beta-TCP) were soaked in the cell suspension. Composites of the block and BMSCs were put immediately into a vacuum desiccator. Low pressure was applied to the low pressure group, while controls were left at atmospheric pressure. Composites were incubated in vitro or subcutaneously implanted into syngeneic rats, then analyzed biologically and histologically. In the in vitro group, cell suspension volume, cell seeding efficiency, alkaline phosphatase (ALP) activity, and DNA content in the beta-TCP blocks were significantly higher in low pressure group than in the controls. Scanning electron microscopy (SEM) demonstrated that a greater number of cells covered the central parts of the composites in the low pressure group. ALP activity in the composites was increased at 3 and 6 weeks after implantation into rats. Histomorphometric analysis revealed more uniform and extensive bone formation in the low pressure group than in the controls. The application of low pressure during the seeding of BMSCs in perfusing medium into a porous scaffold is useful for tissue-engineered bone formation.

Characterization of lentiviral vector production using microwell suspension cultures of HEK293T-derived producer cells.

PubMed

Guy, Heather M; McCloskey, Laura; Lye, Gary J; Mitrophanous, Kyriacos A; Mukhopadhyay, Tarit K

2013-04-01

ProSavin(®) is a lentiviral vector (LV)-based gene therapy for Parkinson's disease. ProSavin(®) is currently in a Phase I/II clinical trial using material that was generated by transient transfection of adherent human embryonic kidney (HEK)293T cells. For future large-scale productions of ProSavin(®), we have previously reported the development and characterization of two inducible producer cell lines, termed PS5.8 and PS46.2. PS46.2 has been successfully adapted to grow in suspension cultures. The present study describes the creation of a small-scale (<2 ml) microwell-based experimental platform for the parallel investigation of ProSavin(®) production using suspension-adapted PS46.2. This is combined with statistical design of experiments (DoE) techniques to enable rapid characterization of the process conditions that impact cell growth and LV production. The effects of postinduction period, microwell liquid fill volume, and concentration of inducer (doxycycline) on ProSavin(®) titer and the particle:infectivity (P:I) ratio was investigated using three rounds of DoE, in order to identify appropriate factor ranges and optimize production conditions. We identified an optimal "harvest window" between approximately 26-46 hr within which maximal titers of around 6×10(4) transducing units (TU)/ml were obtained (an approximately 30-fold improvement compared to starting microwell conditions), providing that the fill volume was maintained at or below 1 ml and the doxycycline concentration was at least 1.0 μg/ml. Insights from the microwell studies were subsequently used to rapidly establish operating conditions for ProSavin(®) production in a 0.5-L wave bioreactor culture. The information presented herein thus aids the design and evaluation of scalable production processes for LVs.

Nanoshell-mediated targeted photothermal therapy of HER2 human breast cancer cells using pulsed and continuous wave lasers: an in vitro study.

PubMed

Khosroshahi, Mohammad E; Hassannejad, Zahra; Firouzi, Masoumeh; Arshi, Ahmad R

2015-09-01

In this study, we report the apoptosis induction in HER2 overexpressed breast cancer cells using pulsed, continuous wave lasers and polyvinylpyrrolidone (PVP)-stabilized magneto-plasmonic nanoshells (PVP-MPNS) delivered by immunoliposomes. The immunoliposomes containing PVP-MPNS were fabricated and characterized. Heating efficiency of the synthesized nanostructures was calculated. The effect of functionalization on cellular uptake of nanoparticles was assessed using two cell lines of BT-474 and Calu-6. The best uptake result was achieved by functionalized liposome (MPNS-LAb) and BT-474. Also, the interaction of 514 nm argon (Ar) and Nd/YAG second harmonic 532-nm lasers with nanoparticles was investigated based on the temperature rise of the nanoshell suspension and the release value of 5(6)-carboxyfluorescein (CF) from CF/MPNS-loaded liposomes. The temperature increase of the suspensions after ten consecutive pulses of 532 nm and 5 min of irradiation by Ar laser were measured approximately 2 and 12 °C, respectively. The irradiation of CF/MPNS-loaded liposomes by Ar laser for 3 min resulted in 24.3 % release of CF, and in the case of 532 nm laser, the release was laser energy dependent. Furthermore, the comparison of CF release showed a higher efficiency for the Ar laser than by direct heating of nanoshell suspension using circulating water. The percentage of cell apoptosis after irradiation by Ar and 532 nm lasers were 44.6 and 42.6 %, respectively. The obtained results suggest that controlling the NP-laser interaction using optical properties of nanoshells and the laser parameters can be used to develop a new cancer therapy modality via targeted nanoshell and drug delivery.

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

PubMed

Dores, C; Rancourt, D; Dobrinski, I

2015-05-01

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling. © 2015 American Society of Andrology and European Academy of Andrology.

Startup of electrophoresis in a suspension of colloidal spheres.

PubMed

Chiang, Chia C; Keh, Huan J

2015-12-01

The transient electrophoretic response of a homogeneous suspension of spherical particles to the step application of an electric field is analyzed. The electric double layer encompassing each particle is assumed to be thin but finite, and the effect of dynamic electroosmosis within it is incorporated. The momentum equation for the fluid outside the double layers is solved through the use of a unit cell model. Closed-form formulas for the time-evolving electrophoretic and settling velocities of the particles in the Laplace transform are obtained in terms of the electrokinetic radius, relative mass density, and volume fraction of the particles. The time scale for the development of electrophoresis and sedimentation is significantly smaller for a suspension with a higher particle volume fraction or a smaller particle-to-fluid density ratio, and the electrophoretic mobility at any instant increases with an increase in the electrokinetic particle radius. The transient electrophoretic mobility is a decreasing function of the particle volume fraction if the particle-to-fluid density ratio is relatively small, but it may increase with an increase in the particle volume fraction if this density ratio is relatively large. The particle interaction effect in a suspension on the transient electrophoresis is much weaker than that on the transient sedimentation of the particles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells.

PubMed

Jiao, Jiao; Sun, Ling; Zhou, Benguo; Gao, Zhengliang; Hao, Yu; Zhu, Xiaoping; Liang, Yuancun

2014-08-15

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H2O2) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H2O2 accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H2O2 production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H2O2 accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

Nielsen, Nathalie; Laustsen, Christoffer; Bertelsen, Lotte Bonde, E-mail: Lotte@clin.au.dk

Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis. With the purpose of enhancing endothelial repair, EPCs could have a potential for future cell therapies. Due to the low amount of EPCs in the peripheral circulating blood, in vitro expansion is needed before administration to recipients and the effects of in vitro culturing is still an under-evaluated field with little knowledge of how the cells change over time in culture. The aim of this study was to use hyperpolarised carbon-13 magnetic resonance spectroscopy to profile important metabolic pathways in a population ofmore » progenitor cells and to show that cell culturing in 3D scaffolds seem to block the metabolic processes that leads to cell senescence. The metabolic breakdown of hyperpolarized [1-{sup 13}C]pyruvate was followed after injection of the substrate to a bioreactor system with EPCs either adhered to 3D printed scaffolds or kept in cell suspension. The pyruvate-to-lactate conversion was elevated in suspension of EPCs compared to the EPCs adhered to scaffolds. Furthermore in the setup with EPCs in suspension, an increase in lactate production was seen over time indicating that the older the cultures of EPCs was before using the cells for cell suspension experiments, the more lactate they produce, compared to a constant lactate level in the cells adhered to scaffolds. It could therefore be stated that cells grown first in 2D culture and subsequent prepared for cell suspension show a metabolism with higher lactate production consistent with cells senescence processes compared to cells grown first at 2D culture and subsequent in the 3D printed scaffolds, where metabolism shows no sign of metabolic shifting during the monitored period. - Highlights: • Hyperpolarized 13C MRS detects EPCs metabolic changes associated with ageing and cultivating conditions. • Increased lactate production in EPC’s correlates positively with aging. • 2D cultivation show an increased lactate production, while 3D cultivation show a maintained lactate production.« less

Influence of cell type and cell culture media on the propagation of foot-and-mouth disease virus with regard to vaccine quality.

PubMed

Dill, Veronika; Hoffmann, Bernd; Zimmer, Aline; Beer, Martin; Eschbaumer, Michael

2018-03-16

Suspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease. Changes to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation. Capsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A 24 -2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media. The selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization profile were unaffected, but the 146S particle yield differed for one of the tested serotypes.

Two-dimensional patterning of colloidal crystals by means of lateral autocloning in edge-patterned cells

NASA Astrophysics Data System (ADS)

Emoto, Akira; Kamei, Tadayoshi; Shioda, Tatsutoshi; Kawatsuki, Nobuhiro; Ono, Hiroshi

2009-06-01

We report the experimental results of two-dimensional patterning of colloidal crystals using edge-patterned cells. Solvent evaporation of a colloidal suspension from the edge of the cell induces self-organized crystallization of spherical colloidal particles. From a reservoir of colloidal suspension in the cell, different colloidal suspensions are injected repetitively. An edge-patterned substrate is introduced into the cell as an upper substrate. As a result, different colloidal crystals are alternately stacked in the lateral direction according to the edge pattern. The characteristics of cloning formation are specifically showed including deformations from the original pattern. This two-dimensional patterning of three-dimensional colloidal crystals by means of lateral autocloning is promising for the development of photonic crystal arrays for use in optic and photonic devices.

Dispersion of nanosized ceramic powders in aqueous suspensions

NASA Astrophysics Data System (ADS)

Chera, L.; Palcevskis, E.; Berzins, M.; Lipe, A.; Jansone, I.

2007-12-01

Seven commercially available dispersants have been applied to produce high concentrated aqueous suspensions of the nanosized alumina and partially stabilized zirconia powders processed by the plasma technique. Simultaneously, the electrokinetic behaviour of powders has been investigated in diluted suspensions by microelectrophoresis method. Zeta potential measurements are used to estimate the influence of selected dispersants on the electrokinetic properties of the powder surface. On the basis of obtained data the correlation between the surface electrokinetic properties in dilute suspensions and reached maximal suspension concentration is discussed.

Suspension survival mediated by PP2A-STAT3-Col XVII determines tumour initiation and metastasis in cancer stem cells

PubMed Central

Liu, Chen-Chi; Lin, Shih-Pei; Hsu, Han-Shui; Yang, Shung-Haur; Lin, Chiu-Hua; Yang, Muh-Hwa; Hung, Mien-Chie; Hung, Shih-Chieh

2016-01-01

Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previously demonstrated that TICs have the capacity to survive under suspension conditions, while other cells undergo anoikis. Here we show that TICs exhibit increased phosphorylation levels of S727STAT3 because of PP2A inactivation. Collagen 17 gene expression is upregulated in a STAT3-dependent manner, which also stabilizes laminin 5 and engages cells to form hemidesmosome-like junctions in response. Blocking the PP2A-S727STAT3-collagen 17 pathway inhibits the suspension survival of TICs and their ability to form tumours in mice, while activation of the same pathway increases the suspension survival and tumour-initiation capacities of bulk cancer cells. The S727STAT3 phosphorylation levels correlate with collagen 17 expression in colon tumour samples, and correlate inversely with survival. Finally, this signalling axis enhances the ability of TIC to form tumours in mouse models of malignant lung cancer pleural effusion and spontaneous colon cancer metastasis. PMID:27306323

Suspension survival mediated by PP2A-STAT3-Col XVII determines tumour initiation and metastasis in cancer stem cells.

PubMed

Liu, Chen-Chi; Lin, Shih-Pei; Hsu, Han-Shui; Yang, Shung-Haur; Lin, Chiu-Hua; Yang, Muh-Hwa; Hung, Mien-Chie; Hung, Shih-Chieh

2016-06-16

Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previously demonstrated that TICs have the capacity to survive under suspension conditions, while other cells undergo anoikis. Here we show that TICs exhibit increased phosphorylation levels of S727STAT3 because of PP2A inactivation. Collagen 17 gene expression is upregulated in a STAT3-dependent manner, which also stabilizes laminin 5 and engages cells to form hemidesmosome-like junctions in response. Blocking the PP2A-S727STAT3-collagen 17 pathway inhibits the suspension survival of TICs and their ability to form tumours in mice, while activation of the same pathway increases the suspension survival and tumour-initiation capacities of bulk cancer cells. The S727STAT3 phosphorylation levels correlate with collagen 17 expression in colon tumour samples, and correlate inversely with survival. Finally, this signalling axis enhances the ability of TIC to form tumours in mouse models of malignant lung cancer pleural effusion and spontaneous colon cancer metastasis.

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines.

PubMed

Binder, Zev A; Wilson, Kelli M; Salmasi, Vafi; Orr, Brent A; Eberhart, Charles G; Siu, I-Mei; Lim, Michael; Weingart, Jon D; Quinones-Hinojosa, Alfredo; Bettegowda, Chetan; Kassam, Amin B; Olivi, Alessandro; Brem, Henry; Riggins, Gregory J; Gallia, Gary L

2016-01-01

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants. Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.

Abiotic elicitation of gymnemic acid in the suspension cultures of Gymnema sylvestre.

PubMed

Ch, Bhuvaneswari; Rao, Kiranmayee; Gandi, Suryakala; Giri, Archana

2012-02-01

Elicitation is one of the few strategies that find commercial application in the enhancement of secondary metabolite production from plants as well as cell culture systems. Due to their immense medicinal value, production of saponins in suspension cultures has been attempted by many researchers. Gymnema sylvestre is a rich source of gymnemic acids (saponins) that find application in the treatment of diabetes. The present study is an attempt to evaluate the effect of various metal salts (cadmium chloride, mercuric chloride, silver nitrate, cupric chloride, cobaltous chloride and calcium chloride) in eliciting the response from G. sylvestre suspension cultures. The maximum gymnemic acid production in the suspensions was achieved on day 12 of culture, though the maximum biomass was obtained on day 16. Among the different salts, CdCl(2) gave maximum response (59.97 mg/gDCW) at 2 mM concentration after a 24 h time period, while, AgNO(3) gave the least response (18.35 mg/gDCW) on incubation of 48 h at 1 mM concentration, in terms of gymnemic acid accumulation. The accumulation of gymnemic acid was found to be dependent on treatment time and concentration of the elicitor. The enhanced gymnemic acid production shown by the suspensions in response to the metal salts indicates their role in evoking the plant defense mechanisms. These elicitation studies help in providing a platform for improved commercial supply of bioactive gymnemic acids.

Highly efficient Agrobacterium-mediated transformation of embryogenic cell suspensions of Musa acuminata cv. Mas (AA) via a liquid co-cultivation system.

PubMed

Huang, Xia; Huang, Xue-Lin; Xiao, Wang; Zhao, Jie-Tang; Dai, Xue-Mei; Chen, Yun-Feng; Li, Xiao-Ju

2007-10-01

A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0-490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.

Formation of Stable Cationic Lipid/DNA Complexes for Gene Transfer

NASA Astrophysics Data System (ADS)

Hofland, Hans E. J.; Shephard, Lee; Sullivan, Sean M.

1996-07-01

Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

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.

A dynamic method for magnetic torque measurement

NASA Technical Reports Server (NTRS)

Lin, C. E.; Jou, H. L.

1994-01-01

In a magnetic suspension system, accurate force measurement will result in better control performance in the test section, especially when a wider range of operation is required. Although many useful methods were developed to obtain the desired model, however, significant error is inevitable since the magnetic field distribution of the large-gap magnetic suspension system is extremely nonlinear. This paper proposed an easy approach to measure the magnetic torque of a magnetic suspension system using an angular photo encoder. Through the measurement of the velocity change data, the magnetic torque is converted. The proposed idea is described and implemented to obtain the desired data. It is useful to the calculation of a magnetic force in the magnetic suspension system.

Direct Visualization of the Hydration Layer on Alumina Nanoparticles with the Fluid Cell STEM in situ

PubMed Central

Firlar, Emre; Çınar, Simge; Kashyap, Sanjay; Akinc, Mufit; Prozorov, Tanya

2015-01-01

Rheological behavior of aqueous suspensions containing nanometer-sized powders is of relevance to many branches of industry. Unusually high viscosities observed for suspensions of nanoparticles compared to those of micron size powders cannot be explained by current viscosity models. Formation of so-called hydration layer on alumina nanoparticles in water was hypothesized, but never observed experimentally. We report here on the direct visualization of aqueous suspensions of alumina with the fluid cell in situ. We observe the hydration layer formed over the particle aggregates and show that such hydrated aggregates constitute new particle assemblies and affect the flow behavior of the suspensions. We discuss how these hydrated nanoclusters alter the effective solid content and the viscosity of nanostructured suspensions. Our findings elucidate the source of high viscosity observed for nanoparticle suspensions and are of direct relevance to many industrial sectors including materials, food, cosmetics, pharmaceutical among others employing colloidal slurries with nanometer-scale particles. PMID:25996055

Direct Visualization of the Hydration Layer on Alumina Nanoparticles with the Fluid Cell STEM in situ.

PubMed

Firlar, Emre; Çınar, Simge; Kashyap, Sanjay; Akinc, Mufit; Prozorov, Tanya

2015-05-21

Rheological behavior of aqueous suspensions containing nanometer-sized powders is of relevance to many branches of industry. Unusually high viscosities observed for suspensions of nanoparticles compared to those of micron size powders cannot be explained by current viscosity models. Formation of so-called hydration layer on alumina nanoparticles in water was hypothesized, but never observed experimentally. We report here on the direct visualization of aqueous suspensions of alumina with the fluid cell in situ. We observe the hydration layer formed over the particle aggregates and show that such hydrated aggregates constitute new particle assemblies and affect the flow behavior of the suspensions. We discuss how these hydrated nanoclusters alter the effective solid content and the viscosity of nanostructured suspensions. Our findings elucidate the source of high viscosity observed for nanoparticle suspensions and are of direct relevance to many industrial sectors including materials, food, cosmetics, pharmaceutical among others employing colloidal slurries with nanometer-scale particles.

Direct visualization of the hydration layer on alumina nanoparticles with the fluid cell STEM in situ

DOE PAGES

Firlar, Emre; Çınar, Simge; Kashyap, Sanjay; ...

2015-05-21

Rheological behavior of aqueous suspensions containing nanometer-sized powders is of relevance to many branches of industry. Unusually high viscosities observed for suspensions of nanoparticles compared to those of micron size powders cannot be explained by current viscosity models. Formation of so-called hydration layer on alumina nanoparticles in water was hypothesized, but never observed experimentally. We report here on the direct visualization of aqueous suspensions of alumina with the fluid cell in situ. We observe the hydration layer formed over the particle aggregates and show that such hydrated aggregates constitute new particle assemblies and affect the flow behavior of the suspensions.more » We discuss how these hydrated nanoclusters alter the effective solid content and the viscosity of nanostructured suspensions. As a result, our findings elucidate the source of high viscosity observed for nanoparticle suspensions and are of direct relevance to many industrial sectors including materials, food, cosmetics, pharmaceutical among others employing colloidal slurries with nanometer-scale particles.« less

The sol-gel route: A versatile process for up-scaling the fabrication of gas-tight thin electrolyte layers

NASA Astrophysics Data System (ADS)

Viazzi, Céline; Rouessac, Vincent; Lenormand, Pascal; Julbe, Anne; Ansart, Florence; Guizard, Christian

2011-03-01

Sol-gel routes are often investigated and adapted to prepare, by suitable chemical modifications, submicronic powders and derived materials with controlled morphology, which cannot be obtained by conventional solid state chemistry paths. Wet chemistry methods provide attractive alternative routes because mixing of species occurs at the atomic scale. In this paper, ultrafine powders were prepared by a novel synthesis method based on the sol-gel process and were dispersed into suspensions before processing. This paper presents new developments for the preparation of functional materials like yttria-stabilized-zirconia (YSZ, 8% Y2O3) used as electrolyte for solid oxide fuel cells. YSZ thick films were coated onto porous Ni-YSZ substrates using a suspension with an optimized formulation deposited by either a dip-coating or a spin-coating process. The suspension composition is based on YSZ particles encapsulated by a zirconium alkoxide which was added with an alkoxide derived colloidal sol. The in situ growth of these colloids increases significantly the layer density after an appropriated heat treatment. The derived films were continuous, homogeneous and around 20 μm thick. The possible up-scaling of this process has been also considered and the suitable processing parameters were defined in order to obtain, at an industrial scale, homogeneous, crack-free, thick and adherent films after heat treatment at 1400 °C.

Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells.

PubMed

Tsuji, Kunikazu; Ojima, Miyoko; Otabe, Koji; Horie, Masafumi; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

2017-06-09

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a nonenzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

Dilatancy and shear thickening of particle suspensions

NASA Astrophysics Data System (ADS)

Bonn, Daniel

2013-03-01

Shear thickening is a fascinating subject, as 99.9% of complex fluids are thinning; thickening systems thus are the ``exception to the rule'' that needs to be understood. Moreover, such tunable systems show very promising applications, e.g. to block large underground pores in oil recovery to maintain a constant oil flow by plugging water filled pores (an approach used in oil recovery by e.g. Shell), or to manufacture bulletproof vests that are comfortable to wear, but stop bullets nonetheless. We study the rheology of non-Brownian particle suspensions (notably, cornstarch) that exhibit shear thickening. Using magnetic resonance imaging (MRI), the local properties of the flow are obtained by the determination of local velocity profiles and concentrations in a Couette cell. We also perform macroscopic rheology experiments in different geometries. The results suggest that the shear thickening is a consequence of dilatancy: the system under flow attempts to dilate but instead undergoes a jamming transition, because it is confined. This proposition is confirmed by an independent measurement of the dilation of the suspension as a function of the shear rate.

Drop-on-Demand Single Cell Isolation and Total RNA Analysis

PubMed Central

Moon, Sangjun; Kim, Yun-Gon; Dong, Lingsheng; Lombardi, Michael; Haeggstrom, Edward; Jensen, Roderick V.; Hsiao, Li-Li; Demirci, Utkan

2011-01-01

Technologies that rapidly isolate viable single cells from heterogeneous solutions have significantly contributed to the field of medical genomics. Challenges remain both to enable efficient extraction, isolation and patterning of single cells from heterogeneous solutions as well as to keep them alive during the process due to a limited degree of control over single cell manipulation. Here, we present a microdroplet based method to isolate and pattern single cells from heterogeneous cell suspensions (10% target cell mixture), preserve viability of the extracted cells (97.0±0.8%), and obtain genomic information from isolated cells compared to the non-patterned controls. The cell encapsulation process is both experimentally and theoretically analyzed. Using the isolated cells, we identified 11 stem cell markers among 1000 genes and compare to the controls. This automated platform enabling high-throughput cell manipulation for subsequent genomic analysis employs fewer handling steps compared to existing methods. PMID:21412416

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

PubMed

Pochechueva, Tatiana; Alam, Shahidul; Schötzau, Andreas; Chinarev, Alexander; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2017-02-10

Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

Iron induction of ferritin synthesis in soybean cell suspensions.

PubMed

Proudhon, D; Briat, J F; Lescure, A M

1989-06-01

In animal cells specialized for iron storage, iron-induced accumulation of ferritin is known to result from a shift of stored mRNA from the ribonucleoprotein fraction to polysomes. Previous reports with bean leaves suggested that in plants iron induction of ferritin synthesis would result from a regulation at the transcriptional level (F van der Mark, F Bienfait, H van der Ende [1983] Biochem Biophys Res Commun 115:463-469). Soybean (Glycine max, cv Mandarin) cell suspension cultures have been used here to support these findings. Ferritin induction is obtained by addition of Fe-citrate to the culture medium. A good correlation is found between cellular iron content and the amount of ferritin accumulation. This protein accumulation corresponds to an increase of in vitro translatable ferritin mRNA. Addition of 4 micrograms actinomycin D per milliliter to the cultures inhibits completely in vivo RNA synthesis, whereas protein synthesis was poorly affected, at least for 24 hours. During the same time, this concentration of actinomycin D strongly inhibits the iron-induced synthesis of ferritin. These results show that in soybean cell cultures, the mechanism of regulation of ferritin synthesis in response to iron does not result from recruitment of preexisting mRNA. They confirm that in plant systems, ferritin synthesis results from increased transcription of the corresponding genes.

Determination of tenogenic differentiation in human mesenchymal stem cells by terahertz waves for measurement of the optical property of cellular suspensions

NASA Astrophysics Data System (ADS)

Morita, Yasuyuki; Azuchi, Kosuke; Ju, Yang; Suzuki, Satoshi; Xu, Baiyao; Yamamoto, Shuhei

2014-06-01

Technology for identifying stem cell-to-tenocyte differentiation that is non-contact and non-destructive in vitro is essential in tissue engineering. It has been found that expression of various RNA and proteins produced by differentiated cells is elevated when human bone marrow mesenchymal stem cells (hBMSCs) differentiate into tenocytes. Also, such biomolecules have absorption bands in the terahertz range. Thus, we attempted to evaluate whether terahertz waves could be used to distinguish hBMSC-to-tenocyte differentiation. Terahertz time-domain spectroscopy (THz-TDS) using femtosecond laser pulses was used for terahertz measurements. HBMSCs differentiated into tenocytes with mechanical stimulation: 10% cyclical uniaxial stretching at 1 Hz for 24 or 48 h. Cellular suspensions before and after differentiation were measured with terahertz waves. Complex refractive index, consisting of a refractive index (real) and an extinction coefficient (imaginary) obtained from the transmitted terahertz signals, was evaluated before and after differentiation at 1.0 THz. As a result, the THz-TDS system enabled discrimination of hBMSC-to-tenocyte differentiation due to the marked contrast in optical parameter before and after differentiation. This is the first report of the potential of a THz-TDS system for the detection of tenogenic differentiation using a non-contact and non-destructive in vitro technique.

Hindlimb suspension reduces muscle regeneration

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Truong, Q.; Macius, A.; Schultz, E.

1998-01-01

Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P < 0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P < 0.05) BrdU labeling in uninjured soleus muscles compared to weight-bearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P > 0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers.

Cosmos: 1989 immunology studies

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald

1991-01-01

The effects of flight on Cosmos mission 2044 on leukocyte subset distribution and the sensitivity of bone marrow cells to colony stimulating factor-GM were determined. A parallel study with antiorthostatic suspension was also carried out. The study involved repetition and expansion of studies performed on Cosmos 1887. Spleen and bone marrow cells were obtained from flown, vivarium control, synchronous control, and suspended rats. The cells were stained with a series of monoclonal antibodies directed against rat leukocyte cell surface antigens. Control cells were stained with a monoclonal antibody directed against an irrelevant species or were unstained. Cells were then analyzed for fluorescence using a FACSCAN flow cytometer. Bone marrow cells were placed in culture with GM-CSF in McCoy's 5a medium and incubated for 5 days. Cultures were then evaluated for the number of colonies of 50 cells or greater.

Effects of method of detachment on electrophoretic mobility of mammalian cells grown in monolayer culture

NASA Technical Reports Server (NTRS)

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

1985-01-01

A variety of proteolytic and micolytic enzumes, mechanical procedures, and changes in the ionic environment, especially Ca chelation, are used for dispersal of monolayer grown cells. If either chelating agents or mechanical dispersion are used alone, the cell yield is often low and suspensions of single cells are difficult to obtain. Confluent monolayers treated with EDTA tend to be released from their surfaces in sheets, and clumps of cells remain even after further incubation in EDTA. Crude trypsin is the most popular dispersal agent and is known to contain a variety of contaminating enzymes which contribute to the dispersal of cells. A variety of cell injuries resulting from the activity of proteolytic enzymes are reported. It is shown that crystalline trypsin is least harmful to cell integrity as judged by trypan blue uptake.

Gamma-irradiated bacterial preparation having anti-tumor activity

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

Vass, A.A.; Tyndall, R.L.; Terzaghi-Howe, P.

1999-11-16

This application describes a bacterial preparation from Pseudomonas species isolated {number{underscore}sign}15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

Co2 + interaction with Azospirillum brasilense Sp7 cells: a 57Co emission Mössbauer spectroscopic study

NASA Astrophysics Data System (ADS)

Kamnev, Alexander A.; Tugarova, Anna V.; Biró, Borbála; Kovács, Krisztina; Homonnay, Zoltán; Kuzmann, Ernő; Vértes, Attila

2012-03-01

Preliminary 57Co emission Mössbauer spectroscopic data were obtained for the soil bacterium Azospirillum brasilense Sp7 ( T = 80 K) in frozen 57Co2 + -containing suspensions and in their dried residues. The Mössbauer parameters were compared with those for A. brasilense strain Sp245 differing from strain Sp7 by ecological behaviour. Live cells of both strains showed metabolic transformations of 57Co2 + within an hour. Differences in the parameters observed for the two strains under similar conditions suggest dissimilarities in their metabolic response to Co2 + .

Oxidative stress, cytoxicity, and cell mortality induced by nano-sized lead in aqueous suspensions.

PubMed

Cornejo-Garrido, Hilda; Kibanova, Daria; Nieto-Camacho, Antonio; Guzmán, José; Ramírez-Apan, Teresa; Fernández-Lomelín, Pilar; Garduño, Maria Laura; Cervini-Silva, Javiera

2011-09-01

This paper reports on the effect of aqueous and nano-particulated Pb on oxidative stress (lipid peroxidation), cytoxicity, and cell mortality. As determined by the Thiobarbituric Acid Reactive Substances (TBARS) method, only 6h after incubation aqueous suspensions bearing nano-sized PbO(2), soluble Pb(II), and brain-homogenate only suspensions, were determined to contain as much as ca. 7, 5, and 1 nmol TBARS mg protein(-1), respectively. Exposure of human cells (central nervous system, prostate, leukemia, colon, breast, lung cells) to nano-PbO(2) led to cell-growth inhibition values (%) ca. ≤18.7%. Finally, as estimated by the Artemia salina test, cell mortality values were found to show high-survival larvae rates. Microscopic observations revealed that Pb particles were swallowed, but caused no mortality, however. Copyright © 2011 Elsevier Ltd. All rights reserved.

Isolation and functional characteristics of adherent phagocytic cells from mouse Peyer's patches.

PubMed Central

MacDonald, T T; Carter, P B

1982-01-01

Attempts were made to isolate adherent phagocytic cells (macrophages) from mouse Peyer's patch cell suspensions. Cell suspensions prepared by teasing apart the Peyer's patches contained no adherent phagocytic cells. However, if Peyer's patch fragments were treated with collagenase to disrupt the tissue matrix, cells prepared in this way contained a subpopulation of adherent phagocytic cells. These cells comprised only 0.1-0.2% of the total nucleated cell population of the Peyer's patch. Similar cells could also be isolated from the Peyer's patches of germ-free mice, but as judged by their ability to ingest opsonized erythrocytes, these cells were less activated than cells from the Peyer's patches of normal mice. Adherent cells from the Peyer's patches of normal mice could present antigen (ovalbumin) to T cells, and Peyer's patches cell suspensions containing adherent cells could be stimulated in vitro to produce an anti-sheep red blood cell plaque-forming cell response in the absence of 2-mercaptoethanol. These studies show that although the frequency of phagocytic adherent cells is extremely low in Peyer's patches, these cells have functions consistent with that of adherent cells in other lymphoid tissues. PMID:7068173

Antiorthostatic suspension as a model for the effects of spaceflight on the immune system

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Mastro, A. M.; Sonnenfeld, G.; Berry, W. D.

1993-01-01

We describe the use and appropriateness of antiorthostatic suspension in immunological investigations. This manuscript describes the model and discusses how well data obtained by using the model correlate with spaceflight data. This review concludes with some suggestions for future experiments using antiorthostatic suspension.

Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station

PubMed Central

Fitzgerald, Wendy; Chen, Silvia; Walz, Carl; Zimmerberg, Joshua; Margolis, Leonid

2013-01-01

The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction. PMID:19609626

Apparent Inhibition of β-Fructosidase Secretion by Tunicamycin May Be Explained by Breakdown of the Unglycosylated Protein during Secretion 1

PubMed Central

Faye, Loïc; Chrispeels, Maarten J.

1989-01-01

Suspension-cultured carrot (Daucus carota) cells synthesize and secrete β-fructosidase, a glycoprotein with asparagine-linked glycans. Treatment of the cells with tunicamycin completely inhibits the apparent secretion of β-fructosidase as measured by the accumulation of the radioactive protein in the cell wall or the culture medium. In the past, such a result has been interpreted as an inhibition of secretion by tunicamycin, but we suggest another explanation based on the following results. In the presence of tunicamycin, unglycosylated β-fructosidase is synthesized and is associated with an endoplasmic-reticulum-rich microsomal fraction. Pulse-chase experiments show that the unglycosylated β-fructosidase does not remain in the cells and appears to be secreted in the same way as glycosylated β-fructosidase; however, no radioactive, unglycosylated β-fructosidase accumulates extracellularly (cell wall or medium). Protoplasts obtained from carrot cells secrete β-fructosidase protein and activity, and treatment of the protoplasts with tunicamycin results in the synthesis of unglycosylated β-fructosidase. In the presence of tunicamycin, there is no accumulation of β-fructosidase activity or unglycosylated β-fructosidase polypeptide in the protoplast incubation medium. These results are consistent with the interpretation that the glycans of β-fructosidase are necessary for its stability, and that in these suspension-cultured cells, the unglycosylated enzyme is degraded during the last stage(s) of secretion, or immediately after its arrival in the wall. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:16666631

Apparent inhibition of. beta. -fructosidase secretion by tunicamycin may be explained by breakdown of the unglycosylated protein during secretion. [Daucus carota

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

Faye, L.; Chrispeels, M.J.

1989-03-01

Suspension-cultured carrot (Daucus carota) cells synthesize and secrete {beta}-fructosidase, a glycoprotein with asparagine-linked glycans. Treatment of the cells with tunicamycin completely inhibits the apparent secretion of {beta}-fructosidase as measured by the accumulation of the {sup 35}S-labelled protein in the cell wall or the culture medium. In the past, such a result has been interpreted as an inhibition of secretion by tunicamycin, but we suggest another explanation based on the following results. In the presence of tunicamycin, unglycosylated {beta}-fructosidase is synthesized and is associated with an endoplasmic-reticulum-rich microsomal fraction. Pulse-chase experiments show that the unglycosylated {beta}-fructosidase does not remain in themore » cells and appears to be secreted in the same way as glycosylated {beta}-fructosidase; however, no radioactive, unglycosylated {beta}-fructosidase accumulates extracellularly (cell wall or medium). Protoplasts obtained from carrot cells secrete {beta}-fructosidase protein and activity, and treatment of the protoplasts with tunicamycin results in the synthesis of unglycosylated {beta}-fructosidase. In the presence of tunicamycin, there is no accumulation of {beta}-fructosidase activity or unglycosylated {beta}-fructosidase polypeptide in the protoplast incubation medium. These results are consistent with the interpretation that the glycans of {beta}-fructosidase are necessary for its stability, and that in these suspension-cultured cells, the unglycosylated enzyme is degraded during the last stage(s) of secretion, or immediately after its arrival in the wall.« less

Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect.

PubMed

Koga, Hideyuki; Shimaya, Masayuki; Muneta, Takeshi; Nimura, Akimoto; Morito, Toshiyuki; Hayashi, Masaya; Suzuki, Shiro; Ju, Young-Jin; Mochizuki, Tomoyuki; Sekiya, Ichiro

2008-01-01

Current cell therapy for cartilage regeneration requires invasive procedures, periosteal coverage and scaffold use. We have developed a novel transplantation method with synovial mesenchymal stem cells (MSCs) to adhere to the cartilage defect. For ex vivo analysis in rabbits, the cartilage defect was faced upward, filled with synovial MSC suspension, and held stationary for 2.5 to 15 minutes. The number of attached cells was examined. For in vivo analysis in rabbits, an autologous synovial MSC suspension was placed on the cartilage defect, and the position was maintained for 10 minutes to adhere the cells to the defect. For the control, either the same cell suspension was injected intra-articularly or the defects were left empty. The three groups were compared macroscopically and histologically. For ex vivo analysis in humans, in addition to the similar experiment in rabbits, the expression and effects of neutralizing antibodies for adhesion molecules were examined. Ex vivo analysis in rabbits demonstrated that the number of attached cells increased in a time-dependent manner, and more than 60% of cells attached within 10 minutes. The in vivo study showed that a large number of transplanted synovial MSCs attached to the defect at 1 day, and the cartilage defect improved at 24 weeks. The histological score was consistently better than the scores of the two control groups (same cell suspension injected intra-articularly or defects left empty) at 4, 12, and 24 weeks. Ex vivo analysis in humans provided similar results to those in rabbits. Intercellular adhesion molecule 1-positive cells increased between 1 minute and 10 minutes, and neutralizing antibodies for intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and activated leukocyte-cell adhesion molecule inhibited the attachment. Placing MSC suspension on the cartilage defect for 10 minutes resulted in adherence of >60% of synovial MSCs to the defect, and promoted cartilage regeneration. This adherent method makes it possible to adhere MSCs with low invasion, without periosteal coverage, and without a scaffold.

Stem Cell Microencapsulation for Phenotypic Control, Bioprocessing, and Transplantation

PubMed Central

Wilson, Jenna L.

2014-01-01

Cell microencapsulation has been utilized for decades as a means to shield cells from the external environment while simultaneously permitting transport of oxygen, nutrients, and secretory molecules. In designing cell therapies, donor primary cells are often difficult to obtain and expand to appropriate numbers, rendering stem cells an attractive alternative due to their capacities for self-renewal, differentiation, and trophic factor secretion. Microencapsulation of stem cells offers several benefits, namely the creation of a defined microenvironment which can be designed to modulate stem cell phenotype, protection from hydrodynamic forces and prevention of agglomeration during expansion in suspension bioreactors, and a means to transplant cells behind a semi-permeable barrier, allowing for molecular secretion while avoiding immune reaction. This review will provide an overview of relevant microencapsulation processes and characterization in the context of maintaining stem cell potency, directing differentiation, investigating scalable production methods, and transplanting stem cells for clinically relevant disorders. PMID:23239279

Mössbauer spectroscopic study of 57Fe metabolic transformations in the rhizobacterium Azospirillum brasilense Sp245

NASA Astrophysics Data System (ADS)

Kamnev, Alexander A.; Tugarova, Anna V.; Kovács, Krisztina; Biró, Borbála; Homonnay, Zoltán; Kuzmann, Ernő

2014-04-01

Preliminary 57Fe transmission Mössbauer spectroscopic data were obtained for the first time for live cells of the plant-growth-promoting rhizobacterium Azospirillum brasilense (wild-type strain Sp245) grown aerobically with 57FeIII-nitrilotriacetate (NTA) complex as a sole source of iron. The results obtained have shown that live cells actively reduce part of the assimilated iron(III) to iron(II), the latter amounting up to 33 % of total cellular iron after 18 h of growth, and 48 % after additional 3 days of storage of the dense wet cell suspension in nutrient-free saline solution in air at room temperature (measured at 80 K). The cellular iron(II) was found to be represented by two quadrupole doublets of different high-spin forms, while the parameters of the cellular iron(III) were close to those typical for bacterioferritins.

Bioactive and biocompatible pieces of HA/sol-gel glass mixtures obtained by the gel-casting method.

PubMed

Padilla, S; Sánchez-Salcedo, S; Vallet-Regí, M

2005-10-01

Hydroxyapatite (HA)/glass mixtures have shown a faster bioactive behaviour than HA itself. On the other hand, the gel-casting method is a simple and reproducible colloidal method to produce ceramic pieces with complex shapes. In this work, pieces of HA/glass mixtures were prepared by the gel-casting method. A study for obtaining concentrated slurries of these mixtures is reported; the bioactivity and biocompatibility of the obtained pieces have been studied also. The influence of pH, dispersant concentration, the content and milling of glass, and the way to prepare the suspensions were investigated. The lowest viscosity and better rheological properties were achieved with the lowest glass content, when the glass was added after the dispersion of the HA powder and when the glass was not milled after calcination. Fluid suspensions with a high solid content (50 vol.%) could be prepared and well-shaped pieces were obtained from these slurries. These pieces showed in vitro bioactive behavior in simulated body fluid; additionally, the proliferation and spreading assays with osteoblastic cells (HOS) showed that the pieces are biocompatible. The results obtained indicate that the gel-casting of HA/glass mixtures produces bioactive and biocompatible pieces with the required shapes. Therefore, these materials could be good candidates for clinical applications and scaffolds for tissue engineering. (c) 2005 Wiley Periodicals, Inc.

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

PubMed

Miki, Hideo; Takagi, Mutsumi

2015-08-01

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

Graphene as a nanocarrier for tamoxifen induces apoptosis in transformed cancer cell lines of different origins.

PubMed

Misra, Santosh K; Kondaiah, Paturu; Bhattacharya, Santanu; Rao, C N R

2012-01-09

A cationic amphiphile, cholest-5en-3β-oxyethyl pyridinium bromide (PY(+) -Chol), is able to efficiently disperse exfoliated graphene (GR) in water by the physical adsorption of PY(+) -Chol on the surface of GR to form stable, dark aqueous suspensions at room temperature. The GR-PY(+) -Chol suspension can then be used to solubilize Tamoxifen Citrate (TmC), a breast cancer drug, in water. The resulting TmC-GR-PY(+) -Chol is stable for a long time without any precipitation. Fluorescence emission and UV absorption spectra indicate the existence of noncovalent interactions between TmC, GR, and PY(+) -Chol in these suspensions. Electron microscopy shows the existence of segregated GR sheets and TmC 'ribbons' in the composite suspensions. Atomic force microscopy indicates the presence of 'extended' structures of GR-PY(+) -Chol, which grows wider in the presence of TmC. The slow time-dependent release of TmC is noticed in a reconstituted cell culture medium, a property useful as a drug carrier. TmC-GR-PY(+) -Chol selectively enhanced the cell death (apoptosis) of the transformed cancer cells compared to normal cells. This potency is found to be true for a wide range of transformed cancer cells viz. HeLa, A549, ras oncogene-transformed NIH3T3, HepG2, MDA-MB231, MCF-7, and HEK293T compared to the normal cell HEK293 in vitro. Confocal microscopy confirmed the high efficiency of TmC-GR-PY(+) -Chol in delivering the drug to the cells, compared to the suspensions devoid of GR. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of indigenous bacterial strains for biocontrol of the frogeye leaf spot of soya bean caused by Cercospora sojina.

PubMed

Simonetti, E; Carmona, M A; Scandiani, M M; García, A F; Luque, A G; Correa, O S; Balestrasse, K B

2012-08-01

Assessment of biological control of Cercospora sojina, causal agent of frogeye leaf spot (FLS) of soya bean, using three indigenous bacterial strains, BNM297 (Pseudomonas fluorescens), BNM340 and BNM122 (Bacillus amyloliquefaciens). From cultures of each bacterial strain, cell suspensions and cell-free supernatants were obtained and assayed to determine their antifungal activity against C. sojina. Both mycelial growth and spore germination in vitro were more strongly inhibited by bacterial cell suspensions than by cell-free supernatants. The Bacillus strains BNM122 and BNM340 inhibited the fungal growth to a similar degree (I ≈ 52-53%), while cells from P. fluorescens BNM297 caused a lesser reduction (I ≈ 32-34%) in the fungus colony diameter. The foliar application of the two Bacillus strains on soya bean seedlings, under greenhouse conditions, significantly reduced the disease severity with respect to control soya bean seedlings and those sprayed with BNM297. This last bacterial strain was not effective in controlling FLS in vivo. Our data demonstrate that the application of antagonistic bacteria may be a promising and environmentally friendly alternative to control the FLS of soya bean.   To our knowledge, this is the first report of biological control of C. sojina by using native Bacillus strains. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Assessment of Cultivation Factors that Affect Biomass and Geraniol Production in Transgenic Tobacco Cell Suspension Cultures

PubMed Central

Vasilev, Nikolay; Schmitz, Christian; Grömping, Ulrike; Fischer, Rainer; Schillberg, Stefan

2014-01-01

A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources. PMID:25117009

Assessment of cultivation factors that affect biomass and geraniol production in transgenic tobacco cell suspension cultures.

PubMed

Vasilev, Nikolay; Schmitz, Christian; Grömping, Ulrike; Fischer, Rainer; Schillberg, Stefan

2014-01-01

A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼ 5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.

[The mutagenic action of the dust of natural zeolites and chrysotile asbestos].

PubMed

Durnev, A D; Suslova, T B; Cheremisina, Z P; Dubovskaia, O Iu; Nigarova, E A; Korkina, L G; Seredenin, S B; Velichkovskiĭ, B T

1990-01-01

The cell chemiluminescence method was used to demonstrate the ability of asbest and zeolite dusts from 8 deposits of the USSR to induce generation of free oxygen radicals in the phagocytosing cells suspension. It has been found that asbest and zeolite (0.01 and 0.05 mg/ml) increase levels of cells with chromosome aberrations in human cell cultures. The cytogenetic effect of asbest was inhibited by superoxide dismutase (50 mg/ml). The damaging effect of zeolite was decreased by the pharmacological drug bemithyl (0.007-0.07 mM) and completely eliminated by catalase (20 mg/ml). The results obtained indicate that mutagenic effect of dust particles of asbest and zeolite is mediated by oxygen radicals.

The housefly Musca domestica as a mechanical vector of Clostridium difficile.

PubMed

Davies, M P; Anderson, M; Hilton, A C

2016-11-01

Clostridium difficile is a bacterial healthcare-associated infection that may be transferred by houseflies (Musca domestica) due to their close ecological association with humans and cosmopolitan nature. To determine the ability of M. domestica to transfer C. difficile both mechanically and following ingestion. M. domestica were exposed to independent suspensions of vegetative cells and spores of C. difficile, then sampled on to selective agar plates immediately postexposure and at 1-h intervals to assess the mechanical transfer of C. difficile. Fly excreta was cultured and alimentary canals were dissected to determine internalization of cells and spores. M. domestica exposed to vegetative cell suspensions and spore suspensions of C. difficile were able to transfer the bacteria mechanically for up to 4h upon subsequent contact with surfaces. The greatest numbers of colony-forming units (CFUs) per fly were transferred immediately following exposure (mean CFUs 123.8 +/- 66.9 for vegetative cell suspension and 288.2 +/- 83.2 for spore suspension). After 1h, this had reduced (21.2 +/- 11.4 for vegetative cell suspension and 19.9 +/- 9 for spores). Mean C. difficile CFUs isolated from the M. domestica alimentary canal was 35 +/- 6.5, and mean C. difficile CFUs per faecal spot was 1.04 +/- 0.58. C. difficile could be recovered from fly excreta for up to 96h. This study describes the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals, highlighting flies as realistic vectors of this micro-organism in clinical areas. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Characterization of Ni-YSZ anodes for solid oxide fuel cells fabricated by suspension plasma spraying with axial feedstock injection

NASA Astrophysics Data System (ADS)

Metcalfe, Craig; Kuhn, Joel; Kesler, Olivera

2013-12-01

Composite Ni-Y0.15Zr0.85O1.925 anodes were fabricated by axial-injection suspension plasma spraying in open atmosphere conditions. The composition of the anode is controllable by adjustment of the plasma gas composition, stand-off distance, and suspension feed rate. The total porosity is controllable through the addition of carbon black to the suspension as a sacrificial pore-forming material as well as by adjustment of the suspension feed rate. The size of the NiO particles in suspension affects both the composition and total porosity, with larger NiO particles leading to increased Ni content and porosity in the deposited coatings. The surface roughness increases with a decrease of the in-flight droplet momentum, which results from both smaller NiO particles in suspension and the addition of low density pore-forming materials. A solid oxide fuel cell was fabricated with both electrodes and electrolyte fabricated by axial-injection plasma spraying. Peak power densities of 0.718 W cm-2 and 1.13 W cm-2 at 750 °C and 850 °C, respectively, were achieved.

Low-Coherence Reflectometry for Refractive Index Measurements of Cells in Micro-Capillaries.

PubMed

Carpignano, Francesca; Rigamonti, Giulia; Mazzini, Giuliano; Merlo, Sabina

2016-10-11

The refractive index of cells provides insights into their composition, organization and function. Moreover, a good knowledge of the cell refractive index would allow an improvement of optical cytometric and diagnostic systems. Although interferometric techniques undoubtedly represent a good solution for quantifying optical path variation, obtaining the refractive index of a population of cells non-invasively remains challenging because of the variability in the geometrical thickness of the sample. In this paper, we demonstrate the use of infrared low-coherence reflectometry for non-invasively quantifying the average refractive index of cell populations gently confined in rectangular glass micro-capillaries. A suspension of human red blood cells in plasma is tested as a reference. As a use example, we apply this technique to estimate the average refractive index of cell populations belonging to epithelial and hematological families.

Low-Coherence Reflectometry for Refractive Index Measurements of Cells in Micro-Capillaries

PubMed Central

Carpignano, Francesca; Rigamonti, Giulia; Mazzini, Giuliano; Merlo, Sabina

2016-01-01

The refractive index of cells provides insights into their composition, organization and function. Moreover, a good knowledge of the cell refractive index would allow an improvement of optical cytometric and diagnostic systems. Although interferometric techniques undoubtedly represent a good solution for quantifying optical path variation, obtaining the refractive index of a population of cells non-invasively remains challenging because of the variability in the geometrical thickness of the sample. In this paper, we demonstrate the use of infrared low-coherence reflectometry for non-invasively quantifying the average refractive index of cell populations gently confined in rectangular glass micro-capillaries. A suspension of human red blood cells in plasma is tested as a reference. As a use example, we apply this technique to estimate the average refractive index of cell populations belonging to epithelial and hematological families. PMID:27727172

24 CFR 5.236 - Procedures for termination, denial, suspension, or reduction of assistance based on information...

Code of Federal Regulations, 2010 CFR

2010-04-01

..., suspension, or reduction of assistance based on information obtained from a SWICA or Federal agency. 5.236 Section 5.236 Housing and Urban Development Office of the Secretary, Department of Housing and Urban... Obtaining Income Information About Applicants and Participants § 5.236 Procedures for termination, denial...

24 CFR 5.236 - Procedures for termination, denial, suspension, or reduction of assistance based on information...

Code of Federal Regulations, 2011 CFR

2011-04-01

..., suspension, or reduction of assistance based on information obtained from a SWICA or Federal agency. 5.236 Section 5.236 Housing and Urban Development Office of the Secretary, Department of Housing and Urban... Obtaining Income Information About Applicants and Participants § 5.236 Procedures for termination, denial...

43 CFR 3287.2 - When may BLM grant a suspension of unit obligations?

Code of Federal Regulations, 2012 CFR

2012-10-01

..., the unit operator is prevented from complying with such obligations, in whole or in part, by: (1) Acts... operator's inability to obtain an electrical sales contract, or when poor economics affect the electrical generation market, limiting the opportunity to obtain a viable sales contract. BLM may grant a suspension of...

43 CFR 3287.2 - When may BLM grant a suspension of unit obligations?

Code of Federal Regulations, 2014 CFR

2014-10-01

..., the unit operator is prevented from complying with such obligations, in whole or in part, by: (1) Acts... operator's inability to obtain an electrical sales contract, or when poor economics affect the electrical generation market, limiting the opportunity to obtain a viable sales contract. BLM may grant a suspension of...

43 CFR 3287.2 - When may BLM grant a suspension of unit obligations?

Code of Federal Regulations, 2013 CFR

2013-10-01

..., the unit operator is prevented from complying with such obligations, in whole or in part, by: (1) Acts... operator's inability to obtain an electrical sales contract, or when poor economics affect the electrical generation market, limiting the opportunity to obtain a viable sales contract. BLM may grant a suspension of...

Selective retension of active cells employing low centrifugal force at the medium change during suspension culture of Chinese hamster ovary cells producing tPA.

PubMed

Takagi, M; Ilias, M; Yoshida, T

2000-01-01

The effect of centrifugal force applied for cell separation at the medium change on the growth, metabolism and tissue plasminogen activator (tPA) productivity of Chinese hamster ovary (CHO) cells suspension culture was investigated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal forces applied for 5 min was less than 67 x g. In cultures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 x g, a centrifugal force lower than 119 x g resulted in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily centrifugation at 67 to 537 x g without discarding the supernatant had no effect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 x g showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifugation at 67 x g have average diameters of 15.5 and 17.4 microm, respectively. The intracellular contents of amino acids, especially nonessential amino acids, of the precipitated cells were markedly higher than those of the cells in the supernatant. These results indicate that large cells with high amino acid content and metabolic activity were selectively retained in the culture by means of centrifugation at low forces such as 67 x g. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture.

Electrochemical analysis in a liposome suspension using lapachol as a hydrophobic electro active species.

PubMed

Okumura, Noriko; Wakamatsu, Shiori; Uno, Bunji

2014-01-01

This study demonstrated that the electro-chemical analysis of hydrophobic quinones can be performed in liposome suspension systems. We prepared and analyzed liposome suspensions containing lapachol, which is a quinone-based anti-tumor activity compound. In this suspension system, a simple one redox couple of lapachol is observed. These results are quite different from those obtained in organic solvents. In addition, the pH dependence of redox behaviors of lapachol could be observed in multilamellar vesicle (MLV) suspension system. This MLV suspension system method may approximate the electrochemical behavior of hydrophobic compounds in aqueous conditions. A benefit of this liposome suspension system for electrochemical analysis is that it enables to observe water-insoluble compounds without using organic solvents.

Enhanced synthesis of andrographolide by Aspergillus niger and Penicillium expansum elicitors in cell suspension culture of Andrographis paniculata (Burm. f.) Nees.

PubMed

Vakil, Moinuddin M A; Mendhulkar, Vijay D

2013-12-01

Andrographis paniculata (Burm. f.) Nees is an important medicinal plant which has enormous applications in pharmaceutical industries. Cell suspension culture of Andrographis paniculata (Burm. f.) Nees. was treated with Aspergillus niger and Penicillium expansum elicitors to enhance the synthesis of andrographolide, the bioactive constituent of A. paniculata. The elicitation treatment with fungal elicitors (A. niger and P. expansum) was observed to be most suitable for eliciting andrographolide production in the culture. The quantification of andrographolide was done using High Performance Liquid Chromatography (HPLC) technique. A. niger extract (1.5 ml with10 days treatment duration) revealed 6.94 fold increase in andrographolide content (132 μg) which was higher than the control (19 μg). P. expansum elicitor (0.6% with 8 days treatment duration) could reveal 6.23 fold enhancement in andrographolide content (81.0 μg) over control (13 μg). The results obtained reveal that the longer treatment duration is most favorable for the elicitation of andrographolide using both the fungal elicitors.

One-dimensional wave propagation in particulate suspensions

NASA Technical Reports Server (NTRS)

Rochelle, S. G.; Peddieson, J., Jr.

1976-01-01

One-dimensional small-amplitude wave motion in a two-phase system consisting of an inviscid gas and a cloud of suspended particles is analyzed using a continuum theory of suspensions. Laplace transform methods are used to obtain several approximate solutions. Properties of acoustic wave motion in particulate suspensions are inferred from these solutions.

[Massive multiplication of coffee (Coffee arabica L. cv. Catimor) through embryogenic cell suspension culture].

PubMed

Flermoso-Gallardo, L; Menóndez-Yuffá, A

2000-01-01

Cell suspensions offer several advantages as a system for massive propagation because of the high rates of multiplication, the higher homogeneity in the culture conditions and the possibility of automatization. In this study, different experimental conditions were analyzed to establish embryogenic cell suspension cultures of coffee. The best conditions to establish the embryogenic cell suspension cultures of coffee were as follows: coffee leaf sections were cultivated during 12 weeks (Stage I) in a solid medium with the Murashige and Skoog salts, 2 mg/l kinetin and 0.5 mg/l 2,4-dichlorophenoxiacetic acid (medium 1). Under these conditions the explants formed a callus tissue that was transferred to a liquid medium containing 5 mg/l of 6-benzylamlno-purine (medium 2). After 12 days in a shaking liquid medium (Stage II), the cultures were sieved and were maintained In the same media, which was renewed every eight days (Stage III). This method yielded 1884 embryos in 50 ml; placing the embryos under conditions for germination yielded plantlets of normal appearance.

Spatial Distribution of Niche and Stem Cells in Ex Vivo Human Limbal Cultures

PubMed Central

Kacham, Santhosh; Purushotham, Jyothi; Maddileti, Savitri; Siamwala, Jamila; Sangwan, Virender Singh

2014-01-01

Stem cells at the limbus mediate corneal epithelial regeneration and regulate normal tissue homeostasis. Ex vivo cultured limbal epithelial transplantations are being widely practiced in the treatment of limbal stem cell deficiency. In this report, we examined whether the limbal niche cells that nurture and regulate epithelial stem cells coexist in ex vivo limbal cultures. We also compared the inherent differences between explant and suspension culture systems in terms of spatial distribution of niche cells and their effect on epithelial stem cell proliferation, migration, and differentiation in vitro. We report that the stem cell content of both culture systems was similar, explaining the comparable clinical outcomes reported using these two methods. We also showed that the niche cells get expanded in culture and the nestin-positive cells migrate at the leading edges to direct epithelial cell migration in suspension cultures, whereas they are limited to the intact niche in explant cultures. We provide evidence that C/EBPδ-positive, p15-positive, and quiescent, label-retaining, early activated stem cells migrate at the leading edges to regulate epithelial cell proliferation in explant cultures, and this position effect is lost in early suspension cultures. However, in confluent suspension cultures, the stem cells and niche cells interact with each another, migrate in spiraling patterns, and self-organize to form three-dimensional niche-like compartments resembling the limbal crypts and thereby reestablish the position effect. These 3D-sphere clusters are enriched with nestin-, vimentin-, S100-, and p27-positive niche cells and p15-, p21-, p63α-, C/EBPδ-, ABCG2-, and Pax6-positive quiescent epithelial stem cells. PMID:25232182

Study of cell killing effect on S180 by ultrasound activating protoporphyrin IX.

PubMed

Wang, Xiao Bing; Liu, Quan Hong; Wang, Pan; Tang, Wei; Hao, Qiao

2008-04-01

The present study was initiated to investigate the potential biological mechanism of cell killing effect on isolate sarcoma 180 (S180) cells induced by ultrasound activating protoporphyrin IX (PPIX). S180 cells were exposed to ultrasound for 30s duration, at a frequency of 2.2 MHz and an acoustic power of 3 W/cm(2) in the presence of 120 microM PPIX. The viability of cells was evaluated using trypan blue staining. The generation of oxygen free radicals in cell suspensions was detected immediately after treatment using a reactive oxygen detection kit. A copper reagent colorimetry method was used to measure the level of FFAs released into cell suspensions by the process of cell damage induced by ultrasound and PPIX treatment. Oxidative stress was assessed by measuring the activities of key antioxidant enzymes (i.e., SOD, CAT, GSH-PX) in S180 tumor cells. Treatment with ultrasound and PPIX together increased the cell damage rate to 50.91%, while treatment with ultrasound alone gave a cell damage rate to 24.24%, and PPIX alone kept this rate unchanged. Colorimetry and enzymatic chemical methods showed that the level of FFAs in cell suspension increased significantly after the treatment, while the activity of all the above enzymes decreased in tumor cells at different levels, and were associated with the generation of oxygen free radicals in cell suspension after treatment. The results indicate that oxygen free radicals may play an important role in improving the membrane lipid peroxidation, degrading membrane phospholipids to release FFAs, and decreasing the activities of the key antioxidant enzymes in cells. This biological mechanism might be involved in mediating the effects on S180 cells and resulting in the cell damage seen with SDT.

[Cell lineage tracing of regenerating cells after partial pancreatectomy using pseudo-type retrovirus].

PubMed

Zhang, Lixin; Ju, Xiaofang; Wang, Fa; Guo, Zhiwei; Piao, Shanhua; Teng, Chunbo

2008-04-01

Pancreas is an important mixed gland having both endocrine and exocrine functions, and has been proven regeneration after injury. To explore the cell lineage tracing methods in pancreas in vivo and the regenerate cells source, we used pseudo-type retrovirus to transfect adult mouse pancreas which had been partially pancreatectomized by rubbing the kerf using a cotton stick saturated with retrovirus suspension then injecting 100 microL retrovirus suspension into pancreas, injecting 100 microL retrovirus by caudal vein, or interperitoneally injecting retrovirus respectively. The results showed that the method of rubbing the kerf then injection of retrovirus suspension into pancreas could more effectively mark the pancreatic cells than the caudal vein injection and the intraperitoneal injection did in vivo. Furthermore, this study also found that some acinus cells could accept injury stimulus signals to regenerate through resuming mitosis after pancreatic injury. This study establishes a cell lineage tracing method in pancreas in vivo using retrovirus and offers a clue for gene therapy of pancreatic diseases using retrovirus vectors.

Detection of early changes in lung-cell cytology by flow-systems analysis techniques. Progress report, January 1--June 30, 1976

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

Steinkamp, J. A.; Hansen, K. M.; Wilson, J. S.

1976-08-01

This report summarizes results of preliminary experiments to develop cytological and biochemical indicators for estimating damage to respiratory epithelium exposed to toxic agents associated with the by-products of nonnuclear energy production using advanced flow-systems cell-analysis technologies. Since initiation of the program one year ago, progress has been made in obtaining adequate numbers of exfoliated lung cells from the Syrian hamster for flow analysis; cytological techniques developed on human exfoliated gynecological samples have been adapted to hamster lung epithelium for obtaining single-cell suspensions; and lung-cell samples have been initially characterized based on DNA content, total protein, nuclear and cytoplasmic size, andmore » multiangle light-scatter measurements. Preliminary results from measurements of the above parameters which recently became available are described in this report. As the flow-systems technology is adapted further to analysis of exfoliated lung cells, measurements of changes in physical and biochemical cellular properties as a function of exposure to toxic agents will be performed.« less

Development and characterization of a monoclonal antibody to human embryonal carcinoma

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

Khazaeli, M.B.; Beierwaltes, W.H.; Pitt, G.S.

1987-06-01

A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components. The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient. An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian,more » 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells. The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines. The antibody did not react with 22 other cell lines or with hCG. The antibody was labeled with /sup 131/I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later. Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization. Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21.« less

Rheological properties of purified illite clays in glycerol/water suspensions

NASA Astrophysics Data System (ADS)

Dusenkova, I.; Malers, J.; Berzina-Cimdina, L.

2015-04-01

There are many studies about rheological properties of clay-water suspensions, but no published investigations about clay-glycerol suspensions. In this work apparent viscosity of previously purified illite containing clay fraction < 2 μm and glycerol/water suspensions were investigated. Carbonates were removed by dissolution in hydrochloric and citric acids and other non-clay minerals were almost totally removed by centrifugation. All obtained suspensions behaved as shear-thinning fluids with multiple times higher viscosity than pure glycerol/water solutions. Reduction of clay fraction concentration by 5% decreased the apparent viscosity of 50% glycerol/water suspensions approximately 5 times. There was basically no difference in apparent viscosity between all four 50% glycerol/water suspensions, but in 90% glycerol/water suspensions samples from Iecava deposit showed slightly higher apparent viscosity, which could be affected by the particle size distribution.

Augmented lymphocyte expansion from solid tumors with engineered cells for costimulatory enhancement

PubMed Central

Friedman, Kevin M; DeVillier, Laura E; Feldman, Steven A; Rosenberg, Steven A; Dudley, Mark E

2011-01-01

Treatment of patients with adoptive T cell therapy requires expansion of unique tumor-infiltrating lymphocyte (TIL) cultures from single cell suspensions processed from melanoma biopsies. Strategies which increase the expansion and reliability of TIL generation from tumor digests are necessary to improve access to TIL therapy. Prior work evaluated artificial antigen presenting cells (aAPCs) for their antigen-specific and costimulatory properties. We investigated engineered cells for co-stimulatory enhancement (ECCE) consisting of K562 cells which express 4-1BBL in the absence of artificial antigen stimulation. ECCE accelerated TIL expansion and significantly improved TIL numbers (p=0.001) from single cell melanoma suspensions. TIL generated with ECCE contain significantly more CD8+CD62L+ and CD8+CD27+ T cells then comparable IL-2-expanded TIL and maintained anti-tumor reactivity. Moreover, ECCE improved TIL expansion from non-melanoma cell suspensions similar to that seen with melanoma tumors. These data demonstrate that ECCE addition to TIL production will enable treatment of patients ineligible using current methods. PMID:21989413

Specific binding of sup 125 I-rErythropoietin to Friend polycythemia virus-transformed erythroleukemia cells purified by centrifugal elutriation

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

Correa, P.N.; Bard, V.; Axelrad, A.A.

1990-01-01

We have used countercurrent centrifugal elutriation (CCE) to determine the distribution of cells with respect to cell volume and buoyant density for an erythroleukemia cell line (JG6) transformed by the polycythemia strain of Friend virus (FV-P), and to determine the effect of inducing the cells to differentiate with dimethylsulfoxide (DMSO) on this distribution. CCE made it possible to obtain suspensions of modal JG6 populations virtually free of dead cells and uniform with respect to volume and buoyant density. These modal populations were assayed for specific binding of erythropoietin (Epo). Between 500 and 550 Epo receptors per cell were detected. Thesemore » belonged to a single class having a dissociation constant of 0.36 nM. DMSO induction of differentiation of the JG6 cells had no effect on the number of Epo receptors expressed.« less

Efficient biotechnological approach for lentiviral transduction of induced pluripotent stem cells.

PubMed

Zare, Mehrak; Soleimani, Masoud; Mohammadian, Mozhdeh; Akbarzadeh, Abolfazl; Havasi, Parvaneh; Zarghami, Nosratollah

2016-01-01

Induced pluripotent stem (iPS) cells are generated from differentiated adult somatic cells by reprogramming them. Unlimited self-renewal, and the potential to differentiate into any cell type, make iPS cells very promising candidates for basic and clinical research. Furthermore, iPS cells can be genetically manipulated for use as therapeutic tools. DNA can be introduced into iPS cells, using lentiviral vectors, which represent a helpful choice for efficient transduction and stable integration of transgenes. In this study, we compare two methods of lentiviral transduction of iPS cells, namely, the suspension method and the hanging drop method. In contrast to the conventional suspension method, in the hanging drop method, embryoid body (EB) formation and transduction occur concurrently. The iPS cells were cultured to form EBs, and then transduced with lentiviruses, using the conventional suspension method and the hanging drop method, to express miR-128 and green fluorescent protein (GFP). The number of transduced cells were assessed by fluorescent microscopy and flow cytometry. MTT assay and real-time PCR were performed to determine the cell viability and transgene expression, respectively. Morphologically, GFP+ cells were more detectable in the hanging drop method, and this finding was quantified by flow cytometric analysis. According to the results of the MTT assay, cell viability was considerably higher in the hanging drop method, and real-time PCR represented a higher relative expression of miR-128 in the iPS cells introduced with lentiviruses in drops. Altogether, it seems that lentiviral transduction of challenging iPS cells using the hanging drop method offers a suitable and sufficient strategy in their gene transfer, with less toxicity than the conventional suspension method.

Technological assumptions for biogas purification.

PubMed

Makareviciene, Violeta; Sendzikiene, Egle

2015-01-01

Biogas can be used in the engines of transport vehicles and blended into natural gas networks, but it also requires the removal of carbon dioxide, hydrogen sulphide, and moisture. Biogas purification process flow diagrams have been developed for a process enabling the use of a dolomite suspension, as well as for solutions obtained by the filtration of the suspension, to obtain biogas free of hydrogen sulphide and with a carbon dioxide content that does not exceed 2%. The cost of biogas purification was evaluated on the basis of data on biogas production capacity and biogas production cost obtained from local water treatment facilities. It has been found that, with the use of dolomite suspension, the cost of biogas purification is approximately six times lower than that in the case of using a chemical sorbent such as monoethanolamine. The results showed travelling costs using biogas purified by dolomite suspension are nearly 1.5 time lower than travelling costs using gasoline and slightly lower than travelling costs using mineral diesel fuel.

Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

PubMed

Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

2015-01-01

Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

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

PubMed

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

2006-01-01

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

An oil-soluble extract of Rubus idaeus cells enhances hydration and water homeostasis in skin cells.

PubMed

Tito, A; Bimonte, M; Carola, A; De Lucia, A; Barbulova, A; Tortora, A; Colucci, G; Apone, F

2015-12-01

Raspberry plants, belonging to the species of Rubus idaeus, are known for their excellent therapeutic properties as they are particularly rich in compounds with strong antioxidant activity, which promote health and well-being of human cells. Besides their high content of phenolic compounds, Rubus plants are rich in oil-soluble compounds, which are also primary components of the hydrolipidic film barrier of the skin. As plant cell cultures represented a valuable system to produce interesting compounds and ingredients for cosmetic applications, we developed liquid suspension cultures from Rubus idaeus leaves and used them to obtain an active ingredient aimed at improving hydration and moisturization capacity in the skin. Rubus idaeus cells, grown in the laboratory under sterile and controlled conditions as liquid suspension cultures, were processed to obtain an oil-soluble (liposoluble) extract, containing phenolic compounds and a wide range of fatty acids. The extract was tested on cultured keratinocytes and fibroblasts and then on the skin in vivo, to assess its cosmetic activities. When tested on skin cell cultures, the extract induced the genes responsible for skin hydration, such as aquaporin 3, filaggrin, involucrin and hyaluronic acid synthase, and stimulated the expression and the activity of the enzyme glucocerebrosidase, involved in ceramide production. Moreover, the liposoluble extract increased the synthesis of the extracellular matrix components in cultured fibroblasts and showed a remarkable skin-hydrating capacity when tested on human skin in vivo. Thanks to these activities, the Rubus idaeus liposoluble extract has several potential applications in skin care cosmetics: it can be used as hydrating and moisturizing ingredient in face and body lotions, and as anti-ageing product in face creams specifically designed to fight wrinkle formation. © 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Monoclonal antibody against human ovarian tumor-associated antigens.

PubMed

Poels, L G; Peters, D; van Megen, Y; Vooijs, G P; Verheyen, R N; Willemen, A; van Niekerk, C C; Jap, P H; Mungyer, G; Kenemans, P

1986-05-01

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by 125I-labeled OV-TL 3 antibodies. Thus OV-TL 3 recognizes a common antigen for most ovarian carcinomas and may be a useful tool for rapid diagnosis of ovarian carcinomas.

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

Poels, L.G.; Peters, D.; van Megen, Y.

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derivedmore » from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by /sup 125/I-labeled OV-TL 3 antibodies.« less

Implications of the stability behavior of zinc oxide nanoparticles for toxicological studies

NASA Astrophysics Data System (ADS)

Meißner, Tobias; Oelschlägel, Kathrin; Potthoff, Annegret

2014-08-01

The increasing use of zinc oxide (ZnO) nanoparticles in sunscreens and other cosmetic products demands a risk assessment that has to be done in toxicological studies. Such investigations require profound knowledge of the behavior of ZnO in cell culture media. The current study was performed to get well-dispersed suspensions of a hydrophilic (ZnO-hydro) and a lipophilic coated (ZnO-lipo) ZnO nanomaterial for use in in vitro tests. Therefore, systematic tests were carried out with common dispersants (phosphate, lecithin, proteins) to elucidate chemical and physical changes of ZnO nanoparticles in water and physiological solutions (PBS, DMEM). Non-physiological stock suspensions were prepared using ultrasonication. Time-dependent changes of pH, conductivity, zeta potential, particle size and dissolution were recorded. Secondly, the stock suspensions were added to physiological media with or without albumin (BSA) or serum (FBS), to examine characteristics such as agglomeration and dissolution. Stable stock suspensions were obtained using phosphate as natural and physiological electrostatic stabilizing agent. Lecithin proved to be an effective wetting agent for ZnO-lipo. Although the particle size remained constant, the suspension changed over time. The pH increased as a result of ZnO dissolution and formation of zinc phosphate complexes. The behavior of ZnO in physiological media was found to depend strongly on the additives used. Applying only phosphate as additive, ZnO-hydro agglomerated within minutes. In the presence of lecithin or BSA/serum, agglomeration was inhibited. ZnO dissolution was higher under physiological conditions than in the stock suspension. Serum especially promoted this process. Using body-related dispersants (phosphate, lecithin) non-agglomerating stock suspensions of hydrophilic and lipophilic ZnO were prepared as a prerequisite to perform meaningful toxicological investigation. Both nanomaterials showed a non-negligible dissolution behavior that strongly depended on the surrounding conditions. Agglomeration of ZnO particles in physiological media is a complex function of particle coating, used dispersants and serum proteins if supplemented. The present study gives a clear guideline how to prepare and handle suspensions with ZnO for in vitro testing and allows the correlation between the chemical-physical particles behavior with findings from toxicological tests.

Thermal and Nonthermal Effects of Discontinuous Microwave Exposure (2.45 Gigahertz) on the Cell Membrane of Escherichia coli

PubMed Central

Rougier, Carole; Chazal, Philippe; Leveque, Philippe; Leprat, Patrick

2014-01-01

The aim of this study was to investigate the effects on the cell membranes of Escherichia coli of 2.45-GHz microwave (MW) treatment under various conditions with an average temperature of the cell suspension maintained at 37°C in order to examine the possible thermal versus nonthermal effects of short-duration MW exposure. To this purpose, microwave irradiation of bacteria was performed under carefully defined and controlled parameters, resulting in a discontinuous MW exposure in order to maintain the average temperature of the bacterial cell suspensions at 37°C. Escherichia coli cells were exposed to 200- to 2,000-W discontinuous microwave (DW) treatments for different periods of time. For each experiment, conventional heating (CH) in a water bath at 37°C was performed as a control. The effects of DW exposure on cell membranes was investigated using flow cytometry (FCM), after propidium iodide (PI) staining of cells, in addition to the assessment of intracellular protein release in bacterial suspensions. No effect was detected when bacteria were exposed to conventional heating or 200 W, whereas cell membrane integrity was slightly altered when cell suspensions were subjected to powers ranging from 400 to 2,000 W. Thermal characterization suggested that the temperature reached by the microwave-exposed samples for the contact time studied was not high enough to explain the measured modifications of cell membrane integrity. Because the results indicated that the cell response is power dependent, the hypothesis of a specific electromagnetic threshold effect, probably related to the temperature increase, can be advanced. PMID:24907330

Plant regeneration from haploid cell suspension-derived protoplasts of Mediterranean rice (Oryza sativa L. cv. Miara).

PubMed

Guiderdoni, E; Chaïr, H

1992-11-01

More than 750 plants were regenerated from protoplasts isolated from microspore callus-derived cell suspensions of the Mediterranean japonica rice Miara, using a nurse-feeder technique and N6-based culture medium. The mean plating efficiency and the mean regeneration ability of the protocalluses were 0.5% and 49% respectively. Flow cytometric evaluation of the DNA contents of 7 month old-cell and protoplast suspensions showed that they were still haploid. Contrastingly, the DNA contents of leaf cell nuclei of the regenerated protoclones ranged from 1C to 5C including 60% 2C plants. This was consistent with the morphological type and the fertility of the mature plants. These results and the absence of chimeric plants suggest that polyploidization occurred during the early phase of protoplast culture.

78 FR 12965 - Suspension of Community Eligibility

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-26

... participation status of a community can be obtained from FEMA's Community Status Book (CSB). The CSB is... for reading third column: Emerg. --Emergency; Reg. --Regular; Susp. --Suspension. Dated: January 30...

Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus suspension cultures

Treesearch

R. Minocha; S.C. Minocha; A. Komamine; W.C. Shortle

1991-01-01

Various inhibitors of polyamine biosynthesis were used to study the role of polyamines in DNA synthesis and cell division in suspension cultures of Catharanthus roseus (L) G. Don. Arginine decarboxylase (ADC; EC 4.1.1.19) was the major enzyme responsible for putrescine production. DL α-difluoromethylarginine inhibited ADC activity, cellular...

Protein Adsorption Alters Hydrophobic Surfaces Used for Suspension Culture of Pluripotent Stem Cells.

PubMed

Jonas, Steven J; Stieg, Adam Z; Richardson, Wade; Guo, Shuling; Powers, David N; Wohlschlegel, James; Dunn, Bruce

2015-02-05

This Letter examines the physical and chemical changes that occur at the interface of methyl-terminated alkanethiol self-assembled monolayers (SAMs) after exposure to cell culture media used to derive embryoid bodies (EBs) from pluripotent stem cells. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy analysis of the SAMs indicates that protein components within the EB cell culture medium preferentially adsorb at the hydrophobic interface. In addition, we examined the adsorption process using surface plasmon resonance and atomic force microscopy. These studies identify the formation of a porous, mat-like adsorbed protein film with an approximate thickness of 2.5 nm. Captive bubble contact angle analysis reveals a shift toward superhydrophilic wetting behavior at the cell culture interface due to adsorption of these proteins. These results show how EBs are able to remain in suspension when derived on hydrophobic materials, which carries implications for the rational design of suspension culture interfaces for lineage specific stem-cell differentiation.

Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production.

PubMed

Cervera, Laura; Kamen, Amine A

2018-01-01

Large-scale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the potential to support preclinical or even clinical studies. Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a transfection reagent. Cells are grown in the bioreactor to 1 × 10 6 cells/mL and transfected with a plasmid DNA-PEI complex at a ratio of 1:2. Dissolved oxygen and pH are controlled and are online monitored during the production phase and cell growth and viability can be measured off line taking samples from the bioreactor. If the product is labeled with a fluorescent marker, transfection efficiency can be also assessed using flow cytometry analysis. Typically, the production phase lasts between 48 and 96 h until the product is harvested.

Suspension-line wave motion during the lines-first parachute unfurling process

NASA Technical Reports Server (NTRS)

Poole, L. R.; Whitesides, J. L.

1974-01-01

A new mathematical approach to modeling the lines-first parachute unfurling process is presented. The unfurling process is treated as two distinct phases: a suspension-line unfurling phase, during which a massless-spring model of the suspension-line elasticity may be employed; and a canopy unfurling phase, during which a formulation considering suspension-line wave mechanics is employed. Histories of unfurled length and tension at the vehicle obtained using the model are compared with flight test data, and generally good agreement is observed.

78 FR 37978 - Suspension of Community Eligibility

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-25

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78 FR 69001 - Suspension of Community Eligibility

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-18

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Study on kinematic and compliance test of suspension

NASA Astrophysics Data System (ADS)

Jing, Lixin; Wu, Liguang; Li, Xuepeng; Zhang, Yu

2017-09-01

Chassis performance development is a major difficulty in vehicle research and development, which is the main factor restricting the independent development of vehicles in China. These years, through a large number of studies, chassis engineers have found that the suspension K&C characteristics as a quasi-static characteristic of the suspension provides a technical route for the suspension performance R&D, and the suspension K&C test has become an important means of vehicle benchmarking, optimization and verification. However, the research on suspension K&C test is less in china, and the test conditions and setting requirements vary greatly from OEM to OEM. In this paper, the influence of different settings on the characteristics of the suspension is obtained through experiments, and the causes of the differences are analyzed; in order to fully reflect the suspension characteristics, the author recommends the appropriate test case and settings.

Kidney cell electrophoresis, continuing task

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated to provide ground support in the form of analytical cell electrophoresis and flow cytometry. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. Cells were prepared in suspension prior to flight in electrophoresis buffer and 10% calf serum. Electrophoretic separation proceeded in electrophoresis buffer without serum in the Continuous Flow Electrophoretic Separator, and fractions were collected into sample bags containing culture medium and concentrated serum. Fractions that yielded enough progeny cells were analyzed for morphology and electrophoretic mobility distributions. It is noted that the lowest mobility fraction studied produced higher mobility progeny while the other fractions produced progeny cells with mobilities related to the fractions from which they were collected.

Treatment of rhinophyma with the Versajet™ Hydrosurgery System and autologous cell suspension (ReCELL®): A case report.

PubMed

K, Yıldız; B R, Kayan; T, Dulgeroglu; E, Guneren

2018-04-01

This is a case report of a 63-year-old male patient who presented with rhinophyma of 17 years duration. Several medical treatments were applied previously, with no response or poor improvement. We present our experience by combining the Versajet™ Hydrosurgery System and ReCELL ® in a heavy smoker patient, which led to a good aesthetic outcome. With the combined technique, we did not encounter any difficulties either within the operation or in the follow-up period. We obtained less complications and faster wound healing, which in return led to higher patient satisfaction.

Reduced Differentiation Efficiency of Murine Embryonic Stem Cells in Stirred Suspension Bioreactors

PubMed Central

Taiani, Jaymi T.; Krawetz, Roman J.; zur Nieden, Nicole I.; Wu, Yiru Elizabeth; Kallos, Michael S.; Matyas, John R.

2010-01-01

The use of embryonic stem cells (ESCs) for regenerative medicine has generated increased attention due to the favorable attributes of these cells; namely, they are pluripotent and possess long-term self-renewal capacity. The initial aims of the present study were: (i) to use stirred suspension bioreactors to expand and differentiate ESCs into osteogenic and chondrogenic cell types and (ii) to explore if these ESC-derived cells influenced skeletal healing in an in vivo fracture model. We show that differentiation protocols used in static culture are insufficient when applied directly to suspension culture bioreactors. Moreover, when bioreactor-differentiated cells are transplanted into a burr-hole defect in bone, severe disruption of the bone architecture was noted at the fracture site, as determined by microcomputed tomography (microCT) imaging and histopathology. Further characterization of the bioreactor-differentiated cultures revealed that a subpopulation of cells in the resulting aggregates expressed the pluripotency marker Oct-4 in the nucleus. Nuclear Oct-4 expression persisted even after 30 days of culture in the absence of leukemia inhibitory factor (LIF). Remarkably, and unlike ESCs differentiated into skeletal cell types in static cultures, bioreactor-differentiated aggregates implanted subcutaneously into SCID mice formed teratomas. The development of effective ESC differentiation protocols for suspension bioreactors will require a more complete understanding of the environmental conditions within these culture systems and the influence that these conditions have on the regulation of pluripotency and differentiation in ESCs. PMID:19775198

Using of dynamic speckled speckles with a small number of scatterers for study of suspension of Chlamydia

NASA Astrophysics Data System (ADS)

Ulyanov, Sergey; Filonova, Nadezhda; Ulianova, Onega; Utz, Sergey; Moiseeva, Yulia; Subbotina, Irina; Kalduzova, Irina; Larionova, Olga; Feodorova, Valentina

2018-04-01

Theory of formation of speckled speckles at diffraction of focused Gaussian beam in the suspension, containing of Chlamydia trachomatis (CT) is presented. Optical model of scattering of light in suspension of Chlamydia is suggested. Formula for bandwidth of spectrum of intensity fluctuations in speckled speckles is derived. It has been demonstrated, that speckle-microscopy can be used for detection of CT bacteria for any concentration of the relevant cells in suspension.

Iron Induction of Ferritin Synthesis in Soybean Cell Suspensions

PubMed Central

Proudhon, Dominique; Briat, Jean-François; Lescure, Anne-Marie

1989-01-01

In animal cells specialized for iron storage, iron-induced accumulation of ferritin is known to result from a shift of stored mRNA from the ribonucleoprotein fraction to polysomes. Previous reports with bean leaves suggested that in plants iron induction of ferritin synthesis would result from a regulation at the transcriptional level (F van der Mark, F Bienfait, H van der Ende [1983] Biochem Biophys Res Commun 115:463-469). Soybean (Glycine max, cv Mandarin) cell suspension cultures have been used here to support these findings. Ferritin induction is obtained by addition of Fe-citrate to the culture medium. A good correlation is found between cellular iron content and the amount of ferritin accumulation. This protein accumulation corresponds to an increase of in vitro translatable ferritin mRNA. Addition of 4 micrograms actinomycin D per milliliter to the cultures inhibits completely in vivo RNA synthesis, whereas protein synthesis was poorly affected, at least for 24 hours. During the same time, this concentration of actinomycin D strongly inhibits the iron-induced synthesis of ferritin. These results show that in soybean cell cultures, the mechanism of regulation of ferritin synthesis in response to iron does not result from recruitment of preexisting mRNA. They confirm that in plant systems, ferritin synthesis results from increased transcription of the corresponding genes. Images Figure 2 Figure 3 Figure 5 PMID:16666812

A photonic crystal hydrogel suspension array for the capture of blood cells from whole blood

NASA Astrophysics Data System (ADS)

Zhang, Bin; Cai, Yunlang; Shang, Luoran; Wang, Huan; Cheng, Yao; Rong, Fei; Gu, Zhongze; Zhao, Yuanjin

2016-02-01

Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells.Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06368j

Formation of peptides from amino acids by single or multiple additions of ATP to suspensions of nucleoproteinoid microparticles

NASA Technical Reports Server (NTRS)

Nakashima, T.; Fox, S. W.

1981-01-01

The synthesis of peptides from individual amino acids or pairs of amino acids and ATP in the presence of catalysis by nucleoproteinoid microparticles is investigated. Experiments were performed with suspensions formed from the condensation of lysine-rich and acidic proteinoids with polyadenylic acid, to which were added glycine, phenylalanine, proline, lysine or glycine-phenylalanine mixtures, and ATP either at once or serially. Peptide yields are found to be greatest for equal amounts of acidic and basic proteinoids. The addition of imidazole is found to alter the preference of glycine-phenylalanine mixtures to form mixed heteropeptides rather than homopeptides. A rapid ATP decay in the peptide synthesis reaction is observed, and a greater yield is obtained for repeated small additions than for a single addition of ATP. The experimental system has properties similar to modern cells, and represents an organizational unit ready for the evolution of associated biochemical pathways.

Spot counting on fluorescence in situ hybridization in suspension images using Gaussian mixture model

NASA Astrophysics Data System (ADS)

Liu, Sijia; Sa, Ruhan; Maguire, Orla; Minderman, Hans; Chaudhary, Vipin

2015-03-01

Cytogenetic abnormalities are important diagnostic and prognostic criteria for acute myeloid leukemia (AML). A flow cytometry-based imaging approach for FISH in suspension (FISH-IS) was established that enables the automated analysis of several log-magnitude higher number of cells compared to the microscopy-based approaches. The rotational positioning can occur leading to discordance between spot count. As a solution of counting error from overlapping spots, in this study, a Gaussian Mixture Model based classification method is proposed. The Akaike information criterion (AIC) and Bayesian information criterion (BIC) of GMM are used as global image features of this classification method. Via Random Forest classifier, the result shows that the proposed method is able to detect closely overlapping spots which cannot be separated by existing image segmentation based spot detection methods. The experiment results show that by the proposed method we can obtain a significant improvement in spot counting accuracy.

Supramolecular Complex Antioxidant Consisting of Vitamins C, E and Hydrophilic-Hydrophobic Silica Nanoparticles

NASA Astrophysics Data System (ADS)

Laguta, I. V.; Kuzema, P. O.; Stavinskaya, O. N.; Kazakova, O. A.

Samples with varied amount of surface trimethylsilyl groups were obtained via gas-phase chemical modification of silica nanoparticles. The biocompatibility tests conducted in erythrocyte suspension have shown that hydrophobization of silica decreases its damaging effect to the cells. Being wettable in aqueous media, partially silylated silicas have higher affinity to hydrophobic bioactive molecules in comparison with the initial silica. Novel antioxidant consisting of vitamins C and E and silica with 40% of surface trimethylsilyl groups was formulated. It was found that supramolecular complexes are formed on the silica surface due to the affinity of water- and fat-soluble antioxidants to hydrophilic silanol and hydrophobic trimethylsilyl groups, respectively. Test reactions (total phenolic index determination, DPPH test) and in vitro studies (spectral analysis of erythrocyte suspensions undergoing UV irradiation) revealed the correlation between antioxidant activity of the complex antioxidant and the vitamins’ content. The antioxidant remained active during long-term storage under standard conditions.

Supramolecular Complex Antioxidant Consisting of Vitamins C, E and Hydrophilic-Hydrophobic Silica Nanoparticles

NASA Astrophysics Data System (ADS)

Laguta, I. V.; Kuzema, P. O.; Stavinskaya, O. N.; Kazakova, O. A.

Samples with varied amount of surface trimethylsilyl groups were obtained via gas-phase chemical modification of silica nanoparticles. The biocompatibility tests conducted in erythrocyte suspension have shown that hydrophobization of silica decreases its damaging effect to the cells. Being wettable in aqueous media, partially silylated silicas have higher affinity to hydrophobic bioactive molecules in comparison with the initial silica. Novel antioxidant consisting of vitamins C and E and silica with 40% of surface trimethylsilyl groups was formulated. It was found that supramolecular complexes are formed on the silica surface due to the affinity of water- and fat-soluble antioxidants to hydrophilic silanol and hydrophobic trimethylsilyl groups, respectively. Test reactions (total phenolic index determination, DPPH test) and in vitro studies (spectral analysis of erythrocyte suspensions undergoing UV irradiation) revealed the correlation between antioxidant activity of the complex antioxidant and the vitamins' content. The antioxidant remained active during long-term storage under standard conditions.

Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b

NASA Astrophysics Data System (ADS)

Li, Jing; Peng, Ying; Wang, Xianghong; Chi, Zhenming

2010-12-01

The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin-1. Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

[The cultivation and identification of lacrimal gland adenoid cystic cancer stem cells].

PubMed

Lyu, Jianmei; He, Yanjin; Xie, Lianfeng; Liu, Xun; Zhu, Limin

2015-10-01

To isolate and cultivate the Lacrimal gland Adenoid Cystic Carcinoma cells line, study Cancer Stem Cells properties. Experimental study. Lacrimal gland adenoid cystic carcinoma cancer stem cells were cultivated in serum-free suspension culture and the morphological changes were observed. Cells were divided into two groups, the LACC-CSC experimental group and the LACC control group. The flow cytometry instrument was used to detect the expression of classical stem cell markers CD133 and ABCG2. Transwell chamber was used to detect the cancer stem cell aggressivity and differentiated into the vascular endothelial cells. The tumorigenic force in vitro xenotransplantation were applied. LACC cells can grow suspensively after vaccinated in serum free medium and form tumor microspheres after 10-12 days. Flow cytometry experiments showed that the expression ratio of stem cell markers CD133 in LACC-CSC was (35.67 ± 6.86)%, significantly different to LACC with (0.46 ± 0.48)%, (t = 8.867, P < 0.05). Similarly, the expression ratio of stem cell marker ABCG2 in LACC-CSC was (39.99 ± 4.54)%, significantly different to LACC with (6.75 ± 1.34)%, (t = -9.932, P < 0.05). In vitro experiment of Matrigel invasion, LACC-CSC went through the matrigel basement membrane averagely (32.60 ± 8.79)/HP contrary to LACC with average (10.20 ± 2.77)/HP after 24 hours, showing statistically significance (t = 5.433, P < 0.05) between the two groups. After training for 48 hours, the difference between two groups was still obvious (t = 5.779, P < 0.05) with LACC-CSC average (62.60 ± 4.83)/HP to LACC (44.00 ± 5.34)/HP. When induced by serum medium containing VEGF and bFGF, LACC-CSC grew adherent gradually and cell morphological changes occurred after continuous induction to long spindle cells. When cultured into three-dimensional matrix structure they formed vessel samples and expressed vascular endothelial marker CD31 and CD34. Transplanted tumor in vitro experiment, mice of LACC-CSC group grew tumors in 9 days with 100% tumorigenic rate, whereas LACC group 12 days with 100% tumorigenic rate. LACC-CSC can be obtained through serum-free culture method. LACC-CSC grew suspensively and expressed classical stem cell markers. LACC-CSC were identified as cancer stem cells with stronger migration and invasion. LACC-CSC have tumorigenic force and multi-directional differentiation potential with general characteristics of the stem cell.

Raman acoustic levitation spectroscopy of red blood cells and Plasmodium falciparum trophozoites.

PubMed

Puskar, Ljiljana; Tuckermann, Rudolf; Frosch, Torsten; Popp, Jürgen; Ly, Vanalysa; McNaughton, Don; Wood, Bayden R

2007-09-01

Methods to probe the molecular structure of living cells are of paramount importance in understanding drug interactions and environmental influences in these complex dynamical systems. The coupling of an acoustic levitation device with a micro-Raman spectrometer provides a direct molecular probe of cellular chemistry in a containerless environment minimizing signal attenuation and eliminating the affects of adhesion to walls and interfaces. We show that the Raman acoustic levitation spectroscopic (RALS) approach can be used to monitor the heme dynamics of a levitated 5 microL suspension of red blood cells and to detect hemozoin in malaria infected cells. The spectra obtained have an excellent signal-to-noise ratio and demonstrate for the first time the utility of the technique as a diagnostic and monitoring tool for minute sample volumes of living animal cells.

Effects of microwaves on the colony-forming capacity of haemopoietic stem cells in mice.

PubMed

Rotkovská, D; Vacek, A; Bartonícková, A

1987-01-01

A suspension of bone marrow cells from femurs of female (CBA X C57Bl)F1 mice was exposed to 2450 MHz CW microwaves in a specially designed waveguide exposure system. The temperature of the suspension rose, during exposure to microwaves, from 20 degrees C to 45 degrees C, and at an interval within 20 degrees C to 45 degrees C the number of haemopoietic stem cells (CFUs) was determined by the spleen exocolony method. The time of exposure of bone marrow cells to each temperature studied was 20 s. Control suspensions of bone marrow cells were exposed to a water bath temperature. There were no significant effects of the CFUs with the water bath temperature, while after exposure to microwaves the number of spleen colonies was elevated with a nadir at the temperature of 37 degrees C. With a microwave-induced increase of the temperature above 41 degrees C the number of CFUs in the bone marrow suspension decreased. The increase in the number of colonies was related to the rise in the seeding rate of the CFUs as well as to a rise in their proliferative activity, while the drop in the number of colonies was influenced also by heat-killing of the CFUs by microwave exposure.

30 CFR 203.47 - What administrative steps do I take to obtain and use the royalty suspension supplement?

Code of Federal Regulations, 2011 CFR

2011-07-01

... RELIEF OR REDUCTION IN ROYALTY RATES OCS Oil, Gas, and Sulfur General Royalty Relief for Drilling Deep... take to obtain and use the royalty suspension supplement? (a) Before you start drilling a well on your... Supervisor for Production and Development of your intent to begin drilling operations and the depth of the...

Impact of fluidic agitation on human pluripotent stem cells in stirred suspension culture.

PubMed

Nampe, Daniel; Joshi, Ronak; Keller, Kevin; Zur Nieden, Nicole I; Tsutsui, Hideaki

2017-09-01

The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters. In particular, detailed characterizations of different agitation rates and their influence on the propagation of hPSCs are often not reported in the literature. In the current study, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and the maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Overall, our results showed that moderate agitation maximized the propagation of hPSCs to approximately 38-fold over 7 days by keeping the cell aggregates below the critical size, beyond which the cells are impacted by the diffusion limit, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. This indicates a new possibility to guide stem cell fate determination by fluidic agitation in stirred suspension cultures. Biotechnol. Bioeng. 2017;114: 2109-2120. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

PubMed

Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2015-12-01

In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

Equations of motion of slung load systems with results for dual lift

NASA Technical Reports Server (NTRS)

Cicolani, Luigi S.; Kanning, Gerd

1990-01-01

General simulation equations are derived for the rigid body motion of slung load systems. These systems are viewed as consisting of several rigid bodies connected by straight-line cables or links. The suspension can be assumed to be elastic or inelastic, both cases being of interest in simulation and control studies. Equations for the general system are obtained via D'Alembert's principle and the introduction of generalized velocity coordinates. Three forms are obtained. Two of these generalize previous case-specific results for single helicopter systems with elastic or inelastic suspensions. The third is a new formulation for inelastic suspensions. It is derived from the elastic suspension equations by choosing the generalized coordinates so as to separate motion due to cable stretching from motion with invariant cable lengths. The result is computationally more efficient than the conventional formulation, and is readily integrated with the elastic suspension formulation and readily applied to the complex dual lift and multilift systems. Equations are derived for dual lift systems. Three proposed suspension arrangements can be integrated in a single equation set. The equations are given in terms of the natural vectors and matrices of three-dimensional rigid body mechanics and are tractable for both analysis and programming.

Hemp (Cannabis sativa L.).

PubMed

Feeney, Mistianne; Punja, Zamir K

2015-01-01

Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection medium were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines, and the presence of the PMI gene was confirmed using PCR and Southern hybridization. Using this method, an average transformation frequency of 31.23% ± 0.14 was obtained for all transformation experiments, with a range of 15.1-55.3%.

Peripolesis followed by cytotoxicity in chronic idiopathic inflammatory bowel disease.

PubMed Central

Wilders, M M; Drexhage, H A; Kokjé, M; Verspaget, H W; Meuwissen, S G

1984-01-01

Antigen presenting veiled cells have recently been described in cell suspensions prepared from the gut wall of patients with chronic idiopathic inflammatory bowel disease (CIBD). The normal gut wall is virtually devoid of these cells. In this report we describe a phenomenon known as peripolesis studied by phase contrast cinematography. This is a process in which lymphocytes are seen to wander around larger target cells. These could be identified ultrastructurally as Ia positive veiled cells. In most cases peripolesis was followed by lysis of the target cell. Peripolesis was recorded in cell suspensions of three out of seven patients with ulcerative colitis and in three out of nine patients with Crohn's disease; furthermore peripolesis was observed in one out of two patients with non-classifiable CIBD. In four cell suspensions showing peripolesis, cell lysis could be recorded and was especially striking in ulcerative colitis. Peripolesis involving veiled cells was previously described in delayed hypersensitivity reactions. This study lends support to the concept that delayed allergic reactivity plays a part in chronic inflammatory bowel disease. The antigens involved are, however, completely unknown. Images Fig. 1 Fig. 2 Fig. 3 PMID:6380839

COOLING DEVICE FOR USE WITH A SONIC OSCILLATOR.

PubMed

ROSETT, T

1965-03-01

A cooling cell is described that facilitates maintenance of biological materials at low temperatures during prolonged sonic treatment. Using this cell and a Branson S-75 Sonifier, I examined temperatures and the release of protein and enzyme activity from suspensions of Saccharomyces cerevisiae. With the Sonifier at full power, it was possible to maintain cell temperature within 9 C of the cooling-bath temperature, and to disrupt 10% (w/v) suspensions of S. cerevisiae in 10 min.

Melatonin-loaded lecithin/chitosan nanoparticles: physicochemical characterisation and permeability through Caco-2 cell monolayers.

PubMed

Hafner, Anita; Lovrić, Jasmina; Voinovich, Dario; Filipović-Grcić, Jelena

2009-11-03

In this study, the potential of lecithin/chitosan nanoparticles (NPs) as a mucoadhesive colloidal nanosystem for transmucosal delivery of melatonin was investigated. The size, zeta potential and melatonin loading of the lecithin/chitosan NPs were investigated as a function of lecithin type (Lipoid S45, S75 and S100) and chitosan content in the preparation. The NPs were characterised by mean diameter and zeta potential ranging between 121.6 and 347.5 nm, and 7.5 and 32.7 mV, respectively, and increasing with lecithin-negative charge and chitosan content in the preparation. Melatonin loadings were up to 7.1%. All NPs were characterised by prolonged release profiles with an initial burst (approximately 25%), followed by a slow release phase. Approximately 60-70% of melatonin was released in 4h. The permeability of melatonin was investigated using Caco-2 cells as an in vitro model of the epithelial barrier. Melatonin permeability from an NP suspension prepared with Lipoid S45 lecithin and a lecithin-to-chitosan weight ratio (L/C) of 20:1 (sample C2) was significantly improved compared to the permeability of melatonin from the solution (P<0.001) and from all other NPs investigated (P<0.05). The results obtained by the cell viability studies (MTT and LDH leakage assays) showed that C2 NP suspension did not induce plasma membrane damage or decrease cell viability and could be safely applied to Caco-2 cells in the concentration range tested (<400 microg/ml).

Suspension plasma spraying of La0.6Sr0.4Co0.2Fe0.8O3-δ cathodes: Influence of carbon black pore former on performance and degradation

NASA Astrophysics Data System (ADS)

Fan, E. S. C.; Kuhn, J.; Kesler, O.

2016-06-01

Suspension plasma spray deposition is utilized to fabricate solid oxide fuel cell cathodes with minimal material decomposition. Adding carbon black as a pore former to the feedstock suspension results in smoother and more porous coatings, but over the range of carbon black concentrations studied, has little impact on the overall symmetrical cell performance. The cathode made with a suspension containing 25 wt% carbon has the highest deposition efficiency and a polarization resistance of 0.062 Ωcm2 at 744 °C. This cathode is tested for 500 h, and it is observed that adding an SDC interlayer between the YSZ electrolyte and the cathode(s) and/or coating the metal substrate with lanthanum chromite decrease the rate of performance degradation.

Control over Silica Particle Growth and Particle–Biomolecule Interactions Facilitates Silica Encapsulation of Mammalian Cells with Thickness Control

DOE PAGES

Johnston, Robert K.; Harper, Jason C.; Tartis, Michaelann S.

2017-07-13

Over the past 20 years, many strategies utilizing sol–gel chemistry to integrate biological cells into silica-based materials have been reported. One such strategy, Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition, shows promise as an efficient encapsulation technique due to the ability to vary the silica encapsulation morphology obtained by this process through variation of SG-CViL reaction conditions. In this report, we develop SG-CViL as a tunable, multi-purpose silica encapsulation strategy by investigating the mechanisms governing both silica particle generation and subsequent interaction with phospholipid assemblies (liposomes and living cells). Using Dynamic Light Scattering (DLS) measurements, linear and exponential silica particlemore » growth dynamics were observed which were dependent on deposition buffer ion constituents and ion concentration. Silica particle growth followed a cluster–cluster growth mechanism at acidic pH, and a monomer-cluster growth mechanism at neutral to basic pH. Increasing silica sol aging temperature resulted in higher rates of particle growth and larger particles. DLS measurements employing PEG-coated liposomes and cationic liposomes, serving as model phospholipid assemblies, revealed that electrostatic interactions promote more stable liposome–silica interactions than hydrogen bonding and facilitate silica coating on suspension cells. However, continued silica reactivity leads to aggregation of silica-coated suspension cells, revealing the need for cell isolation to tune deposited silica thickness. As a result, utilizing these mechanistic study insights, silica was deposited onto adherent HeLa cells under biocompatible conditions with micrometer-scale control over silica thickness, minimal cell manipulation steps, and retained cell viability over several days.« less

Control over Silica Particle Growth and Particle–Biomolecule Interactions Facilitates Silica Encapsulation of Mammalian Cells with Thickness Control

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

Johnston, Robert K.; Harper, Jason C.; Tartis, Michaelann S.

Over the past 20 years, many strategies utilizing sol–gel chemistry to integrate biological cells into silica-based materials have been reported. One such strategy, Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition, shows promise as an efficient encapsulation technique due to the ability to vary the silica encapsulation morphology obtained by this process through variation of SG-CViL reaction conditions. In this report, we develop SG-CViL as a tunable, multi-purpose silica encapsulation strategy by investigating the mechanisms governing both silica particle generation and subsequent interaction with phospholipid assemblies (liposomes and living cells). Using Dynamic Light Scattering (DLS) measurements, linear and exponential silica particlemore » growth dynamics were observed which were dependent on deposition buffer ion constituents and ion concentration. Silica particle growth followed a cluster–cluster growth mechanism at acidic pH, and a monomer-cluster growth mechanism at neutral to basic pH. Increasing silica sol aging temperature resulted in higher rates of particle growth and larger particles. DLS measurements employing PEG-coated liposomes and cationic liposomes, serving as model phospholipid assemblies, revealed that electrostatic interactions promote more stable liposome–silica interactions than hydrogen bonding and facilitate silica coating on suspension cells. However, continued silica reactivity leads to aggregation of silica-coated suspension cells, revealing the need for cell isolation to tune deposited silica thickness. As a result, utilizing these mechanistic study insights, silica was deposited onto adherent HeLa cells under biocompatible conditions with micrometer-scale control over silica thickness, minimal cell manipulation steps, and retained cell viability over several days.« less

Interaction of Francisella tularensis bacterial cells with dynamic speckles

NASA Astrophysics Data System (ADS)

Ulianova, Onega V.; Ulyanov, Sergey S.; Sazanova, Elena V.; Zudina, Irina; Zhang, Zhihong; Sibo, Zhou; Luo, Qingming

2006-08-01

Influence of low-coherent speckles on the colonies grows is investigated. It has been demonstrated that effects of light on the inhibition of cells (Francisella Tularensis) are caused by speckle dynamics. The regimes of illumination of cell suspension with purpose of devitalization of hazard bacteria, caused very dangerous infections, such as tularemia, are found. Mathematical model of interaction of low-coherent laser radiation with bacteria suspension has been proposed. Computer simulations of the processes of laser-cells interaction have been carried out. Role of coherence of light in the processes of laser-cell interaction is analyzed.

Xenobiotics and loss of cell adhesion drive distinct transcriptional outcomes by aryl hydrocarbon receptor signaling.

PubMed

Hao, Nan; Lee, Kian Leong; Furness, Sebastian G B; Bosdotter, Cecilia; Poellinger, Lorenz; Whitelaw, Murray L

2012-12-01

The aryl hydrocarbon receptor (AhR) is a signal-regulated transcription factor, which is canonically activated by the direct binding of xenobiotics. In addition, switching cells from adherent to suspension culture also activates the AhR, representing a nonxenobiotic, physiological activation of AhR signaling. Here, we show that the AhR is recruited to target gene enhancers in both ligand [isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439)]-treated and suspension cells, suggesting a common mechanism of target gene induction between these two routes of AhR activation. However, gene expression profiles critically differ between xenobiotic- and suspension-activated AhR signaling. Por and Cldnd1 were regulated predominantly by ligand treatments, whereas, in contrast, ApoER2 and Ganc were regulated predominantly by the suspension condition. Classic xenobiotic-metabolizing AhR targets such as Cyp1a1, Cyp1b1, and Nqo1 were regulated by both ligand and suspension conditions. Temporal expression patterns of AhR target genes were also found to vary, with examples of transient activation, transient repression, or sustained alterations in expression. Furthermore, sequence analysis coupled with chromatin immunoprecipitation assays and reporter gene analysis identified a functional xenobiotic response element (XRE) in the intron 1 of the mouse Tiparp gene, which was also bound by hypoxia-inducible factor-1α during hypoxia and features a concatemer of four XRE cores (GCGTG). Our data suggest that this XRE concatemer site concurrently regulates the expression of both the Tiparp gene and its cis antisense noncoding RNA after ligand- or suspension-induced AhR activation. This work provides novel insights into how AhR signaling drives different transcriptional programs via the ligand versus suspension modes of activation.

Experimental Study of Membrane Fouling during Crossflow Microfiltration of Yeast and Bacteria Suspensions: Towards an Analysis at the Microscopic Level

PubMed Central

Ben Hassan, Ines; Ennouri, Monia; Lafforgue, Christine; Schmitz, Philippe; Ayadi, Abdelmoneim

2013-01-01

Microfiltration of model cell suspensions combining macroscopic and microscopic approaches was studied in order to better understand microbial membrane fouling mechanisms. The respective impact of Saccharomyces cerevisiae yeast and Escherichia coli bacteria on crossflow microfiltration performances was investigated using a multichannel ceramic 0.2 µm membrane. Pure yeast suspensions (5 µm ovoid cells) and mixtures of yeast and bacteria (1 to 2.5 µm rod shape cells) were considered in order to analyse the effect of interaction between these two microorganisms on fouling reversibility. The resistances varied significantly with the concentration and characteristics of the microorganisms. Membrane fouling with pure yeast suspension was mainly reversible. For yeast and bacteria mixed suspensions (6 g L−1 yeast concentration) the increase in bacteria from 0.15 to 0.30 g L−1 increased the percentage of normalized reversible resistance. At 10 g L−1 yeast concentration, the addition of bacteria tends to increase the percentage of normalized irreversible resistance. For the objective of performing local analysis of fouling, an original filtration chamber allowing direct in situ observation of the cake by confocal laser scanning microscopy (CLSM) was designed, developed and validated. This device will be used in future studies to characterize cake structure at the microscopic scale. PMID:24958619

Semi-microdroplet assay for cell adhesion molecules. M.S. Thesis

NASA Technical Reports Server (NTRS)

Tawa, Lawrence Shinzo

1988-01-01

A new cell-to-cell adhesion assay was devised. Using dissociated embryos of the sea urchin, this procedure involves rotating a 0.100 ml suspension of single cells with 0.100 ml of the solution to be tested in the bulb portion of a transfer pipet with the tip removed. After 1 hour of rotation at 60 rpm at 15 C, the contents of each bulb were transferred into individual wells of a 96 well flat bottom plate. After the plate was incubated for 1 hour at 15 C, black and white photographs were taken with a 35 mm camera attached to an inverted photomicroscope. Examining a proof sheet of the negatives directly allowed a rapid evaluation of suspected cell adhesion promoting factors. A ranking system was used to evaluate all samples. The assay was tested by examining the effect of specific solutions on the aggregation of single cells obtained from dissociated 23 hour embryos.

Cell-Specific Multifunctional Processing of Heterogeneous Cell Systems in a Single Laser Pulse Treatment

PubMed Central

Lukianova-Hleb, Ekaterina Y.; Mutonga, Martin B. G.; Lapotko, Dmitri O.

2012-01-01

Current methods of cell processing for gene and cell therapies use several separate procedures for gene transfer and cell separation or elimination, because no current technology can offer simultaneous multi-functional processing of specific cell sub-sets in highly heterogeneous cell systems. Using the cell-specific generation of plasmonic nanobubbles of different sizes around cell-targeted gold nanoshells and nanospheres, we achieved simultaneous multifunctional cell-specific processing in a rapid single 70 ps laser pulse bulk treatment of heterogeneous cell suspension. This method supported the detection of cells, delivery of external molecular cargo to one type of cells and the concomitant destruction of another type of cells without damaging other cells in suspension, and real-time guidance of the two above cellular effects. PMID:23167546

Effects of Selected Physicochemical Parameters on Zerumbone Production of Zingiber zerumbet Smith Cell Suspension Culture.

PubMed

Jalil, Mahanom; Annuar, Mohamad Suffian Mohamad; Tan, Boon Chin; Khalid, Norzulaani

2015-01-01

Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement.

Effects of Selected Physicochemical Parameters on Zerumbone Production of Zingiber zerumbet Smith Cell Suspension Culture

PubMed Central

Annuar, Mohamad Suffian Mohamad; Khalid, Norzulaani

2015-01-01

Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement. PMID:25767555

[Determination of Azospirillum Brasilense Cells With Bacteriophages via Electrooptical Analysis of Microbial Suspensions].

PubMed

Gulii, O I; Karavayeva, O A; Pavlii, S A; Sokolov, O I; Bunin, V D; Ignatov, O V

2015-01-01

The dependence-of changes in the electrooptical properties of Azospirillum brasilense cell suspension Sp7 during interaction with bacteriophage ΦAb-Sp7 on the number and time of interactions was studied. Incubation of cells with bacteriophage significantly changed the electrooptical signal within one minute. The selective effect of bacteriophage ΦAb on 18 strains of bacteria of the genus Azospirillum was studied: A. amazonense Ami4, A. brasilense Sp7, Cd, Sp107, Sp245, Jm6B2, Brl4, KR77, S17, S27, SR55, SR75, A. halopraeferans Au4, A. irakense KBC1, K A3, A. lipoferum Sp59b, SR65 and RG20a. We determined the limit of reliable determination of microbial cells infected with bacteriophage: - 10(4) cells/mL. The presence of foreign cell cultures of E. coli B-878 and E. coli XL-1 did not complicate the detection of A brasilense Sp7 cells with the use of bacteriophage ΦAb-Sp7. The results demonstrated that bacteriophage (ΦAb-Sp7 can be used for the detection of Azospirillum microbial cells via t electrooptical analysis of cell suspensions.

Composite anode for lithium ion batteries

DOEpatents

de Guzman, Rhet C.; Ng, K.Y. Simon; Salley, Steven O.

2018-03-06

A composite anode for a lithium-ion battery is manufactured from silicon nanoparticles having diameters mostly under 10 nm; providing an oxide layer on the silicon nanoparticles; dispersing the silicon nanoparticles in a polar liquid; providing a graphene oxide suspension; mixing the polar liquid containing the dispersed silicone nanoparticles with the graphene oxide suspension to obtain a composite mixture; probe-sonicating the mixture for a predetermined time; filtering the composite mixture to obtain a solid composite; drying the composite; and reducing the composite to obtain graphene and silicon.

Advances in the measurement of mud flocs within turbulent suspensions in both the laboratory and field

NASA Astrophysics Data System (ADS)

Strom, K.; Tran, D. A.; Dillon, B.

2017-12-01

Predicting the size and settling velocity of mud suspensions under the influence of flocculation is crucial for the accurate prediction of mud movement and deposition in sediment transport modeling of environments such as agricultural streams, large coastal rivers, estuaries, river plumes, boundary currents, and turbidity currents. Yet, collecting accurate and high resolution data on mud flocs is difficult. For example, measurement of flocs with camera systems generally provide the best avenue for preserving floc structure and obtaining accurate information about true floc sizes. However, capturing images of flocs in swirling turbulent flows can be difficult and often limited to suspensions where concentrations are low (< 100 mg/L). As a result, models that account for the influence of flocculation on mud settling velocity are based on sparse data that often present non-congruent functionality in floc size and settling velocity with basic influencers such as suspended sediment concentration. Here we present developments in image acquisition systems and processing methods that allow for measurement of floc size distributions in turbulent suspensions approaching 500 mg/L. At the heart of these developments are the introduction of a simple flow-through cell, modern digital sensor technology, and automated image processing. The combination of these elements allows for high-resolution times series of floc size populations to be measured in turbulent suspensions over a much broader range of suspended sediment concentration than has previously been possible — all without the need to transfer samples to a separate imaging container. We show applications and results from these developments in laboratory experiments and highlight their use in a newly-developed, low-cost, and field-deployable floc camera system.

Physiological responses of suspension cultures of Catharanthus roseus to aluminum: changes in polyamines and inorganic ions

Treesearch

Xinhua Zhou; Rakesh Minocha; Subhash C. Minocha

1995-01-01

The effects of aluminum (Al) treatment on polyamines were studied using suspension cultures of Madagascar periwinkle [Catharanthus roseus (L.) G. Don]. The addition of A1 (0.2, 0.5, 1.0 mM) to the suspension cultures caused a significant increase in putrescine within 24h only in freshly transferred cells. By contrast, Al treatment reduced putrescine...

Particle dynamics and particle-cell interaction in microfluidic systems

NASA Astrophysics Data System (ADS)

Stamm, Matthew T.

Particle-laden flow in a microchannel resulting in aggregation of microparticles was investigated to determine the dependence of the cluster growth rate on the following parameters: suspension void fraction, shear strain rate, and channel-height to particle-diameter ratio. The growth rate of an average cluster was found to increase linearly with suspension void fraction, and to obey a power-law relationships with shear strain rate as S 0.9 and channel-height to particle-diameter ratio as (h/d )--3.5. Ceramic liposomal nanoparticles and silica microparticles were functionalized with antibodies that act as targeting ligands. The bio-functionality and physical integrity of the cerasomes were characterized. Surface functionalization allows cerasomes to deliver drugs with selectivity and specificity that is not possible using standard liposomes. The functionalized particle-target cell binding process was characterized using BT-20 breast cancer cells. Two microfluidic systems were used; one with both species in suspension, the other with cells immobilized inside a microchannel and particle suspension as the mobile phase. Effects of incubation time, particle concentration, and shear strain rate on particle-cell binding were investigated. With both species in suspension, the particle-cell binding process was found to be reasonably well-described by a first-order model. Particle desorption and cellular loss of binding affinity in time were found to be negligible; cell-particle-cell interaction was identified as the limiting mechanism in particle-cell binding. Findings suggest that separation of a bound particle from a cell may be detrimental to cellular binding affinity. Cell-particle-cell interactions were prevented by immobilizing cells inside a microchannel. The initial stage of particle-cell binding was investigated and was again found to be reasonably well-described by a first-order model. For both systems, the time constant was found to be inversely proportional to particle concentration. The second system revealed the time constant to obey a power-law relationship with shear strain rate as tau∝ S-.37+/-.06. Under appropriate scaling, the behavior displayed in both systems is well-described by the same exponential curve. Identification of the appropriate scaling parameters allows for extrapolation and requires only two empirical values. This could provide a major head-start in any dosage optimization studies.

Changes in phytochelatins and their biosynthetic intermediates in red spruce (Picea rubens Sarg.) cell suspension cultures under cadmium and zinc stress

Treesearch

P. Thangavel; Stephanie Long; Rakesh Minocha

2007-01-01

Cell suspension cultures of red spruce (Picea rubens Sarg.) were selected to study the effects of cadmium (Cd) and zinc (Zn) on phytochelatins (PCs) and related metabolites after 24 h exposure. The PC2 and its precursor, γ-glutamylcysteine (γ-EC) increased two to fourfold with Cd concentrations ranging from 12...

Improved understanding of the acoustophoretic focusing of dense suspensions in a microchannel

NASA Astrophysics Data System (ADS)

Karthick, S.; Sen, A. K.

2017-11-01

We provide improved understanding of acoustophoretic focusing of a dense suspension (volume fraction φ >10 % ) in a microchannel subjected to an acoustic standing wave using a proposed theoretical model and experiments. The model is based on the theory of interacting continua and utilizes a momentum transport equation for the mixture, continuity equation, and transport equation for the solid phase. The model demonstrates the interplay between acoustic radiation and shear-induced diffusion (SID) forces that is critical in the focusing of dense suspensions. The shear-induced particle migration model of Leighton and Acrivos, coupled with the acoustic radiation force, is employed to simulate the continuum behavior of particles. In the literature, various closures for the diffusion coefficient Dφ* are available for rigid spheres at high concentrations and nonspherical deformable particles [e.g., red blood cells (RBCs)] at low concentrations. Here we propose a closure for Dφ* for dense suspension of RBCs and validate the proposed model with experimental data. While the available closures for Dφ* fail to predict the acoustic focusing of a dense suspension of nonspherical deformable particles like RBCs, the predictions of the proposed model match experimental data within 15%. Both the model and experiments reveal a competition between acoustic radiation and SID forces that gives rise to an equilibrium width w* of a focused stream of particles at some distance Leq* along the flow direction. Using different shear rates, acoustic energy densities, and particle concentrations, we show that the equilibrium width is governed by Péclet number Pe and Strouhal number Stas w*=1.4(PeSt) -0.5 while the length required to obtain the equilibrium-focused width depends on St as Leq*=3.8 /(St)0.6 . The proposed model and correlations would find significance in the design of microchannels for acoustic focusing of dense suspensions such as undiluted blood.

CELL SHAPE AND HEXOSE TRANSPORT IN NORMAL AND VIRUS-TRANSFORMED CELLS IN CULTURE

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

Bissell, M.J.; Farson, D.; Tung, A.S.C.

1976-07-01

The rate of hexose transport was compared in normal and virus-transformed cells on a monolayer and in suspension. It was shown that: (1) Both trypsin-removed cells and those suspended for an additional day in methyl cellulose had decreased rates of transport and lower available water space when compared with cells on a monolayer. Thus, cell shape affects the overall rate of hexose transport, especially at higher sugar concentrations. (2) Even in suspension, the initial transport rates remained higher in transformed cells with reference to normal cells. Scanning electron micrographs of normal and transformed chick cells revealed morphological differences only inmore » the flat state. This indicates that the increased rate of hexose transport after transformation is not due to a difference in the shape of these cells on a monolayer. The relation between the geometry of cells, transport rates, and growth regulation is undoubtedly very complex, and our knowledge of these relationships is still very elementary. In a recent review on the influence of geometry on control of cell growth, Folkman and Greenspan (1) pointed out that the permeability of cells in a flat versus a spherical state may indeed be very different. The growth properties of cells on a surface and in suspension have been compared often (1-5). However, with one exception. little is known about the changes in transport properties when cell shape is changed. Foster and Pardee (6) demonstrated that the active transport of a-aminoisobutyric acid was reduced 2.5 times in suspension cultures of Chinese hamster cells with respect to the cells grown on a coverslip. They attributed this to the smaller surface area of suspended cells. While it is not clear why active transport should be dependent on the surface area available, it is possible that once the cells assume a spherical configuration, the carrier proteins are redistributed in such a way as to make them less accessible to the substrate. What happens to facilitated and nonmediated diffusion when cells are placed in suspension has not been determined. The transport of hexoses into animal cells in culture has been shown to occur by facilitated diffusion (7). In chick embryo fibroblasts in culture, there is more than one component to this transport system: a saturable carrier-mediated transport with a Km for 2-deoxy-D-glucose (2DG) of about 1-5 mM (8-1 1) and a nonsaturable component which may include a low affinity transport site and/or nonmediated diffusion (8, 10, 11). It was shown previously (1 1) that growth rate, cell density, glucose deprivation, and virus transformation may alter not only the overall transport rate but also the rate of transport of one mode relative to the other. The rate of the nonmediated uptake, at least, is dependent upon the surface area available, and in addition total level of transport is dependent on the available water space (which in turn is roughly related to cell volume). Since it is estimated that the surface area may change as much as tenfold when cells go from a flat configuration to a spherical one (I), it is apparent that the cell shape could play a role in overall transport characteristics of cultured cells. Using glucose analogues, 2DG and 3-0-methylglucose (3MG), we compared the transport properties of normal and virus-transformed cells in suspension and on monolayers. It was found that both the rates as well as the total levels of transport were decreased after the cells were placed in suspension, with the nonsaturable component being most affected. Nevertheless, a difference in the initial rates of transport between normal and transformed cells remained, indicating that the difference is independent of cell shape.« less

Reading a Suspenseful Literary Text Activates Brain Areas Related to Social Cognition and Predictive Inference

PubMed Central

Lehne, Moritz; Engel, Philipp; Rohrmeier, Martin; Menninghaus, Winfried; Jacobs, Arthur M.; Koelsch, Stefan

2015-01-01

Stories can elicit powerful emotions. A key emotional response to narrative plots (e.g., novels, movies, etc.) is suspense. Suspense appears to build on basic aspects of human cognition such as processes of expectation, anticipation, and prediction. However, the neural processes underlying emotional experiences of suspense have not been previously investigated. We acquired functional magnetic resonance imaging (fMRI) data while participants read a suspenseful literary text (E.T.A. Hoffmann's “The Sandman”) subdivided into short text passages. Individual ratings of experienced suspense obtained after each text passage were found to be related to activation in the medial frontal cortex, bilateral frontal regions (along the inferior frontal sulcus), lateral premotor cortex, as well as posterior temporal and temporo-parietal areas. The results indicate that the emotional experience of suspense depends on brain areas associated with social cognition and predictive inference. PMID:25946306

Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes.

PubMed

Guliy, Olga I; Matora, Larisa Yu; Dykman, Lev A; Staroverov, Sergey A; Burygin, Gennady L; Bunin, Viktor D; Burov, Andrei M; Ignatov, Oleg V

2015-01-01

The exposure of Azospirillum brasilense carbohydrate epitopes was investigated by electro-optical analysis of bacterial cell suspensions. To study changes in the electro-optical (EO) properties of the suspensions, we used antibodies generated to the complete lipopolysaccharide of A. brasilense type strain Sp7 and also antibodies to the smooth and rough O polysaccharides of Sp7. After 18 hr of culture growth, the EO signal of the suspension treated with antibodies to smooth O polysaccharide was approximately 20% lower than that of the suspension treated with antibodies to complete lipopolysaccharide (control). After 72 hr of culture growth, the strongest EO signal was observed for the cells treated with antibodies to rough O polysaccharide (approximately 46% greater than the control), whereas for the cells treated with antibodies to smooth O polysaccharide, it was much lower (approximately 23% of the control). These data were confirmed by electron microscopy. The results of the study may have importance for the rapid evaluation of changes in lipopolysaccharide form in microbial biotechnology, when the antigenic composition of the bacterial surface requires close control.

Latest status of the clinical and industrial applications of cell sheet engineering and regenerative medicine.

PubMed

Egami, Mime; Haraguchi, Yuji; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2014-01-01

Cell sheet engineering, which allows tissue engineering to be realized without the use of biodegradable scaffolds as an original approach, using a temperature-responsive intelligent surface, has been applied in regenerative medicine for various tissues, and a number of clinical studies have been already performed for life-threatening diseases. By using the results and findings obtained from the initial clinical studies, additional investigative clinical studies in several tissues with cell sheet engineering are currently in preparation stage. For treating many patients effectively by cell sheet engineering, an automated system integrating cell culture, cell-sheet fabrication, and layering is essential, and the system should include an advanced three-dimensional suspension cell culture system and an in vitro bioreactor system to scale up the production of cultured cells and fabricate thicker vascularized tissues. In this paper, cell sheet engineering, its clinical application, and further the authors' challenge to develop innovative cell culture systems under newly legislated regulatory platform in Japan are summarized and discussed.

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

PubMed

Shackney, Stanley; Emlet, David R; Pollice, Agnese; Smith, Charles; Brown, Kathryn; Kociban, Deborah

2006-01-01

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. Copyright (c) 2005 Wiley-Liss, Inc.

NMR relaxometric properties and cytotoxicity of Gd2O3 nanoparticle suspensions in an organic liquid

NASA Astrophysics Data System (ADS)

Babić-Stojić, Branka; Jokanović, Vukoman; Milivojević, Dušan; Požek, Miroslav; Jagličić, Zvonko; Makovec, Darko; Arsikin, Katarina; Paunović, Verica

2014-10-01

Gd2O3 nanoparticles and their agglomerates from approximately 10 to 80 nm in size suspended in an organic liquid were synthesized via polyol route. The reaction between diethylene glycol and added acetic acid, which occurred simultaneously with the synthesis of Gd2O3 nanoparticles, was catalyzed by sodium bisulfate to transform as much as possible diethylene glycol in corresponding ester at the end of complete reaction. The produced nanosized material of gadolinium oxide was investigated by TEM, DLS, FTIR spectroscopy, and NMR relaxometry. Biological evaluation of this material was done by MTT and crystal violet assays to determine the cell viability. Longitudinal and transverse relaxivities of water-diluted Gd2O3 nanoparticle suspensions estimated to be r 1 = 13.6 and r 2 = 14.7 s-1 mM-1 are about three times higher compared to the relaxivities obtained for standard contrast agent Gd-DTPA (Magnevist). Good MRI signal intensities of the water-diluted Gd2O3 nanoparticle suspensions were recorded in the Gd concentration range 0.2-0.3 mM for which the suspensions were not toxic exhibiting simultaneously higher signal intensities than those for Magnevist in the Gd concentration range 0.4-1 mM for which this standard contrast agent was not toxic. These properties make the produced Gd2O3 nanoparticle material promising for potential application as MRI contrast agent.

Stress modeling in colloidal dispersions undergoing non-viscometric flows

NASA Astrophysics Data System (ADS)

Dolata, Benjamin; Zia, Roseanna

2017-11-01

We present a theoretical study of the stress tensor for a colloidal dispersion undergoing non-viscometric flow. In such flows, the non-homogeneous suspension stress depends on not only the local average total stresslet-the sum of symmetric first moments of both the hydrodynamic traction and the interparticle force-but also on the average quadrupole, octupole, and higher-order moments. To compute the average moments, we formulate a six dimensional Smoluchowski equation governing the microstructural evolution of a suspension in an arbitrary fluid velocity field. Under the conditions of rheologically slow flow, where the Brownian relaxation of the particles is much faster than the spatiotemporal evolution of the flow, the Smoluchowski equation permits asymptotic solution, revealing a suspension stress that follows a second-order fluid constitutive model. We obtain a reciprocal theorem and utilize it to show that all constitutive parameters of the second-order fluid model may be obtained from two simpler linear-response problems: a suspension undergoing simple shear and a suspension undergoing isotropic expansion. The consequences of relaxing the assumption of rheologically slow flow, including the appearance of memory and microcontinuum behaviors, are discussed.

Photomixing of chlamydomonas rheinhardtii suspensions

NASA Astrophysics Data System (ADS)

Dervaux, Julien; Capellazzi Resta, Marina; Abou, Bérengère; Brunet, Philippe

2014-11-01

Chlamydomonas rheinhardtii is a fast swimming unicellular alga able to bias its swimming direction in gradients of light intensity, an ability know as phototaxis. We have investigated experimentally both the swimming behavior of individual cells and the macroscopic response of shallow suspensions of these micro-organisms in response to a localized light source. At low light intensity, algae exhibit positive phototaxis and accumulate beneath the excitation light. In weakly concentrated thin layers, the balance between phototaxis and cell motility results in steady symmetrical patterns compatible with a purely diffusive model using effective diffusion coefficients extracted from the analysis of individual cell trajectories. However, at higher cell density and layer depth, collective effects induce convective flows around the light source. These flows disturb the cell concentration patterns which spread and may then becomes unstable. Using large passive tracer particles, we have characterized the velocity fields associated with this forced bioconvection and their dependence on the cell density and layer depth. By tuning the light distribution, this mechanism of photo-bioconvection allows a fine control over the local fluid flows, and thus the mixing efficiency, in algal suspensions.

Gravisensing, apoptosis, and drug recovery in Taxus cell suspensions

NASA Technical Reports Server (NTRS)

Durzan, D. J.

1999-01-01

Haploid and diploid cell suspensions of Taxus spp. were examined for their adaptive plasticity in response to simulated microgravity, unit gravity, and hypergravity. Cell suspensions produced the taxane, paclitaxel, (TAXOL (R)), which is useful for the treatment of various cancers. Amyloplasts contributed to taxane ring biosynthesis and to drug release at the cell wall. Drug-producing cells reacted as gravisensing osmotic tensiometers. In stressed cells, amyloplasts docked and fused in clusters to sites on the plasmalemma before taxane discharge into the culture medium. In simulated microgravity and compared to all other treatments, taxane production was reduced nearly 100-fold. The percent paclitaxel of total taxanes remained 3-to 6-fold greater, and biomass doubled. When p53-independent programmed cell death was induced, taxanes were released into the culture medium as free molecules (soluble and insoluble) or bound to membranes, nuclear fragments, xylan residues, and other particulate materials. Unit gravity and especially hypergravity promoted xylogenesis and significant drug overproduction. A model relating families of >touch = (TCH), taxane early response (TER), nuclear cycling, and apoptosis-regulating genes to gravisensing, cell wall modifications, and to taxane recovery accounted for most but not all of the observations.

Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor.

PubMed

Yan, Yuanwei; Song, Liqing; Tsai, Ang-Chen; Ma, Teng; Li, Yan

2016-01-01

Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.

Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

PubMed

Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

2016-05-15

Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

Dynamic changes in transcriptome and cell wall composition underlying brassinosteroid-mediated lignification of switchgrass suspension cells.

PubMed

Rao, Xiaolan; Shen, Hui; Pattathil, Sivakumar; Hahn, Michael G; Gelineo-Albersheim, Ivana; Mohnen, Debra; Pu, Yunqiao; Ragauskas, Arthur J; Chen, Xin; Chen, Fang; Dixon, Richard A

2017-01-01

Plant cell walls contribute the majority of plant biomass that can be used to produce transportation fuels. However, the complexity and variability in composition and structure of cell walls, particularly the presence of lignin, negatively impacts their deconstruction for bioenergy. Metabolic and genetic changes associated with secondary wall development in the biofuel crop switchgrass ( Panicum virgatum ) have yet to be reported. Our previous studies have established a cell suspension system for switchgrass, in which cell wall lignification can be induced by application of brassinolide (BL). We have now collected cell wall composition and microarray-based transcriptome profiles for BL-induced and non-induced suspension cultures to provide an overview of the dynamic changes in transcriptional reprogramming during BL-induced cell wall modification. From this analysis, we have identified changes in candidate genes involved in cell wall precursor synthesis, cellulose, hemicellulose, and pectin formation and ester-linkage generation. We have also identified a large number of transcription factors with expression correlated with lignin biosynthesis genes, among which are candidates for control of syringyl (S) lignin accumulation. Together, this work provides an overview of the dynamic compositional changes during brassinosteroid-induced cell wall remodeling, and identifies candidate genes for future plant genetic engineering to overcome cell wall recalcitrance.

Descriptive parameters of the erythrocyte aggregation phenomenon using a laser transmission optical chip

NASA Astrophysics Data System (ADS)

Toderi, Martín A.; Castellini, Horacio V.; Riquelme, Bibiana D.

2017-01-01

The study of red blood cell (RBC) aggregation is of great interest because of its implications for human health. Altered RBC aggregation can lead to microcirculatory problems as in vascular pathologies, such as hypertension and diabetes, due to a decrease in the erythrocyte surface electric charge and an increase in the ligands present in plasma. The process of erythrocyte aggregation was studied in stasis situation (free shear stresses), using an optical chip based on the laser transmission technique. Kinetic curves of erythrocyte aggregation under different conditions were obtained, allowing evaluation and characterization of this process. Two main characteristics of blood that influence erythrocyte aggregation were analyzed: the erythrocyte surface anionic charge (EAC) after digestion with the enzyme trypsin and plasmatic protein concentration in suspension medium using plasma dissolutions in physiological saline with human albumin. A theoretical approach was evaluated to obtain aggregation and disaggregation ratios by syllectograms data fitting. Sensible parameters (Amp100, t) regarding a reduced erythrocyte EAC were determined, and other parameters (AI, M-Index) resulted that are representative of a variation in the plasmatic protein content of the suspension medium. These results are very useful for further applications in biomedicine.

Continuous Microfluidics (Ecology-on-a-Chip) Experiments for Long Term Observation of Bacteria at Liquid-Liquid Interfaces

NASA Astrophysics Data System (ADS)

Miranda, Michael; White, Andrew; Jalali, Maryam; Sheng, Jian

2017-11-01

A microfluidic bioassay incorporating a peristaltic pump and chemostat capable of continuously culturing a bacterial suspension through a microchannel for an extended period of time relevant to ecological processes is presented. A single crude oil droplet is dispensed on-chip and subsequently pinned to the top and bottom surfaces of the microchannel to establish a vertical curved oil-water interface to observe bacteria without boundary interference. The accumulation of extracellular polymeric substances (EPS), microbial film formation, and aggregation is provided by DIC microscopy with an EMCCD camera at an interval of 30 sec. Cell-interface interactions such as cell translational and angular motilities as well as encountering, attachment, detachment to the interface are obtained by a high speed camera at 1000 fps with a sampling interval of 10 min. Experiments on Pseudomonas sp. (P62) and isolated EPS suspensions from Sagitulla Stelleta and Roseobacter show rapid formation of bacterial aggregates including EPS streamers stretching tens of drop diameters long. These results provide crucial insights into environmentally relevant processes such as the initiation of marine oil snow, an alternative mode of biodegradation to conventional bioconsumption. Funded by GoMRI, NSF, ARO.

Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis.

PubMed

Shrestha, B R; Tokuhara, K; Mii, M

2007-06-01

Protoplasts isolated from cell suspension culture of Phalaenopsis "Wataboushi" were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l L-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to greenhouse conditions.

Coupling Deep Transcriptome Analysis with Untargeted Metabolic Profiling in Ophiorrhiza pumila to Further the Understanding of the Biosynthesis of the Anti-Cancer Alkaloid Camptothecin and Anthraquinones

PubMed Central

Yamazaki, Mami; Mochida, Keiichi; Asano, Takashi; Nakabayashi, Ryo; Chiba, Motoaki; Udomson, Nirin; Yamazaki, Yasuyo; Goodenowe, Dayan B.; Sankawa, Ushio; Yoshida, Takuhiro; Toyoda, Atsushi; Totoki, Yasushi; Sakaki, Yoshiyuki; Góngora-Castillo, Elsa; Buell, C. Robin; Sakurai, Tetsuya; Saito, Kazuki

2013-01-01

The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila. PMID:23503598

Evaluation of analgesic, anti-inflammatory, anti-depressant and anti-coagulant properties of Lactuca sativa (CV. Grand Rapids) plant tissues and cell suspension in rats.

PubMed

Ismail, Hammad; Mirza, Bushra

2015-06-27

Lactuca sativa (lettuce) has been traditionally used for relieving pain, inflammation, stomach problems including indigestion and lack of appetite. Moreover, the therapeutic significance of L. sativa includes its anticonvulsant, sedative-hypnotic and antioxidant properties. In the present study, the MC (methanol and chloroform; 1:1) and aqueous extracts of seed and leaf along with cell suspension exudate were prepared. These extracts were explored for their analgesic, anti-inflammatory, antidepressant and anticoagulant effects by hot plate analgesic assay; carrageenan induced hind paw edema test, forced swimming test and capillary method for blood clotting respectively in a rat model. The results were analyzed using one-way Analysis of Variance (ANOVA) followed by Turkey multiple comparison test. Interestingly, the extracts and the cell suspension exudate showed dual inhibition by reducing pain and inflammation. The results indicated that the aqueous extracts of leaf exhibited highest analgesic and anti-inflammatory activities followed by leaf MC, cell suspension exudate, seed aqueous and seed MC extracts. The current findings show that aqueous and MC extracts of seed have the least immobility time in the forced swimming test, which could act as an anti-depressant on the central nervous system. The leaf extracts and cell suspension exudate also expressed moderate anti-depressant activities. In anticoagulant assay, the coagulation time of aspirin (positive control) and MC extract of leaf was comparable, suggesting strong anti-coagulant effect. Additionally, no abnormal behavior or lethality was observed in any animal tested. Taken together, L. sativa can potentially act as a strong herbal drug due to its multiple pharmaceutical effects and is therefore of interest in drug discovery and development of formulations.

Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

PubMed

Vera-Estrella, Rosario; Miranda-Vergara, Maria Cristina; Barkla, Bronwyn J

2009-03-01

Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L(-1) 2,4-dichlorophenoxyacetic acid, and 0.05 mg L(-1) benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 micromoles zinc g(-1) FW, and cell suspension cultures 30.9 micromoles zinc g(-1) DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.

Characterization of metal-supported axial injection plasma sprayed solid oxide fuel cells with aqueous suspension plasma sprayed electrolyte layers

NASA Astrophysics Data System (ADS)

Waldbillig, D.; Kesler, O.

A method for manufacturing metal-supported SOFCs with atmospheric plasma spraying (APS) is presented, making use of aqueous suspension feedstock for the electrolyte layer and dry powder feedstock for the anode and cathode layers. The cathode layer was deposited first directly onto a metal support, in order to minimize contact resistance, and to allow the introduction of added porosity. The electrolyte layers produced by suspension plasma spraying (SPS) were characterized in terms of thickness, permeability, and microstructure, and the impact of substrate morphology on electrolyte properties was investigated. Fuel cells produced by APS were electrochemically tested at temperatures ranging from 650 to 750 °C. The substrate morphology had little effect on open circuit voltage, but substrates with finer porosity resulted in lower kinetic losses in the fuel cell polarization.

Computer simulation of the processes of inactivation of bacterial cells by dynamic low-coherent speckles

NASA Astrophysics Data System (ADS)

Ulianova, Onega V.; Ulyanov, Sergey S.; Sazanova, Elena V.; Zhihong, Zhang; Sibo, Zhou; Luo, Qingming; Zudina, Irina; Bednov, Andrey

2006-05-01

Biochemical, biophysical and optical aspects of interaction of low-coherent light with bacterial cells have been discussed. Influence of low-coherent speckles on the colonies grows is investigated. It has been demonstrated that effects of light on the inhibition of cells (Francisella Tularensis) are connected with speckle dynamics. The regimes of illumination of cell suspension with purpose of devitalization of hazard bacteria, caused very dangerous infections, such as tularemia, are found. Mathematical model of interaction of low-coherent laser radiation with bacteria suspension has been proposed. Computer simulations of the processes of laser-cells interaction have been carried out.

Differentiating Pseudomonas sp. strain ADP cells in suspensions and biofilms using Raman spectroscopy and scanning electron microscopy.

PubMed

Henry, Victoria A; Jessop, Julie L P; Peeples, Tonya L

2017-02-01

High quality spectra of Pseudomonas sp. strain ADP in the planktonic and biofilm state were obtained using Raman microspectroscopy. These spectra enabled the identification of key differences between free and biofilm cells in the fingerprint region of Raman spectra in the nucleic acid, carbohydrate, and protein regions. Scanning electron microscopy (SEM) enabled detailed visualization of ADP biofilm with confirmation of associated extracellular matrix structure. Following extraction and Raman analysis of extracellular polymeric substances, Raman spectral differences between free and biofilm cells were largely attributed to the contribution of extracellular matrix components produced in mature biofilms. Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species. Graphical Abstract Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species.

Fast filtration sampling protocol for mammalian suspension cells tailored for phosphometabolome profiling by capillary ion chromatography - tandem mass spectrometry.

PubMed

Kvitvang, Hans F N; Bruheim, Per

2015-08-15

Capillary ion chromatography (capIC) is the premium separation technology for low molecular phosphometabolites and nucleotides in biological extracts. Removal of excessive amounts of salt during sample preparation stages is a prerequisite to enable high quality capIC separation in combination with reproducible and sensitive MS detection. Existing sampling protocols for mammalian cells used for GC-MS and LC-MS metabolic profiling can therefore not be directly applied to capIC separations. Here, the development of a fast filtration sampling protocol for mammalian suspension cells tailored for quantitative profiling of the phosphometabolome on capIC-MS/MS is presented. The whole procedure from sampling the culture to transfer of filter to quenching and extraction solution takes less than 10s. To prevent leakage it is critical that a low vacuum pressure is applied, and satisfactorily reproducibility was only obtained by usage of a vacuum pressure controlling device. A vacuum of 60mbar was optimal for filtration of multiple myeloma Jjn-3 cell cultures through 5μm polyvinylidene (PVDF) filters. A quick deionized water (DI-water) rinse step prior to extraction was tested, and significantly higher metabolite yields were obtained during capIC-MS/MS analyses in this extract compared to extracts prepared by saline and reduced saline (25%) washing steps only. In addition, chromatographic performance was dramatically improved. Thus, it was verified that a quick DI-water rinse is tolerated by the cells and can be included as the final stage during filtration. Over 30 metabolites were quantitated in JJN-3 cell extracts by using the optimized sampling protocol with subsequent capIC-MS/MS analysis, and up to 2 million cells can be used in a single filtration step for the chosen filter and vacuum pressure. The technical set-up is also highly advantageous for microbial metabolome filtration protocols after optimization of vacuum pressure and washing solutions, and the reduced salt content of the extract will also improve the quality of LC-MS analysis due to lower salt adduct ion formation. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression of a highly basic peroxidase gene in NaCl-adapted tomato cell suspensions.

PubMed

Medina, M I; Botella, M A; Quesada, M A; Valpuesta, V

1997-05-05

A tomato peroxidase gene, TPX2, that is only weakly expressed in the roots of young tomato seedlings is highly expressed in tomato suspension cells adapted to high external NaCl concentration. The protein encoded by this gene, with an isolectric point value of approximately 9.6, is found in the culture medium of the growing cells. Our data suggest that the expression of TPX2 in the salt-adapted cells is not the result of the elicitation imposed by the in vitro culture or the presence of high NaCl concentration in the medium.

Role of cellular adhesions in tissue dynamics spectroscopy

NASA Astrophysics Data System (ADS)

Merrill, Daniel A.; An, Ran; Turek, John; Nolte, David

2014-02-01

Cellular adhesions play a critical role in cell behavior, and modified expression of cellular adhesion compounds has been linked to various cancers. We tested the role of cellular adhesions in drug response by studying three cellular culture models: three-dimensional tumor spheroids with well-developed cellular adhesions and extracellular matrix (ECM), dense three-dimensional cell pellets with moderate numbers of adhesions, and dilute three-dimensional cell suspensions in agarose having few adhesions. Our technique for measuring the drug response for the spheroids and cell pellets was biodynamic imaging (BDI), and for the suspensions was quasi-elastic light scattering (QELS). We tested several cytoskeletal chemotherapeutic drugs (nocodazole, cytochalasin-D, paclitaxel, and colchicine) on three cancer cell lines chosen from human colorectal adenocarcinoma (HT-29), human pancreatic carcinoma (MIA PaCa-2), and rat osteosarcoma (UMR-106) to exhibit differences in adhesion strength. Comparing tumor spheroid behavior to that of cell suspensions showed shifts in the spectral motion of the cancer tissues that match predictions based on different degrees of cell-cell contacts. The HT-29 cell line, which has the strongest adhesions in the spheroid model, exhibits anomalous behavior in some cases. These results highlight the importance of using three-dimensional tissue models in drug screening with cellular adhesions being a contributory factor in phenotypic differences between the drug responses of tissue and cells.

Myosin II Activity Softens Cells in Suspension.

PubMed

Chan, Chii J; Ekpenyong, Andrew E; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

2015-04-21

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Activity-induced regulation of myosin isoform distribution - Comparison of two contractile activity programs

NASA Technical Reports Server (NTRS)

Diffee, Gary M.; Caiozzo, Vince J.; Mccue, Samuel A.; Herrick, Robert E.; Baldwin, Kenneth M.

1993-01-01

This study examined the role of specific types of contractile activity in regulating myosin heavy chain (MHC) isoform expression in rodent soleus. A combination of hindlimb suspension (SN) and two programmed contractile training activity paradigms, either isometric contractile activity (ST-IM) or high-load slowly shortening isovelocity activity, were utilized. Both training paradigms increased muscle mass compared with SN alone. However, only ST-IM resulted in a partial prevention of the suspension-induced decrease in type I MHC. With the use of a fluorescently labeled antibody to type IIa MHC, the distribution of MHCs among fibers was examined immunohistochemically. In SN, the percentage of cells staining positive for type IIa MHC was increased but the staining intensity of the positively staining cells was unchanged compared with control cells. In the ST-IM soleus, the percentage of positively staining fibers was unchanged but the intensity of the positively staining cells was decreased compared with SN values. These results suggest that 1) isometric contractile activity is more effective than isovelocity activity in preventing suspension-induced shifts in soleus MHC distribution and 2) changes associated with both suspension and training occur in only a small number of fibers, with the majority of fibers apparently unresponsive to these interventions.

Methods for suspension culture, protoplast extraction, and transformation of high-biomass yielding perennial grass Arundo donax.

PubMed

Pigna, Gaia; Dhillon, Taniya; Dlugosz, Elizabeth M; Yuan, Joshua S; Gorman, Connor; Morandini, Piero; Lenaghan, Scott C; Stewart, C Neal

2016-12-01

Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in Arundo. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 µM 2,4-D and 4.4 µM BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a five-fold increase in tissue mass over 14 days. In contrast, CIM medium containing 13 µM 2,4-D, resulted in just a 1.4-fold increase in mass over the same period. Optimized suspension cultures were superior to previously-described solidified medium-based callus culture methods for tissue mass increase. Suspension cultures proved to be very effective for subsequent protoplast isolation. Protoplast electroporation resulted in a 3.3 ± 1.5% transformation efficiency. A dual fluorescent reporter gene vector enabled the direct comparison of the CAMV 35S promoter with the switchgrass ubi2 promoter in single cells of Arundo. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in Arundo. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effective viscosity of a suspension of flagellar-beating microswimmers: Three-dimensional modeling

NASA Astrophysics Data System (ADS)

Jibuti, Levan; Zimmermann, Walter; Rafaï, Salima; Peyla, Philippe

2017-11-01

Micro-organisms usually can swim in their liquid environment by flagellar or ciliary beating. In this numerical work, we analyze the influence of flagellar beating on the orbits of a swimming cell in a shear flow. We also calculate the effect of the flagellar beating on the rheology of a dilute suspension of microswimmers. A three-dimensional model is proposed for Chlamydomonas Reinhardtii swimming with a breaststroke-like beating of two anterior flagella modeled by two counter-rotating fore beads. The active swimmer model reveals unusual angular orbits in a linear shear flow. Namely, the swimmer sustains orientations transiently across the flow. Such behavior is a result of the interplay between shear flow and the swimmer's periodic beating motion of flagella, which exert internal torques on the cell body. This peculiar behavior has some significant consequences on the rheological properties of the suspension. We calculate Einstein's viscosity of the suspension composed of such isolated modeled microswimmers (dilute case) in a shear flow. We use numerical simulations based on a Rotne-Prager-like approximation for hydrodynamic interaction between simplified flagella and the cell body. The results show an increased intrinsic viscosity for active swimmer suspensions in comparison to nonactive ones as well as a shear thinning behavior in accordance with previous experimental measurements [Phys. Rev. Lett. 104, 098102 (2010), 10.1103/PhysRevLett.104.098102].

Survival of salmonella transformed to express green fluorescent protein on Italian parsley as affected by processing and storage.

PubMed

Duffy, E A; Cisneros-Zevallos, L; Castillo, A; Pillai, S D; Ricke, S C; Acuff, G R

2005-04-01

To study the effect of processing and storage parameters on the survival of Salmonella on fresh Italian parsley, parsley bunches were dipped for 3 or 15 min in suspensions that were preequilibrated to 5, 25, or 35 degrees C and inoculated with Salmonella transformed to express enhanced green fluorescent protein. Loosely attached and/or associated, strongly attached and/or associated, and internalized and/or entrapped Salmonella cells were enumerated over 0, 1, and 7 days of storage at 25 degrees C and over 0, 1, 7, 14, and 30 days of storage at 4 degrees C using surface-plating procedures. Leaf sections obtained from samples after 0, 1, and 7 days of storage were examined using confocal scanning laser microscopy. Temperature of the dip suspension had little effect on the attachment and survival of Salmonella cells on parsley. Regardless of the temperature or duration of dip, Salmonella was internalized. Immersion for longer times resulted in higher numbers of attached and internalized cells. Microscopic observations supported these results and revealed Salmonella cells near the stomata and within cracks in the cuticle. Storage temperature had the greatest impact on the survival of Salmonella cells on parsley. When stored at 25 degrees C, parsley had a shelf life of 7 days, and Salmonella populations significantly increased over the 7 days of storage. For parsley stored at 4 degrees C, numbers of Salmonella cells decreased over days 0, 1, and 7. After 7 days of storage, there were no viable internalized Salmonella cells detected. Storage temperature represents an important control point for the safety of fresh parsley.

Stilbene production in cell cultures of Vitis vinifera L. cvs Red Globe and Michele Palieri elicited by methyl jasmonate.

PubMed

Santamaria, A R; Antonacci, D; Caruso, G; Cavaliere, C; Gubbiotti, R; Lagana, A; Valletta, A; Pasqua, G

2010-09-01

Cell cultures obtained from Vitis vinifera cvs Michele Palieri and Red Globe were cultured in order to stimulate stilbene production. In the calli, stilbene production peaked at day 22 of culture for both cultivars; the main compound was trans-piceid, followed by cis-piceid. Methyl jasmonate, which was added to cell suspensions in the first half of the exponential growth phase, enhanced stilbene accumulation, producing mainly trans-piceid and epsilon-viniferin. Other stilbenoids, though in lower quantities, were identified by liquid chromatography/positive electrospray mass spectrometry. epsilon-Viniferin and trans-resveratrol were the main compounds released into the culture medium. The total quantity of stilbenes was genotype dependent, with a better response found for the cv Red Globe.

Colloidal spray method for low cost thin coating deposition

DOEpatents

Pham, Ai-Quoc; Glass, Robert S.; Lee, Tae H.

2005-01-25

A dense or porous coating of material is deposited onto a substrate by forcing a colloidal suspension through an ultrasonic nebulizer and spraying a fine mist of particles in a carrier medium onto a sufficiently heated substrate. The spraying rate is essentially matched to the evaporation rate of the carrier liquid from the substrate to produce a coating that is uniformly distributed over the surface of the substrate. Following deposition to a sufficient coating thickness, a single sintering step may be used to produce a dense ceramic coating. Using this method, coatings ranging in thickness from about one to several hundred microns can be obtained. By using a plurality of compounds in the colloidal suspension, coatings of mixed composition can be obtained. By using a plurality of solutions and separate pumps and a single or multiple ultrasonic nebulizer(s), and varying the individual pumping rates and/or the concentrations of the solutions, a coating of mixed and discontinuously graded (e.g., stepped) or continuously graded layers may be obtained. This method is particularly useful for depositing ceramic coatings. Dense ceramic coating materials on porous substrates are useful in providing improved electrode performance in devices such as high power density solid oxide fuel cells. Dense ceramic coatings obtained by the invention are also useful for gas turbine blade coatings, sensors, steam electrolyzers, etc. The invention has general use in preparation of systems requiring durable and chemically resistant coatings, or coatings having other specific chemical or physical properties.

Colloidal spray method for low cost thin coating deposition

DOEpatents

Pham, Ai-Quoc; Glass, Robert S.; Lee, Tae H.

2002-01-01

A dense or porous coating of material is deposited onto a substrate by forcing a colloidal suspension through an ultrasonic nebulizer and spraying a fine mist of particles in a carrier medium onto a sufficiently heated substrate. The spraying rate is essentially matched to the evaporation rate of the carrier liquid from the substrate to produce a coating that is uniformly distributed over the surface of the substrate. Following deposition to a sufficient coating thickness, a single sintering step may be used to produce a dense ceramic coating. Using this method, coatings ranging in thickness from about one to several hundred microns can be obtained. By using a plurality of compounds in the colloidal suspension, coatings of mixed composition can be obtained. By using a plurality of solutions and separate pumps and a single or multiple ultrasonic nebulizer(s), and varying the individual pumping rates and/or the concentrations of the solutions, a coating of mixed and discontinuously graded (e.g., stepped) or continuously graded layers may be obtained. This method is particularly useful for depositing ceramic coatings. Dense ceramic coating materials on porous substrates are useful in providing improved electrode performance in devices such as high power density solid oxide fuel cells. Dense ceramic coatings obtained by the invention are also useful for gas turbine blade coatings, sensors, steam electrolyzers, etc. The invention has general use in preparation of systems requiring durable and chemically resistant coatings, or coatings having other specific chemical or physical properties.

Second-harmonic generation of ZnO nanoparticles synthesized by laser ablation of solids in liquids

NASA Astrophysics Data System (ADS)

Rocha-Mendoza, Israel; Camacho-López, Santiago; Luna-Palacios, Yryx Y.; Esqueda-Barrón, Yasmín; Camacho-López, Miguel A.; Camacho-López, Marco; Aguilar, Guillermo

2018-02-01

We report the synthesis of small zinc oxide nanoparticles (ZnO NPs) based colloidal suspensions and the study of second-harmonic generation from aggregated ZnO NPs deposited on glass substrates. The colloidal suspensions were obtained using the laser ablation of solids in liquids technique, ablating a Zn solid target immersed in acetone as the liquid medium, with ns-laser pulses (1064 nm) of a Nd-YAG laser. The per pulse laser fluence, the laser repetition rate frequency and the ablation time were kept constant. The absorption evolution of the obtained suspensions was optically characterized through absorption spectroscopy until stabilization. Raman spectroscopy, SEM and HRTEM were used to provide evidence of the ZnO NPs structure. HRTEM results showed that 5-8 nm spheroids ZnO NPs were obtained. Strong second-harmonic signal is obtained from random ZnO monocrystalline NPs and from aggregated ZnO NPs, suggesting that the high efficiency of the nonlinear process may not depend on the NPs size or aggregation state.

PHYSIOLOGY OF NITROGEN FIXATION BY BACILLUS POLYMYXA

PubMed Central

Grau, F. H.; Wilson, P. W.

1962-01-01

Grau, F. H. (University of Wisconsin, Madison) and P. W. Wilson. Physiology of nitrogen fixation by Bacillus polymyxa. J. Bacteriol. 83:490–496. 1962.—Of 17 strains of Bacillus polymyxa tested for fixation of molecular nitrogen, 15 fixed considerable quantities (30 to 150 μg N/ml). Two strains of the closely related B. macerans did not use N2, but possibly other members of this species may do so. Confirmation of fixation was obtained by showing incorporation of N15 into cell material. Both iron and molybdenum are specifically required for fixation; without the addition of these metals to the nitrogen-free medium, the growth rate and the total nitrogen fixed were reduced about 30 to 50%. No requirement for added molybdenum could be shown when ammonia was the nitrogen source, and the absence of iron caused only a slight decrease in growth. Washed-cell suspensions of B. polymyxa containing an active hydrogenase readily incorporated N15 into cell materials when provided with mannitol, glucose, or pyruvate but not when formate was the substrate. Hydrogen is a specific inhibitor of fixation, reducing both the rate and final amount of nitrogen fixed; it did not reduce growth on ammonia. Fixation was strictly anaerobic, 1% oxygen in the gas phase being sufficient to stop fixation. Arsenate is a powerful inhibitor of fixation of N2 by washed-cell suspensions of B. polymyxa, indicating that high-energy phosphate may be significant for this process. PMID:13901244

Design of covalently functionalized carbon nanotubes filled with metal oxide nanoparticles for imaging, therapy, and magnetic manipulation.

PubMed

Liu, Xiaojie; Marangon, Iris; Melinte, Georgian; Wilhelm, Claire; Ménard-Moyon, Cécilia; Pichon, Benoit P; Ersen, Ovidiu; Aubertin, Kelly; Baaziz, Walid; Pham-Huu, Cuong; Bégin-Colin, Sylvie; Bianco, Alberto; Gazeau, Florence; Bégin, Dominique

2014-11-25

Nanocomposites combining multiple functionalities in one single nano-object hold great promise for biomedical applications. In this work, carbon nanotubes (CNTs) were filled with ferrite nanoparticles (NPs) to develop the magnetic manipulation of the nanotubes and their theranostic applications. The challenges were both the filling of CNTs with a high amount of magnetic NPs and their functionalization to form biocompatible water suspensions. We propose here a filling process using CNTs as nanoreactors for high-yield in situ growth of ferrite NPs into the inner carbon cavity. At first, NPs were formed inside the nanotubes by thermal decomposition of an iron stearate precursor. A second filling step was then performed with iron or cobalt stearate precursors to enhance the encapsulation yield and block the formed NPs inside the tubes. Water suspensions were then obtained by addition of amino groups via the covalent functionalization of the external surface of the nanotubes. Microstructural and magnetic characterizations confirmed the confinement of NPs into the anisotropic structure of CNTs making them suitable for magnetic manipulations and MRI detection. Interactions of highly water-dispersible CNTs with tumor cells could be modulated by magnetic fields without toxicity, allowing control of their orientation within the cell and inducing submicron magnetic stirring. The magnetic properties were also used to quantify CNTs cellular uptake by measuring the cell magnetophoretic mobility. Finally, the photothermal ablation of tumor cells could be enhanced by magnetic stimulus, harnessing the hybrid properties of NP loaded-CNTs.

Engineering Multifunctional Living Paints: Thin, Convectively-Assembled Biocomposite Coatings of Live Cells and Colloidal Latex Particles Deposited by Continuous Convective-Sedimentation Assembly

NASA Astrophysics Data System (ADS)

Jenkins, Jessica Shawn

Advanced composite materials could be revolutionized by the development of methods to incorporate living cells into functional materials and devices. This could be accomplished by continuously and rapidly depositing thin ordered arrays of adhesive colloidal latex particles and live cells that maintain stability and preserve microbial reactivity. Convective assembly is one method of rapidly assembling colloidal particles into thin (<10 microm thick), ordered films with engineered compositions, thicknesses, and particle packing that offer several advantages over thicker randomly ordered composites, including enhanced cell stability and increased reactivity through minimized diffusion resistance to nutrients and reduced light scattering. This method can be used to precisely deposit live bacteria, cyanobacteria, yeast, and algae into biocomposite coatings, forming reactive biosensors, photoabsorbers, or advanced biocatalysts. This dissertation developed new continuous deposition and coating characterization methods for fabricating and characterizing <10 microm thick colloid coatings---monodispersed latex particle or cell suspensions, bimodal blends of latex particles or live cells and microspheres, and trimodal formulations of biomodal latex and live cells on substrates such as aluminum foil, glass, porous Kraft paper, polyester, and polypropylene. Continuous convective-sedimentation assembly (CSA) is introduced to enable fabrication of larger surface area and long coatings by constantly feeding coating suspension to the meniscus, thus expanding the utility of convective assembly to deposit monolayer or very thin films or multi-layer coatings composed of thin layers on a large scale. Results show thin, tunable coatings can be fabricated from diverse coating suspensions and critical coating parameters that control thickness and structure. Particle size ratio and charge influence deposition, convective mixing or demixing and relative particle locations. Substrate wettability and suspension composition influence coating microstructure by controlling suspension delivery and spreading across the substrate. Microbes behave like colloidal particles during CSA, allowing for deposition of very thin stable biocomposite coatings of latex-live cell blends. CSA of particle-cell blends result in open-packed structures (15-45% mean void space), instead of tightly packed coatings attainable with single component systems, confirming the existence of significant polymer particle-cell interactions and formation of particle aggregates that disrupt coating microstructure during deposition. Tunable process parameters, such as particle concentration, fluid sonication, and fluid density, influence coating homogeneity when the meniscus is continuously supplied. Fluid density modification and fluid sonication affect particle sedimentation and distribution in the coating growth front whereas the suspended particle concentration strongly affects coating thickness, but has almost no effect on void space. Changing the suspension delivery mode (topside versus underside CCSA) yields disparate meniscus volumes and uneven particle delivery to the drying front, which enables control of the coating microstructure by varying the total number of particles available for deposition. The judicious combination of all these parameters will enable deposition of uniform, thin, latex-cell monolayers over areas on the order of tens of square centimeters or larger. To demonstrate the utility of biocomposite coatings, this dissertation investigated photoreactive coatings (artificial leaves) from suspensions of latex particles and nitrogen-limited Rps. palustris CGA009 or sulfur-limited C. reinhardtii CC-124. These coatings demonstrated stable, sustained (>90 hours) photohydrogen production under anoxygenic conditions. Nutrient reduction slows cell division, minimizing coating outgrowth, and promotes photohydrogen generation, improving coating reactivity. Scanning electron microscopy of microstructure revealed how coating reactivity can be controlled by the size and distribution of the nanopores in the biocomposite layers. Variations in colloid microsphere size and suspension composition do not affect coating reactivity, but both parameters alter coating microstructure. Porous paper coated with thin coatings of colloidal particles and cells to enable coatings to be used in a gas-phase without dehydration may offer higher volumetric productivity for hydrogen production. Future work should focus on optimization of cell density, light intensity, media cycling, and acetate concentration.

Induction of high mitotic index in Petunia suspension cultures by sequential treatment with aphidicolin and colchicine.

PubMed

Guri, A; Zelcer, A; Izhar, S

1984-12-01

Significantly higher than normal mitotic index (MI) values were induced in Petunia cell suspensions following treatments with colchicine, aphidicolin, drastic medium replacement, or a sequential application of aphidicolin and colchicine. This last treatment yielded the highest MI values: cells incubated with 30 μg/ml aphidicolin for 18 h, then cultured in drug-free medium for 8 h and finally exposed to 0.1% colchicine for 8 additional hours exhibited MI of 62.8% and 65.7% respectively, for the two cell lines in study.

High aspect reactor vessel and method of use

NASA Technical Reports Server (NTRS)

Wolf, David A. (Inventor); Sams, Clarence F. (Inventor); Schwarz, Ray P. (Inventor)

1992-01-01

An improved bio-reactor vessel and system useful for carrying out mammalian cell growth in suspension in a culture media are presented. The main goal of the invention is to grow and maintain cells under a homogeneous distribution under acceptable biochemical environment of gas partial pressures and nutrient levels without introducing direct agitation mechanisms or associated disruptive mechanical forces. The culture chamber rotates to maintain an even distribution of cells in suspension and minimizes the length of a gas diffusion path. The culture chamber design is presented and discussed.

Behavior of yeast cells in aqueous suspension affected by pulsed electric field.

PubMed

El Zakhem, H; Lanoisellé, J-L; Lebovka, N I; Nonus, M; Vorobiev, E

2006-08-15

This work discusses pulsed electric fields (PEF) induced effects in treatment of aqueous suspensions of concentrated yeast cells (S. cerevisiae). The PEF treatment was done using pulses of near-rectangular shape, electric field strength was within E=2-5 kV/cm and the total time of treatment was t(PEF)=10(-4)-0.1 s. The concentration of aqueous yeast suspensions was in the interval of C(Y)=0-22 (wt%), where 1% concentration corresponds to the cellular density of 2x10(8) cells/mL. Triton X-100 was used for studying non-ionic surfactant additive effects. The electric current peak value I was measured during each pulse application, and from these data the electrical conductivity sigma was estimated. The PEF-induced damage results in increase of sigma with t(PEF) increasing and attains its saturation level sigma approximately sigma(max) at long time of PEF treatment. The value of sigma(max) reflects the efficiency of damage. The reduced efficiency of damage at suspension volume concentration higher than phi(Y) approximately 32 vol% is explained by the percolation phenomenon in the randomly packed suspension of near-spherical cells. The higher cytoplasmic ions leakage was observed in presence of surfactant. Experiments were carried out in the static and continuous flow treatment chambers in order to reveal the effects of mixing in PEF-treatment efficiency. A noticeable aggregation of the yeast cells was observed in the static flow chamber during the PEF treatment, while aggregation was not so pronounced in the continuous flow chamber. The nature of the enhanced aggregation under the PEF treatment was revealed by the zeta-potential measurements: these data demonstrate different zeta-potential signs for alive and dead cells. The effect of the electric field strength on the PEF-induced extraction of the intracellular components of S. cerevisiae is discussed.

The Current Status on the Overseas Development of Magnetic Suspension Railroads,

DTIC Science & Technology

1986-02-04

country appears to make all possible efforts to gain new technologies. Technologies obtained during the development stages are being applied to other...lQ63-A - 224 & FOREIGN TECHNOLOGY DIVISION IV-. THE CURRENT STATUS ON THE OVERSEAS DEVELOPMENT OF MAGNETIC SUSPENSION RAILROADS by Yoshiyasu 1Kyani...86 MICOFIHE R:FTD-86-C-001454 IV. THE CURRENT STATUS ON THE OVERSEAS DEVELOPMENT OF MAGNETIC SUSPENSION RAILROADS By: Yoshiyasu Kyotani English pages

Evaluation of massless-spring modeling of suspension-line elasticity during the parachute unfurling process

NASA Technical Reports Server (NTRS)

Poole, L. R.; Huckins, E. K., III

1972-01-01

A general theory on mathematical modeling of elastic parachute suspension lines during the unfurling process was developed. Massless-spring modeling of suspension-line elasticity was evaluated in detail. For this simple model, equations which govern the motion were developed and numerically integrated. The results were compared with flight test data. In most regions, agreement was satisfactory. However, poor agreement was obtained during periods of rapid fluctuations in line tension.

Rheological Characterization of Polyoxyethylene (POE) and Carboxymethyl Cellulose (CMC) Suspensions with Added Solids

NASA Astrophysics Data System (ADS)

Mora, A.; Skurtys, O.; Osorio, F.

2015-04-01

The rheological properties of high molecular weight POE and CMC suspensions by adding micro-metric solid particles such as fibers or spheres were studied. The volume fraction, Φ, was varied between 0 and 0.4. Their rheological properties were obtained after fitting a Cross model. For POE suspending fluid with spherical particle, the behavior of the normalized steady shear viscosity, μ/μ0, as function of the fraction volume followed a Thomas model. However, for CMC suspensions, μ/μ0 seems to be lineal with Φ. For a pure fluid or a suspension with Φ = 0; 2, the suspension presented an elastic behavior whereas it was observed a viscous behavior when the volume fraction was increased.

A zeta potential value determines the aggregate's size of penta-substituted [60]fullerene derivatives in aqueous suspension whereas positive charge is required for toxicity against bacterial cells.

PubMed

Deryabin, Dmitry G; Efremova, Ludmila V; Vasilchenko, Alexey S; Saidakova, Evgeniya V; Sizova, Elena A; Troshin, Pavel A; Zhilenkov, Alexander V; Khakina, Ekaterina A; Khakina, Ekaterina E

2015-08-08

The cause-effect relationships between physicochemical properties of amphiphilic [60]fullerene derivatives and their toxicity against bacterial cells have not yet been clarified. In this study, we report how the differences in the chemical structure of organic addends in 10 originally synthesized penta-substituted [60]fullerene derivatives modulate their zeta potential and aggregate's size in salt-free and salt-added aqueous suspensions as well as how these physicochemical characteristics affect the bioenergetics of freshwater Escherichia coli and marine Photobacterium phosphoreum bacteria. Dynamic light scattering, laser Doppler micro-electrophoresis, agarose gel electrophoresis, atomic force microscopy, and bioluminescence inhibition assay were used to characterize the fullerene aggregation behavior in aqueous solution and their interaction with the bacterial cell surface, following zeta potential changes and toxic effects. Dynamic light scattering results indicated the formation of self-assembled [60]fullerene aggregates in aqueous suspensions. The measurement of the zeta potential of the particles revealed that they have different surface charges. The relationship between these physicochemical characteristics was presented as an exponential regression that correctly described the dependence of the aggregate's size of penta-substituted [60]fullerene derivatives in salt-free aqueous suspension from zeta potential value. The prevalence of DLVO-related effects was shown in salt-added aqueous suspension that decreased zeta potential values and affected the aggregation of [60]fullerene derivatives expressed differently for individual compounds. A bioluminescence inhibition assay demonstrated that the toxic effect of [60]fullerene derivatives against E. coli cells was strictly determined by their positive zeta potential charge value being weakened against P. phosphoreum cells in an aquatic system of high salinity. Atomic force microscopy data suggested that the activity of positively charged [60]fullerene derivatives against bacterial cells required their direct interaction. The following zeta potential inversion on the bacterial cells surface was observed as an early stage of toxicity mechanism that violates the membrane-associated energetic functions. The novel data about interrelations between physicochemical parameters and toxic properties of amphiphilic [60]fullerene derivatives make possible predicting their behavior in aquatic environment and their activity against bacterial cells.

Induction of a photomixotrophic plant cell culture of Helianthus annuus and optimization of culture conditions for improved α-tocopherol production.

PubMed

Geipel, Katja; Song, Xue; Socher, Maria Lisa; Kümmritz, Sibylle; Püschel, Joachim; Bley, Thomas; Ludwig-Müller, Jutta; Steingroewer, Juliane

2014-03-01

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used, e.g., as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower (Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l(-1) sucrose, 0.5 mg l(-1) of the auxin 1-naphthalene acetic acid, and 0.5 mg l(-1) of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration, and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230%) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures.

Thumb-size ultrasonic-assisted spectroscopic imager for in-situ glucose monitoring as optional sensor of conventional dialyzers

NASA Astrophysics Data System (ADS)

Nogo, Kosuke; Mori, Keita; Qi, Wei; Hosono, Satsuki; Kawashima, Natsumi; Nishiyama, Akira; Wada, Kenji; Ishimaru, Ichiro

2016-03-01

We proposed the ultrasonic-assisted spectroscopic imaging for the realization of blood-glucose-level monitoring during dialytic therapy. Optical scattering and absorption caused by blood cells deteriorate the detection accuracy of glucose dissolved in plasma. Ultrasonic standing waves can agglomerate blood cells at nodes. In contrast, around anti-node regions, the amount of transmitted light increases because relatively clear plasma appears due to decline the number of blood cells. Proposed method can disperse the transmitted light of plasma without time-consuming pretreatment such as centrifugation. To realize the thumb-size glucose sensor which can be easily attached to dialysis tubes, an ultrasonic standing wave generator and a spectroscopic imager are required to be small. Ultrasonic oscillators are ∅30[mm]. A drive circuit of oscillators, which now size is 41×55×45[mm], is expected to become small. The trial apparatus of proposed one-shot Fourier spectroscopic imager, whose size is 30×30×48[mm], also can be little-finger size in principal. In the experiment, we separated the suspension mixed water and micro spheres (Θ10[mm) into particles and liquid regions with the ultrasonic standing wave (frequency: 2[MHz]). Furthermore, the spectrum of transmitted light through the suspension could be obtained in visible light regions with a white LED.

Magnetic suspension - Today's marvel, tomorrow's tool

NASA Technical Reports Server (NTRS)

Lawing, Pierce L.

1989-01-01

NASA's Langley facility has through constant advocacy of magnetic suspension systems (MSSs) for wind-tunnel model positioning obtained a technology-development status for the requisite large magnets, computers, automatic control techniques, and apparatus configurations, to contemplate the construction of MSSs for large wind tunnels. Attention is presently given to the prospects for MSSs in wind tunnels employing superfluid helium atmospheres to obtain very high Reynolds numbers, where the MSS can yield substantial enhancements of wind tunnel productivity.

On the dynamic response at the wheel axle of a pneumatic tire

NASA Astrophysics Data System (ADS)

Kung, L. E.; Soedel, W.; Yang, T. Y.

1986-06-01

A method for calculating the steady state displacement response and force transmission at the wheel axle of a pneumatic tire-suspension system due to a steady state force or displacement excitation at the tire to ground contact point is developed. The method requires the frequency responses (or receptances)_of both tire-wheel and suspension units. The frequency response of the tire-wheel unit is obtained by using the modal expansion method. The natural frequencies and mode shapes of the tire-wheel unit are obtained by using a geometrically non-linear, ring type, thin shell finite element of laminate composite. The frequency response of the suspension unit is obtained analytically. These frequency responses are used to calculate the force-input and the displacement-input responses at the wheel axle. This method allows the freedom of designing a vehicle and its tires independently and still achieving optimum dynamic performance.

Role of Abscisic Acid in the Induction of Freezing Tolerance in Brassica napus Suspension-Cultured Cells 1

PubMed Central

Johnson-Flanagan, Anne M.; Huiwen, Zhong; Thiagarajah, Mohan R.; Saini, Hargurdeep S.

1991-01-01

Brassica napus suspension-cultured cells could be hardened in 6 days at 25°C by the addition of mefluidide or ABA to the culture medium. Cells treated with mefluidide (10 milligrams per liter) or ABA (50 micromolar) attained an LT50 of −17.5°C or −18°C, respectively, while the LT50 for the comparable nonhardened control (sucrose) was −10°C. The increased freezing tolerance of mefluidide-treated cells was paralleled by a 4- to 23-fold increase in ABA, as measured by gas-liquid chromatography using electron capture detection. Application of 1 milligram per liter of fluridone, an inhibitor of abscisic acid biosynthesis, prevented the mefluidide-induced increase in freezing tolerance and the accumulation of ABA. Both these inhibitory effects of fluridone were overridden by 50 micromolar ABA in the culture medium. On the basis of these results, we concluded that increased ABA levels are important for the induction of freezing tolerance in suspension-cultured cells. PMID:16668089

Preservation of capsular material of streptococcal cells by specific lectins determined by immunoelectron microscopy.

PubMed

Molinari, A; Orefici, G; Donelli, G; Von Hunolstein, C; Paradisi, S; Arancia, G

1988-09-01

We describe the use of lectins as specific stabilizing agents for the polysaccharide capsular components of two Gram-positive bacteria, Streptococcus agalactiae and Streptococcus bovis. Treatment of bacterial suspensions with wheatgerm agglutinin and concanavalin A allowed better morphological preservation as well as immunoelectron microscopic localization of a capsular component (lipoteichoic acid) by employing specific antibodies and the protein A-gold technique. Data obtained indicate that lectins are useful agents in preserving highly water-soluble capsular components during the electron microscopy procedures for both unembedded and embedded samples.

Effects of aluminum on growth, polyamine metabolism, and inorganic ions in suspension cultures of red spruce (Picea rubens)

Treesearch

Rakesh Minocha; Walter C. Shortle; Daniel J. Jr. Coughin; Subhash C. Minocha

1996-01-01

The influence of age of red spruce (Picea rubens Sarg.) cell suspensions on aluminum (Al) effects was studied by adding AICI3 (0.2, 0.5, and 1.0 mM) to the media on each day of a 7-day culture period and analyzing for changes in total cell mass, polyamines, arginine decarboxylase activity, and inorganic ions after 24 h of...

Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

Treesearch

Rakesh Minocha; Subhash C. Minocha; Stephanie L. Long; Walter C. Shortle

1992-01-01

Increased aluminum (Al) solubility in soil waters due to acid precipitation has aroused considerable interest in the problem of Al toxicity in plants. In the present study, an in vitro suspension culture system of Catharanthus roseus (L.) G. Don was used to analyze the effects of aluminum on several biochemical processes in these cells. The aliphatic...

Optical trapping for complex fluid microfluidics

NASA Astrophysics Data System (ADS)

Vestad, Tor; Oakey, John; Marr, David W. M.

2004-10-01

Many proposed applications of microfluidics involve the manipulation of complex fluid mixtures such as blood or bacterial suspensions. To sort and handle the constituent particles within these suspensions, we have developed a miniaturized automated cell sorter using optical traps. This microfluidic cell sorter offers the potential to perform chip-top microbiology more rapidly and with less associated hardware and preparation time than other techniques currently available. To realize the potential of this technology in practical clinical and consumer lab-on-a-chip devices however, microscale control of not only particulates but also the fluid phase must be achieved. To address this, we have developed a mechanical fluid control scheme that integrates well with our optical separations approach. We demonstrate here a combined technique, one that employs both mechanical actuation and optical trapping for the precise control of complex suspensions. This approach enables both cell and particle separations as well as the subsequent fluid control required for the completion of complex analyses.

Plasminogen Activator Production Accompanies Loss of Anchorage Regulation in Transformation of Primary Rat Embryo Cells by Simian Virus 40

PubMed Central

Pollack, R.; Risser, R.; Conlon, S.; Rifkin, D.

1974-01-01

We have isolated several lines of rat embryo cells transformed by simian virus 40. All these lines are fully transformed with regard to saturation density and serum sensitivity, but they differ greatly in their anchorage dependence, as assayed by efficiency of plating in methyl cellulose suspension. This set of lines reveals a consistent relation of plasminogen activator production to plating efficiency in methyl cellulose. T-antigen-positive transformed lines that synthesize activator grow in methyl cellulose suspension, while T-antigen-positive transformed lines that do not synthesize activator fail to form colonies in suspension. Normal rat embryo cells produce very little plasminogen activator and do not grow in methyl cellulose. Sera that permit high levels of plasmin formation and activity support growth in semi-solid medium better than sera whose plasminogen is activated poorly and/or sera that contain inhibitors to plasmin. PMID:4373730

A technical feasibility study of surfactant-free drug suspensions using octenyl succinate-modified starches.

PubMed

Kuentz, Martin; Egloff, Peter; Röthlisberger, Dieter

2006-05-01

Many new drugs exhibit poor wetting behaviour and low aqueous solubility. This is particularly an issue for preclinical studies like toxicological trials, in which considerably higher doses and volumes are being administered compared to clinical studies. Preclinical vehicles typically contain high levels of surfactants that can exert biological effects. However, the biological inertness of vehicles is pivotal for the application in preclinical studies stressing the need in finding new excipients to solve formulation problems of today's drug discovery. The present study investigated the technical feasibility of surfactant-free suspensions using a new poorly soluble drug as model. It was shown that octenyl succinate-modified starches adequately wetted the drug and homogenous tasteless suspensions were obtained. The polymer xanthan gum was identified as macroscopically compatible gelling agent. Concentration effects of xanthan, drug and different modified starches were studied in a D-optimal design with respect to rheological properties. The suspensions were also tested in an analytical centrifuge using NIR transmission profiles to obtain a measure of sedimentation stability under accelerated conditions. The modified starches exhibited only little influence on the viscosity as well as on the yield point in contrast to the rheological effects of xanthan gum. This gelling agent was the main stabilising excipient as the modified starches hindered to a lesser extent sedimentation. The most stable suspensions displayed convenient flow properties. The viscosity at 100 s(-1) and 25 degrees C was in technically acceptable range of 120-140 mPa s in view of a application via gavage or a syringe in animal studies. The results demonstrated that surfactant-free drug suspensions with excellent technical performance can be obtained using octenyl succinate-modified starches. The vehicles were tasteless and based on the experience of modified starches in the food industry, the vehicles should exhibit good tolerability. The future use of such surfactant-free drug suspensions in toxicological, pharmacokinetic and pharmacodynamic studies will have to determine their advantage in terms of biological inertness.

The effect of crystal shape, size and bimodality on the maximum packing and the rheology of crystal bearing magma

NASA Astrophysics Data System (ADS)

Moitra, Pranabendu; Gonnermann, Helge

2014-05-01

Magma often contains crystals of various shapes and sizes. We present experimental results on the effect of the shape- and size-distribution of solid particles on the rheological properties of solid-liquid suspensions, which are hydrodynamically analogous to crystal-bearing magmas. The suspensions were comprised of either a single particle shape and size (unimodal) or a mixture of two different particle shapes and sizes (bimodal). For each type of suspension we characterized the dry maximum packing fraction of the particle mixture using the tap density method. We then systematically varied the total volume fraction of particles in the suspension, as well as the relative proportion of the two different particle types in the bimodal suspensions. For each of the resultant mixtures (suspensions) we performed controlled shear stress experiments using a rotational rheometer in parallel-plate geometry spanning 4 orders of magnitude in shear stress. The resultant data curves of shear stress as a function of shear rate were fitted using a Herschel-Bulkley rheological model. We find that the dry maximum packing decreases with increasing particle aspect ratio (ar) and decreasing particle size ratio (Λ). The highest dry maximum packing was obtained at 60-75% volume of larger particles for bimodal spherical particle mixture. Normalized consistency, Kr, defined as the ratio of the consistency of the suspension and the viscosity of the suspending liquid, was fitted using a Krieger-Dougherty model as a function of the total solid volume fraction (φ). The maximum packing fractions (φm) obtained from the shear experimental data fitting of the unimodal suspensions were similar in magnitude with the dry maximum packing fractions of the unimodal particles. Subsequently, we used the dry maximum packing fractions of the bimodal particle mixtures to fit Kr as a function of φ for the bimodal suspensions. We find that Kr increases rapidly for suspensions with larger ar and smaller Λ. We also find that both the apparent yield stress and the shear thinning behavior of the suspensions increase with increasing ar and become significant at φ/φm ≥ 0.4.

Cell Size Clues for the Allee Effect in Vegetative Amoeba Suspension Culture

NASA Astrophysics Data System (ADS)

Franck, Carl; Rappazzo, Brendan; Wang, Xiaoning; Segota, Igor

That cells proliferate at higher rates with increasing density helps us appreciate and understand the development of multicellular behavior through the study of dilute cell systems. However, arduous cell counting with a microscope reveals that in the model eukaryote, Dictyostelium discoideum this transition is difficult to ascertain and thereby further explore despite our earlier progress (Phys. Rev. E 77, 041905, (2008)). Here we report preliminary evidence that the slow proliferation phase is well characterized by reduced cell size compared to the wide distribution of cell sizes in the familiar exponential proliferation phase of moderate densities. This observation is enabled by a new system for characterizing cells in stirred suspension cultures. Our technique relies on quickly acquiring magnitude distributions of detected flashes of laser light scattered in situ by cell targets.

In-line mixing states monitoring of suspensions using ultrasonic reflection technique.

PubMed

Zhan, Xiaobin; Yang, Yili; Liang, Jian; Zou, Dajun; Zhang, Jiaqi; Feng, Luyi; Shi, Tielin; Li, Xiwen

2016-02-01

Based on the measurement of echo signal changes caused by different concentration distributions in the mixing process, a simple ultrasonic reflection technique is proposed for in-line monitoring of the mixing states of suspensions in an agitated tank in this study. The relation between the echo signals and the concentration of suspensions is studied, and the mixing process of suspensions is tracked by in-line measurement of ultrasonic echo signals using two ultrasonic sensors. Through the analysis of echo signals over time, the mixing states of suspensions are obtained, and the homogeneity of suspensions is quantified. With the proposed technique, the effects of impeller diameter and agitation speed on the mixing process are studied, and the optimal agitation speed and the minimum mixing time to achieve the maximum homogeneity are acquired under different operating conditions and design parameters. The proposed technique is stable and feasible and shows great potential for in-line monitoring of mixing states of suspensions. Copyright © 2015 Elsevier B.V. All rights reserved.

The effect of linear spring number at side load of McPherson suspension in electric city car

NASA Astrophysics Data System (ADS)

Budi, Sigit Setijo; Suprihadi, Agus; Makhrojan, Agus; Ismail, Rifky; Jamari, J.

2017-01-01

The function of the spring suspension on Mc Pherson type is to control vehicle stability and increase ride convenience although having tendencies of side load presence. The purpose of this study is to obtain simulation results of Mc Pherson suspension spring in the electric city car by using the finite element method and determining the side load that appears on the spring suspension. This research is conducted in several stages; they are linear spring designing models with various spring coil and spring suspension modeling using FEM software. Suspension spring is compressed in the vertical direction (z-axis) and at the upper part of the suspension springs will be seen the force that arises towards the x, y, and z-axis to simulate the side load arising on the upper part of the spring. The results of FEM simulation that the side load on the spring toward the x and y-axis which the value gets close to zero is the most stable spring.

Quantitative Evaluation of Cisplatin Uptake in Sensitive and Resistant Individual Cells by Single-Cell ICP-MS (SC-ICP-MS).

PubMed

Corte Rodríguez, M; Álvarez-Fernández García, R; Blanco, E; Bettmer, J; Montes-Bayón, M

2017-11-07

One of the main limitations to the Pt-therapy in cancer is the development of associated drug resistance that can be associated with a significant reduction of the intracellular platinum concentration. Thus, intracellular Pt concentration could be considered as a biomarker of cisplatin resistance. In this work, an alternative method to address intracellular Pt concentration in individual cells is explored to permit the evaluation of different cell models and alternative therapies in a relatively fast way. For this aim, total Pt analysis in single cells has been implemented using a total consumption nebulizer coupled to inductively coupled plasma mass spectrometric detection (ICP-MS). The efficiency of the proposed device has been evaluated in combination with flow cytometry and turned out to be around 25% (cells entering the ICP-MS from the cells in suspension). Quantitative uptake studies of a nontoxic Tb-containing compound by individual cells were conducted and the results compared to those obtained by bulk analysis of the same cells. Both sets of data were statistically comparable. Thus, final application of the developed methodology to the comparative uptake of Pt-species in cisplatin resistant and sensitive cell lines (A2780cis and A2780) was conducted. The results obtained revealed the potential of this analytical strategy to differentiate between different cell lines of different sensitivity to the drug which might be of high medical interest.

Characterization of highly scattering media by measurement of diffusely backscattered polarized light

DOEpatents

Hielscher, Andreas H.; Mourant, Judith R.; Bigio, Irving J.

2000-01-01

An apparatus and method for recording spatially dependent intensity patterns of polarized light that is diffusely backscattered from highly scattering media are described. These intensity patterns can be used to differentiate different turbid media, such as polystyrene-sphere and biological-cell suspensions. Polarized light from a He-Ne laser (.lambda.=543 nm) is focused onto the surface of the scattering medium, and a surface area of approximately 4.times.4 cm centered on the light input point is imaged through polarization analysis optics onto a CCD camera. A variety of intensity patterns may be observed by varying the polarization state of the incident laser light and changing the analyzer configuration to detect different polarization components of the backscattered light. Experimental results for polystyrene-sphere and Intralipid suspensions demonstrate that the radial and azimuthal variations of the observed pattern depend on the concentration, size, and anisotropy factor, g, of the particles constituting the scattering medium. Measurements performed on biological cell suspensions show that intensity patterns can be used to differentiate between suspensions of cancerous and non-cancerous cells. Introduction of the Mueller-matrix for diffusely backscattered light, permits the selection of a subset of measurements which comprehensively describes the optical properties of backscattering media.

Growth inhibition of heat-injured Enterococcus faecium by oligophosphates in a cured meat model.

PubMed

Houben, J H; Tjeerdsma-van Bokhoven, J L M

2004-12-01

Cells of two heat-resistant strains of Enterococcus faecium were heated and incubated in meat suspensions containing curing ingredients. The concentrations of the curing ingredients were those frequently used for pasteurized ham-type products, except that the concentrations of the oligophosphates (triphosphate and diphosphate) varied. Heating tests at 69 degrees C were performed with inoculated meat suspensions in heat-sealed plastic pouches. Numbers of bacteria were counted immediately after heating and in parallel series of heated pouches incubated at 37 degrees C. Plating was performed in Tryptone Dextrose Yeast Meat Peptonised Milk Agar (TDYMP); in TDYMP Agar to which the curing ingredients were added; and in TDYMP Agar to which the curing ingredients except oligophosphates were added. The inclusion of oligophosphates in the heating medium increased the heat-injury sustained by the E. faecium cells, and in combination with rather severe heat treatment even completely blocked the growth of surviving organisms in the meat suspension incubated at 37 degrees C. The presence of oligophosphates in the culture medium TDYMP Agar severely reduced the counts of freshly heated cells; however, this effect disappeared after repair and growth of the surviving organisms in the meat suspension.

THE ACTION OF EXTREME COLD ON LEUKEMIC CELLS OF MICE

PubMed Central

Breedis, Charles

1942-01-01

Suspensions of leukemic cells of mice from three different strains of leukemia were subjected to rapid or slow freezing and rapid or slow thawing. Suspensions rapidly frozen to –196°C. were in all cases innocuous, whereas those frozen slowly were capable of transmitting leukemia. The infectivity of slowly frozen material varied from an estimated 0.0001 per cent to 1 per cent of that of fresh material, and this figure probably represents the percentage of surviving leukemic cells. Particles of spleen and lymph node reacted to slow and rapid freezing in the same manner as suspensions prepared from them. For one of the strains rapid thawing was less injurious than slow thawing; for the other two the rate of thawing seemed to be immaterial. Infectivity was equally well preserved after freezing to –21°C. whether freezing occurred spontaneously after supercooling or was initiated near the freezing point by inoculation with ice, or whether thawing was slow or rapid. Suspensions already slowly frozen at temperatures of –2° or lower, whether spontaneously or by inoculation with ice, could no longer be completely inactivated by subsequent rapid cooling to –196°C. Unfrozen suspensions initially above the freezing point or supercooled to –2°C. or –8°C. and then rapidly cooled to –196°C. were inactivated. This protective action of previous slow freezing was most marked when the initial temperature of the frozen suspension was –15°C. or lower; when it was –2°C. protection was barely detected. These observations indicate that the changes which are peculiar to rapid freezing alone and lead to complete inactivation take place during rapid transition from the liquid to the solid state, in a range of temperature lying between –15°C. and the freezing point. Temperature measurements carried out in this range showed that suspensions were about equally infections whether the temperature at their centers dropped from 0°C. to –15°C. in 30 minutes or in 1 minute; when the drop occurred in 12 seconds or less, the suspensions became innocuous. PMID:19871231

Dynamic shear jamming in granular suspensions

NASA Astrophysics Data System (ADS)

Peters, Ivo; Majumdar, Sayantan; Jaeger, Heinrich

2014-11-01

Jamming by shear allows a frictional granular packing to transition from an unjammed state into a jammed state while keeping the system volume and average packing fraction constant. Shear jamming of dry granular media can occur quasi-statically, but boundaries are crucial to confine the material. We perform experiments in aqueous starch suspension where we apply shear using a rheometer with a large volume (400 ml) cylindrical Couette cell. In our suspensions the packing fraction is sufficiently low that quasi-static deformation does not induce a shear jammed state. Applying a shock-like deformation however, will turn the suspension into a jammed solid. A fully jammed state is reached within tens of microseconds, and can be sustained for at least several seconds. High speed imaging of the initial process reveals a jamming front propagating radially outward through the suspension, while the suspension near the outer boundary remains quiescent. This indicates that granular suspensions can be shear jammed without the need of confining solid boundaries. Instead, confinement is most likely provided by the dynamics in the front region.

Impedance spectroscopy assisted by magnetic nanoparticles as a potential biosensor principle for breast cancer cells in suspension.

PubMed

Silva, Jesús G; Cárdenas, Rey A; Quiróz, Alan R; Sánchez, Virginia; Lozano, Lucila M; Pérez, Nadia M; López, Jaime; Villanueva, Cleva; González, César A

2014-06-01

Breast cancer (BC) is the leading cause of cancer death in women worldwide, with a higher mortality reported in undeveloped countries. Ideal adjuvant therapeutic strategies require the continuous monitoring of patients by regular blood tests to detect circulating cancer cells, in order to determine whether additional treatment is necessary to prevent cancer dissemination. This circumstance requires a non-complex design of tumor cell biosensor in whole blood with feasibility for use in poor regions. In this work we have evaluated an inexpensive and simple technique of relative bioimpedance measurement, assisted by magnetic nanoparticles, as a potential biosensor of BC cells in suspension. Measurements represent the relative impedance changes caused by the magnetic holding of an interphase of tumor cells versus a homogenous condition in the frequency range of 10-100 kHz. The results indicate that use of a magnet to separate tumor cells in suspension, coupled to magnetic nanoparticles, is a feasible technique to fix an interphase of tumor cells in close proximity to gold electrodes. Relative impedance changes were shown to have potential value as a biosensor method for BC cells in whole blood, at frequencies around 20 kHz. Additional studies are warranted with respect to electrode design and sensitivity at micro-scale levels, according to the proposed technique.

Separation of sperm and epithelial cells based on the hydrodynamic effect for forensic analysis

PubMed Central

Liu, Weiran; Chen, Weixing; Liu, Ran; Ou, Yuan; Liu, Haoran; Xie, Lan; Lu, Ying; Li, Caixia; Li, Bin; Cheng, Jing

2015-01-01

In sexual assault cases, forensic samples are a mixture of sperm from the perpetrator and epithelial cells from the victim. To obtain an independent short tandem repeat (STR) profile of the perpetrator, sperm cells must be separated from the mixture of cells. However, the current method used in crime laboratories, namely, differential extraction, is a time-consuming and labor-intensive process. To achieve a rapid and automated sample pretreatment process, we fabricated a microdevice for hydrodynamic and size-based separation of sperm and epithelial cells. When cells in suspension were introduced into the device's microfluidic channels, they were forced to flow along different streamlines and into different outlets due to their different diameters. With the proposed microdevice, sperm can be separated within a short period of time (0.5 h for a 50-μl mock sample). The STR profiles of the products in the sperm outlet reservoir demonstrated that a highly purified male DNA fraction could be obtained (94.0% male fraction). This microdevice is of low-cost and can be easily integrated with other subsequent analysis units, providing great potential in the process of analyzing sexual assault evidence as well as in other areas requiring cell sorting. PMID:26392829

DC conductivity of a suspension of insulating particles with internal rotation

NASA Astrophysics Data System (ADS)

Pannacci, N.; Lemaire, E.; Lobry, L.

2009-04-01

We analyse the consequences of Quincke rotation on the conductivity of a suspension. Quincke rotation refers to the spontaneous rotation of insulating particles dispersed in a slightly conducting liquid and subject to a high DC electric field: above a critical field, each particle rotates continuously around itself with an axis pointing in any direction perpendicular to the DC field. When the suspension is subject to an electric field lower than the threshold one, the presence of insulating particles in the host liquid decreases the bulk conductivity since the particles form obstacles to ion migration. But for electric fields higher than the critical one, the particles rotate and facilitate ion migration: the effective conductivity of the suspension is increased. We provide a theoretical analysis of the impact of Quincke rotation on the apparent conductivity of a suspension and we present experimental results obtained with a suspension of PMMA particles dispersed in weakly conducting liquids.

In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions

PubMed Central

Akenhead, Michael L.; Horrall, Nolan M.; Rowe, Dylan; Sethu, Palaniappan; Shin, Hainsworth Y.

2015-01-01

Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s−1 shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia). PMID:26065495

In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions.

PubMed

Akenhead, Michael L; Horrall, Nolan M; Rowe, Dylan; Sethu, Palaniappan; Shin, Hainsworth Y

2015-09-01

Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s(-1) shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia).

Lipid extraction from microalgae using a single ionic liquid

DOEpatents

Salvo, Roberto Di; Reich, Alton; Dykes, Jr., H. Waite H.; Teixeira, Rodrigo

2013-05-28

A one-step process for the lysis of microalgae cell walls and separation of the cellular lipids for use in biofuel production by utilizing a hydrophilic ionic liquid, 1-butyl-3-methylimidazolium. The hydrophilic ionic liquid both lyses the microalgae cell walls and forms two immiscible layers, one of which consists of the lipid contents of the lysed cells. After mixture of the hydrophilic ionic liquid with a suspension of microalgae cells, gravity causes a hydrophobic lipid phase to move to a top phase where it is removed from the mixture and purified. The hydrophilic ionic liquid is recycled to lyse new microalgae suspensions.

Large-Scale Transient Transfection of Chinese Hamster Ovary Cells in Suspension.

PubMed

Rajendra, Yashas; Balasubramanian, Sowmya; Hacker, David L

2017-01-01

We describe a one-liter transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells using polyethyleneimine (PEI) for DNA delivery. The method involves transfection at a high cell density (5 × 10 6 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. We also describe an alternative method in which 90% of the pDNA is replaced by nonspecific (filler) DNA, and the production phase is performed at 31 °C in the presence of 0.25% N, N-dimethylacetamide (DMA).

An efficient sensitivity analysis method for modified geometry of Macpherson suspension based on Pearson correlation coefficient

NASA Astrophysics Data System (ADS)

Shojaeefard, Mohammad Hasan; Khalkhali, Abolfazl; Yarmohammadisatri, Sadegh

2017-06-01

The main purpose of this paper is to propose a new method for designing Macpherson suspension, based on the Sobol indices in terms of Pearson correlation which determines the importance of each member on the behaviour of vehicle suspension. The formulation of dynamic analysis of Macpherson suspension system is developed using the suspension members as the modified links in order to achieve the desired kinematic behaviour. The mechanical system is replaced with an equivalent constrained links and then kinematic laws are utilised to obtain a new modified geometry of Macpherson suspension. The equivalent mechanism of Macpherson suspension increased the speed of analysis and reduced its complexity. The ADAMS/CAR software is utilised to simulate a full vehicle, Renault Logan car, in order to analyse the accuracy of modified geometry model. An experimental 4-poster test rig is considered for validating both ADAMS/CAR simulation and analytical geometry model. Pearson correlation coefficient is applied to analyse the sensitivity of each suspension member according to vehicle objective functions such as sprung mass acceleration, etc. Besides this matter, the estimation of Pearson correlation coefficient between variables is analysed in this method. It is understood that the Pearson correlation coefficient is an efficient method for analysing the vehicle suspension which leads to a better design of Macpherson suspension system.

Source of cytotoxicity in a colloidal silver nanoparticle suspension.

PubMed

Hatipoglu, Manolya Kukut; Keleştemur, Seda; Altunbek, Mine; Culha, Mustafa

2015-05-15

Silver nanoparticles (AgNPs) are increasingly used in a variety of applications because of their potential antimicrobial activity and their plasmonic and conductivity properties. In this study, we investigated the source of cytotoxicity, genotoxicity, and reactive oxygen species (ROS) production on human dermal fibroblast and human lung cancer (A549) cell lines upon exposure to AgNP colloidal suspensions prepared with the simplest and most commonly used Lee–Meisel method with a variety of reaction times and the concentrations of the reducing agent. The AgNPs synthesized with shorter reaction times were more cytotoxic and genotoxic due to the presence of a few nanometer-sized AgNP seeds. The suspensions prepared with an increased citrate concentration were not cytotoxic, but they induced more ROS generation on A549 cells due to the high citrate concentration. The genotoxicity of the suspension decreased significantly at the higher citrate concentrations. The analysis of both transmission electron microscopy images from the dried droplet areas of the colloidal suspensions and toxicity data indicated that the AgNP seeds were the major source of toxicity. The completion of the nucleation step and the formation of larger AgNPs effectively decreased the toxicity.

Allelopathy of small everlasting (Antennaria microphylla) : Phytotoxicity to leafy spurge (Euphorbia esula) in tissue culture.

PubMed

Hogan, M E; Manners, G D

1990-03-01

Media and media extracts from callus cultures of small everlasting (Antennaria microphylla) inhibited leafy spurge (Euphorbia esula L.) callus tissue and suspension culture growth (50 and 70% of control, respectively) and were phytotoxic in lettuce and leafy spurge root elongation bioassays (64 and 77% of control, respectively). Hydroquinone, a phytotoxic compound previously isolated from small everlasting, was also biosynthesized by callus and suspension cultures of this species. Exogenously supplied hydroquinone (0.5 mM) was toxic to leafy spurge suspension culture cells and was only partially biotransformed to its nontoxic water-soluble monoglucoside, arbutin, by these cells. This report confirms the chronic involvement of hydroquinone in the allelopathic interaction between small everlasting and leafy spurge.

Gas hydrate suspensions formation and transportation research

NASA Astrophysics Data System (ADS)

Gulkov, A. N.; Gulkova, S.; Zemenkov, Yu D.; Lapshin, V. D.

2018-05-01

An experimental unit for studying the formation of gas hydrate suspensions and their transport properties is considered. The scheme of installation and the basic processes, which can be studied, are described. The results of studies of gas hydrates and a gas hydrate suspension’ formation in an adiabatic process in a stream of seawater are given. The adiabatic method of obtaining gas hydrates and forming gas hydrate suspensions is offered to use. Directions for further research are outlined.

Langerhans' cell expression of the selectin ligand, sialyl Lewis x.

PubMed Central

Ross, E L; Barker, J N; Allen, M H; Chu, A C; Groves, R W; MacDonald, D M

1994-01-01

Cellular adhesion molecules play a central role in leucocyte migration through peripheral blood and tissues. A crucial stage in these events in selectin-mediated adhesion involving E-selectin expressed on activated endothelium interacting with a range of carbohydrate ligands expressed by specific subpopulations of leucocytes. As such mechanisms may be relevant to bone marrow-derived dendritic epidermal Langerhans' cell (LC) migration, expression of these carbohydrate ligands was assessed immunocytochemically in whole skin biopsies and in epidermal cell suspensions obtained from adult humans. Double-labelling experiments revealed that sialyl Lewis x, recognized by the monoclonal antibody CSLEX1, was expressed on epidermal LC (n = 9). Furthermore, expression was enhanced at 24 hr following epicutaneous application of antigen and in the inflammatory disorder psoriasis (n = 10). E-selectin was concomitantly strongly expressed on dermal endothelium in psoriasis and allergic contact dermatitis. Intradermal injection of the T-cell-derived cytokine interferon-gamma (IFN-gamma) led to increased LC expression of sialyl Lewis x. In epidermal cell suspensions, in contrast to keratinocytes, CD1a+ cells expressed sialyl Lewis x, intensity of which was enhanced after 4 days in culture. CSLEX1 staining could be abolished and CD15 (non-sialated Lewis x) expression induced by saponification and treatment with neuraminidase. Expression of other selectin ligands was also examined. While the cutaneous lymphocyte antigen defined by the monoclonal antibody HECA-452 reacted with a small minority of LC, sialyl Lewis a and sulphatide were not expressed under any experimental conditions. These studies indicate that E-selectin-sialyl Lewis x interactions are potentially important in LC migration, both into and out of skin. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:7512530

Modulation of the Effect of l‐β‐D‐Arabinofuranosylcytosine by 6‐Mercaptopurine in L1210 Cells

PubMed Central

Kawai, Yasukazu; Ueda, Takanori; Nakamura, Toru

1994-01-01

In L1210 cells incubated with l‐β‐D‐arabinofuranosylcytosine (ara‐C), 6‐mercaptopurine (6‐MP) significantly potentiated 1‐β‐D‐arabinofuranosylcytosme 5′‐triphosphate (ara‐CTP) accumulation and ara‐C incorporation into DNA (ara‐C/DNA). The cytotoxicity of these two drugs was assessed to be at least additive by clonogenic assay. l‐β‐D‐Arabinofuranosylnracil (ara‐U) level in a cell suspension was suppressed by 6‐MP in a concentration‐dependent fashion, though intracellular cytidine deaminase (CDD) activity was not affected by 6‐MP. In addition, extracted CDD activity was not directly inhibited by 6‐MP or by its intracellular metabolites in vitro. After preincubation in the presence or absence of 6‐MP, the cell suspension was fractionated to obtain the spent medium and cell pellet. Then, each fraction was incubated with ara‐C. Ara‐U formation in the spent medium was found to increase conspicuously in relation to the time of preincubation in the control and it was suppressed by 6‐MP pretreatment. Ara‐U formation in the cell compartment increased slightly in relation to the time of preincubation in the control and substantially no suppression of ara‐U formation was observed in spite of 6‐MP pretreatment. In conclusion, intracellularly synthesized CDD was thought to be rapidly shed into the medium and the released CDD could play an important role in ara‐C inactivation. 6‐MP interrupted some step between synthesis and shedding of CDD, resulting in a decrease of the ara‐C deamination in the medium and enhancement of its antileukemic effect. PMID:7961129

Combination of medical needling and non-cultured autologous skin cell transplantation (ReNovaCell) for repigmentation of hypopigmented burn scars.

PubMed

Busch, K H; Bender, R; Walezko, N; Aziz, H; Altintas, M A; Aust, M C

2016-11-01

Burn scars remain a serious physical and psychological problem for the affected people. Clinical studies as well as basic scientific research have shown that medical needling can significantly increase the quality of burn scars with comparatively low risk and stress for the patient with regards to skin elasticity, moisture, erythema and transepidermal water loss. However, medical needling has no influence on repigmentation of large hypopigmented scars. The goal of this study is to evaluate whether two established methods - needling (for improvement of scar quality) and non-cultured autologous skin cell suspension (for repigmentation) - can be successfully combined. Twenty subjects with mean age of 33 years (6-60 years) with scars from deep second and third degree burns have been treated. The average treated surface area was 94cm 2 (15-250cm 2 ) and was focused on prominent areas such as the face, neck, chest and arm. Percutaneous collagen induction or "medical needling" was performed using a roller covered with 3mm long needles. The roller is vertically, horizontally and diagonally rolled over the scar, inducing microtrauma. Then, non-cultured autologous skin cell suspension (NCASCS) was produced and applied using the ReNovaCell Autologous Cell Harvesting Device (Avita Medical), according to the manufacturer's instructions. The patients were followed 12 months postoperatively. Pigmentation changes were measured objectively, as well as with patient and observer ratings. Patient satisfaction/preference was also obtained. Taken together, the pigmentation ratings and objective measures indicate individual improvement in 17 of the study participants. The melanin increases seen 12 months after NCASCS treatment are statistically significant. Medical needling in combination with NCASCS shows promise for repigmentation of burn cars. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase.

PubMed

Becerra-Arteaga, Alejandro; Shuler, Michael L

2007-08-15

We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins. (c) 2007 Wiley Periodicals, Inc.

Evidence that pulsed electric field treatment enhances the cell wall porosity of yeast cells.

PubMed

Ganeva, Valentina; Galutzov, Bojidar; Teissie, Justin

2014-02-01

The application of rectangular electric pulses, with 0.1-2 ms duration and field intensity of 2.5-4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2-8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.

Stochastic Approach to Determine CO2 Hydrate Induction Time in Clay Mineral Suspensions

NASA Astrophysics Data System (ADS)

Lee, K.; Lee, S.; Lee, W.

2008-12-01

A large number of induction time data for carbon dioxide hydrate formation were obtained from a batch reactor consisting of four independent reaction cells. Using resistance temperature detector(RTD)s and a digital microscope, we successfully monitored the whole process of hydrate formation (i.e., nucleation and crystal growth) and detected the induction time. The experiments were carried out in kaolinite and montmorillonite suspensions at temperatures between 274 and 277 K and pressures ranging from 3.0 to 4.0 MPa. Each set of data was analyzed beforehand whether to be treated by stochastic manner or not. Geochemical factors potentially influencing the hydrate induction time under different experimental conditions were investigated by stochastic analyses. We observed that clay mineral type, pressure, and temperature significantly affect the stochastic behavior of the induction times for CO2 hydrate formation in this study. The hydrate formation kinetics along with stochastic analyses can provide basic understanding for CO2 hydrate storage in deep-sea sediment and geologic formation, securing its stability under the environments.

Dual-frequency dielectrophoretic levitation of Canola protoplasts.

PubMed

Kaler, K V; Xie, J P; Jones, T B; Paul, R

1992-07-01

A novel dual-frequency excitation technique is introduced which permits investigation of the low-frequency dispersion of Canola plant protoplasts using feedback-controlled dielectrophoretic levitation. The upper and intermediate frequency spectra obtained using the new technique are generally consistent with previous work. However, below some cross-over frequency f(OL), the protoplasts exhibit an apparent positive dielectrophoretic response that is not predicted by conventional theory. This cross-over frequency is linearly related to suspension conductivity, virtually independent of the suspension pH, and inversely proportional to the square of the cell radius. Examination of the complex Clausius-Mossotti polarization coefficient reveals that the observed positive dielectrophoretic response can not be accounted for in terms of Maxwell-Wagner polarization associated with a conventional layered model for the protoplast. The failure of straightforward enhancements to the protoplast model in explaining the low frequency behavior may indicate the presence of an electrophoretic contribution to the net observable force on the particle. To account for such fluid mechanical effects, it will be necessary to modify the existing dielectrophoretic force formulation.

Bioactive Glass-Ceramic Foam Scaffolds from ‘Inorganic Gel Casting’ and Sinter-Crystallization

PubMed Central

Molino, Giulia; Vitale Brovarone, Chiara

2018-01-01

Highly porous bioactive glass-ceramic scaffolds were effectively fabricated by an inorganic gel casting technique, based on alkali activation and gelification, followed by viscous flow sintering. Glass powders, already known to yield a bioactive sintered glass-ceramic (CEL2) were dispersed in an alkaline solution, with partial dissolution of glass powders. The obtained glass suspensions underwent progressive hardening, by curing at low temperature (40 °C), owing to the formation of a C–S–H (calcium silicate hydrate) gel. As successful direct foaming was achieved by vigorous mechanical stirring of gelified suspensions, comprising also a surfactant. The developed cellular structures were later heat-treated at 900–1000 °C, to form CEL2 glass-ceramic foams, featuring an abundant total porosity (from 60% to 80%) and well-interconnected macro- and micro-sized cells. The developed foams possessed a compressive strength from 2.5 to 5 MPa, which is in the range of human trabecular bone strength. Therefore, CEL2 glass-ceramics can be proposed for bone substitutions. PMID:29495498

Colony formation by normal and malignant human B-lymphocytes.

PubMed Central

Izaguirre, C. A.; Minden, M. D.; Howatson, A. F.; McCulloch, E. A.

1980-01-01

A method is described that permits colony formation in culture by B lymphocytes from normal blood and from blood, marrow or lymph nodes of patients with myeloma or lymphoma. The method depends on: (1) exhaustively depleting cell suspensions of T lymphocytes, (2) a medium conditioned by T lymphocytes in the presence of phytohaemagglutinin (PHA-TCM), and (3) irradiated autologous or homologous T lymphocytes. Under these conditions the assay is linear. Cellular development of B lymphocytes can be followed; differentiation to plasma cells is seen in cultures of cells from normal individuals and myeloma patients, but not lymphoma patients. Malignant B lymphocytes in culture produced immunoglobulin of the class identified in the patient's blood, or in freshly obtained cells. We conclude that the assay is suitable for studying the growth, differentiation and regulation of normal and malignant B lymphocytes in culture. Images Fig. 1 Fig. 2 PMID:6968572

Overexpression of OsEXPA8, a Root-Specific Gene, Improves Rice Growth and Root System Architecture by Facilitating Cell Extension

PubMed Central

Ma, Nana; Wang, Ying; Qiu, Shichun; Kang, Zhenhui; Che, Shugang; Wang, Guixue; Huang, Junli

2013-01-01

Expansins are unique plant cell wall proteins that are involved in cell wall modifications underlying many plant developmental processes. In this work, we investigated the possible biological role of the root-specific α-expansin gene OsEXPA8 in rice growth and development by generating transgenic plants. Overexpression of OsEXPA8 in rice plants yielded pleiotropic phenotypes of improved root system architecture (longer primary roots, more lateral roots and root hairs), increased plant height, enhanced leaf number and enlarged leaf size. Further study indicated that the average cell length in both leaf and root vascular bundles was enhanced, and the cell growth in suspension cultures was increased, which revealed the cellular basis for OsEXPA8-mediated rice plant growth acceleration. Expansins are thought to be a key factor required for cell enlargement and wall loosening. Atomic force microscopy (AFM) technology revealed that average wall stiffness values for 35S::OsEXPA8 transgenic suspension-cultured cells decreased over six-fold compared to wild-type counterparts during different growth phases. Moreover, a prominent change in the wall polymer composition of suspension cells was observed, and Fourier-transform infrared (FTIR) spectra revealed a relative increase in the ratios of the polysaccharide/lignin content in cell wall compositions of OsEXPA8 overexpressors. These results support a role for expansins in cell expansion and plant growth. PMID:24124527

Use of Non-Conventional Cell Disruption Method for Extraction of Proteins from Black Yeasts

PubMed Central

Čolnik, Maja; Primožič, Mateja; Knez, Željko; Leitgeb, Maja

2016-01-01

The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum, and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2) by varying pressure at fixed temperature (35°C). The black yeasts cell walls were disrupted, and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase, and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV–Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2-treated cells. The advantages of the proposed method are in a simple use, which is also possible for heat-sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions. PMID:27148527

Differentiation and Cell-Cell Interactions of Neural Progenitor Cells Transplanted into Intact Adult Brain.

PubMed

Sukhinich, K K; Kosykh, A V; Aleksandrova, M A

2015-11-01

We studied the behavior and cell-cell interactions of embryonic brain cell from GFP-reporter mice after their transplantation into the intact adult brain. Fragments or cell suspensions of fetal neocortical cells at different stages of development were transplanted into the neocortex and striatum of adult recipients. Even in intact brain, the processes of transplanted neurons formed extensive networks in the striatum and neocortical layers I and V-VI. Processes of transplanted cells at different stages of development attained the rostral areas of the frontal cortex and some of them reached the internal capsule. However, the cells transplanted in suspension had lower process growth potency than cells from tissue fragments. Tyrosine hydroxylase fibers penetrated from the recipient brain into grafts at both early and late stages of development. Our experiments demonstrated the formation of extensive reciprocal networks between the transplanted fetal neural cells and recipient brain neurons even in intact brain.

Defined three-dimensional culture conditions mediate efficient induction of definitive endoderm lineage from human umbilical cord Wharton's jelly mesenchymal stem cells.

PubMed

Al Madhoun, Ashraf; Ali, Hamad; AlKandari, Sarah; Atizado, Valerie Lopez; Akhter, Nadeem; Al-Mulla, Fahd; Atari, Maher

2016-11-16

Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are gaining increasing interest as an alternative source of stem cells for regenerative medicine applications. Definitive endoderm (DE) specification is a prerequisite for the development of vital organs such as liver and pancreas. Hence, efficient induction of the DE lineage from stem cells is crucial for subsequent generation of clinically relevant cell types. Here we present a defined 3D differentiation protocol of WJ-MSCs into DE cells. WJ-MSCs were cultured in suspension to generate spheroids, about 1500 cells each, for 7 days. The serum-free differentiation media contained specific growth factors, cytokines, and small molecules that specifically regulate signaling pathways including sonic hedgehog, bone morphogenetic protein, Activin/Wnt, and Notch. We obtained more than 85 % DE cells as shown with FACS analysis using antibodies directed against the DE marker CXCR4. In addition, biochemical and molecular analysis of bona-fide DE markers revealed a time-course induction of Sox17, CXCR4, and FoxA2. Focused PCR-based array also indicated a specific induction into the DE lineage. In this study, we report an efficient serum-free protocol to differentiate WJ-MSCs into DE cells utilizing 3D spheroid formation. Our approach might aid in the development of new protocols to obtain DE-derivative lineages including liver-like and pancreatic insulin-producing cells.

Osteoarthritic human chondrocytes proliferate in 3D co-culture with mesenchymal stem cells in suspension bioreactors.

PubMed

Khurshid, Madiha; Mulet-Sierra, Aillette; Adesida, Adetola; Sen, Arindom

2018-03-01

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage-generating cells. Previous studies in static culture have shown that hACs co-cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co-culture of hMSCs and OA hACs under serum-free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125-ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75-fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co-culture as a 2D monolayer in static culture flasks, bioreactor co-culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum-free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell-based cartilage repair. Copyright © 2017 John Wiley & Sons, Ltd.

Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures 1

PubMed Central

Thomas, John C.; De Armond, Richard L.; Bohnert, Hans J.

1992-01-01

The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs. ImagesFigure 4Figure 5 PMID:16668687

Effects of magnetic cobalt ferrite nanoparticles on biological and artificial lipid membranes

PubMed Central

Drašler, Barbara; Drobne, Damjana; Novak, Sara; Valant, Janez; Boljte, Sabina; Otrin, Lado; Rappolt, Michael; Sartori, Barbara; Iglič, Aleš; Kralj-Iglič, Veronika; Šuštar, Vid; Makovec, Darko; Gyergyek, Sašo; Hočevar, Matej; Godec, Matjaž; Zupanc, Jernej

2014-01-01

Background The purpose of this work is to provide experimental evidence on the interactions of suspended nanoparticles with artificial or biological membranes and to assess the possibility of suspended nanoparticles interacting with the lipid component of biological membranes. Methods 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicles and human red blood cells were incubated in suspensions of magnetic bare cobalt ferrite (CoFe2O4) or citric acid (CA)-adsorbed CoFe2O4 nanoparticles dispersed in phosphate-buffered saline and glucose solution. The stability of POPC giant unilamellar vesicles after incubation in the tested nanoparticle suspensions was assessed by phase-contrast light microscopy and analyzed with computer-aided imaging. Structural changes in the POPC multilamellar vesicles were assessed by small angle X-ray scattering, and the shape transformation of red blood cells after incubation in tested suspensions of nanoparticles was observed using scanning electron microscopy and sedimentation, agglutination, and hemolysis assays. Results Artificial lipid membranes were disturbed more by CA-adsorbed CoFe2O4 nanoparticle suspensions than by bare CoFe2O4 nanoparticle suspensions. CA-adsorbed CoFe2O4-CA nanoparticles caused more significant shape transformation in red blood cells than bare CoFe2O4 nanoparticles. Conclusion Consistent with their smaller sized agglomerates, CA-adsorbed CoFe2O4 nanoparticles demonstrate more pronounced effects on artificial and biological membranes. Larger agglomerates of nanoparticles were confirmed to be reactive against lipid membranes and thus not acceptable for use with red blood cells. This finding is significant with respect to the efficient and safe application of nanoparticles as medicinal agents. PMID:24741305

Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures

PubMed Central

Nakano, Tomoyuki; Kanai, Yoshihiko; Amano, Yusuke; Yoshimoto, Taichiro; Matsubara, Daisuke; Shibano, Tomoki; Tamura, Tomoko; Oguni, Sachiko; Katashiba, Shizuka; Ito, Takeshi; Murakami, Yoshinori; Fukayama, Masashi; Murakami, Takashi; Endo, Shunsuke; Niki, Toshiro

2017-01-01

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3–4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion. PMID:28786996

Phagocytes in cell suspensions of human colon mucosa.

PubMed Central

Beeken, W; Northwood, I; Beliveau, C; Gump, D

1987-01-01

Because little is known of the phagocytes of the human colon we enumerated these cells in mucosal suspensions and studied their phagocytic activity. Phagocyte rich suspensions were made by EDTA collagenase dissociation followed by elutriation centrifugation. Phagocytosis was evaluated by measuring cellular radioactivity after incubation of phagocytes with 3H-adenine labelled E coli ON2 and checked microscopically. Dissociation of normal mucosa from colorectal neoplasms yielded means of 1.9 X 10(6) eosinophils, 1.4 X 10(6) macrophages and 2 X 10(5) neutrophils per gram of mucosa. Visually normal mucosa of inflammatory states yielded 2.2 X 10(6) eosinophils, 2.3 X 10(6) macrophages and 7 X 10(5) neutrophils per gram of mucosa. Phagocyte rich suspensions of normal mucosa from tumour patients phagocytosed 21.8% of a pool of opsonised tritiated E coli ON2 and by microscopy 100% of mucosal neutrophils ingested bacteria, 83% of eosinophils were phagocytic, and 53% of macrophages contained bacteria. These results suggest that in the human colonic mucosa, the eosinophil is more abundant than the macrophage and the per cent of those cells exhibiting phagocytosis is intermediate between that of the macrophage and the neutrophil. Thus these three types of cells are actively phagocytic and share the potential for a major role in host defence against invasive enteric bacteria. PMID:3666566

Evaluation of treatment response to autologous transplantation of noncultured melanocyte/keratinocyte cell suspension in patients with stable vitiligo.

PubMed

Ramos, Mariana Gontijo; Ramos, Daniel Gontijo; Ramos, Camila Gontijo

2017-01-01

Vitiligo is a chronic disease characterized by the appearance of achromic macules caused by melanocyte destruction. Surgical treatments with melanocyte transplantation can be used for stable vitiligo cases. To evaluate treatment response to the autologous transplantation of noncultured epidermal cell suspension in patients with stable vitiligo. Case series study in patients with stable vitiligo submitted to noncultured epidermal cell suspension transplantation and evaluated at least once, between 3 and 6 months after the procedure, to observe repigmentation and possible adverse effects. The maximum follow-up period for some patients was 24 months. Of the 20 patients who underwent 24 procedures, 25% showed an excellent rate of repigmentation, 50% good repigmentation, 15% regular, and 10% poor response. The best results were observed in face and neck lesions, while the worst in extremity lesions (88% and 33% of satisfactory responses, respectively). Patients with segmental vitiligo had a better response (84%) compared to non-segmental ones (63%). As side effects were observed hyperpigmentation of the treated area and the appearance of Koebner phenomenon in the donor area. Some limitations of the study included the small number of patients, a subjective evaluation, and the lack of long-term follow-up on the results. CONCLUSION: Noncultured epidermal cell suspension transplantation is efficient and well tolerated for stable vitiligo treatment, especially for segmental vitiligo on the face and neck.

Effect of space flight on interferon production - mechanistic studies

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald

1991-01-01

Ground-based models were studied for the effects of space flight on immune responses. Most time was spent on the model for the antiorthostatic, hypokinetic, hypodynamic suspension model for rats. Results indicate that suspension is useful for modeling the effects of spaceflight on functional immune responses, such as interferon and interleukin production. It does not appear to be useful for modeling shifts in leukocyte sub-populations. Calcium and 1,25-dihydroxyvitamin D sub 3 appear to play a pivitol role in regulating shifts in immune responses due to suspension. The macrophage appears to be an important target cell for the effects of suspension on immune responses.

Cell compaction influences the regenerative potential of passaged bovine articular chondrocytes in an ex vivo cartilage defect model.

PubMed

Schmutzer, Michael; Aszodi, Attila

2017-04-01

The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in ex vivo systems than in in vitro cell cultures. I have therefore established an ex vivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p = 0.0083) and P2 (p < 0.05). Suspension-cultured cells did not promote cartilage repair when compared with implanted monolayer-cultured chondrocytes and pellets: 24.0 ± 0.66% for suspension cells, 46.4 ± 2.9% for monolayer cells, and 127.64 ± 0.90% for pellets (p < 0.0001) of the original defect volume (percentage of defect). Additional cultivation with chondrogenesis-promoting growth factors TGF-β1 and BMP-2 revealed an enhancing effect on cartilage repair in all settings. The advantage and innovation of this system over in vitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such ex vivo explants might serve as a better model system to mimic clinical situations. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation.

PubMed

Fukai, Katsuhiko; Morioka, Kazuki; Yamada, Manabu; Nishi, Tatsuya; Yoshida, Kazuo; Kitano, Rie; Yamazoe, Reiko; Kanno, Toru

2015-07-01

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-α(v)β(6) have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-α(v)β(6) cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-α(v)β(6) cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same. © 2015 The Author(s).

Effect of complexation conditions on microcapsulation of Lactobacillus acidophilus in xanthan-chitosan polyelectrolyte complex gels.

PubMed

Chen, He; Song, Yajuan; Liu, Nina; Wan, Hongchang; Shu, Guowei; Liao, Na

2015-01-01

Lactobacillus acidophilus has become increasingly popular because of their beneficial effects on health of their host, and are called proboscis. In order to exert beneficial effects for probiotics, they must be able to tolerate the acidic conditions of the stomach environment and the bile in the small intestine. Microencapsulated form has received reasonable attention, since it can protect probiotic organisms against an unfavourable environment, and to allow their release in a viable and metabolically active state in the intestine. The aim of this study was to investigate some factores, such as chitosan solution pH and concentration, xanthan concentration, cell suspension-xanthan ratio, mixed bacteria glue liquid-chitosan ratio, which impacted the process of microencapsulation of L. acidophilus. In this study, L. acidophilus was immobilized with xanthan⁄chitosan gel using extrusion method. The viable counts and encapsulation yield of L. acidophilus encapsulated in different chitosan solution pH (4.5, 5, 5.5 and 6), in different chitosan concentration (0.5%, 0.7%, 0.9% and 1.1%), in different xanthan concentration (0.5%, 0.7%, 0.9% and 1.1%), in different cell suspension-xanthan ratios (1:5, 1:10, 1:15 and 1:20), in different mixed bacteria glue liquid-chitosan ratios (1:3, 1:4, 1:5 and 1:6), have been investigated by single factor experiment method. The optimum conditions of microencapsulated L. acidophilus have been observed. The optimum chitosan solution pH for L. acidophilus was 5.5; the optimum chitosan concentration was 0.9%; the optimum xanthan concentration was 0.7%; the optimum cell suspension-xanthan ratio was 1:10; the optimum mixed bacteria glue liquid-chitosan ratio was 1:3. These results will be helpful to further optimize the process of L. acidophilus microencapsulation, and provide reference for obtaining higher viable counts and entrapped yield of L. acidophilus microcapsules.

METHOD FOR THE PREPARATION OF STABLE ACTINIDE METAL OXIDE-CONTAINING SLURRIES AND OF THE OXIDES THEREFOR

DOEpatents

Hansen, R.S.; Minturn, R.E.

1958-02-25

This patent deals with a method of preparing actinide metal oxides of a very fine particle size and of forming stable suspensions therefrom. The process consists of dissolving the nitrate of the actinide element in a combustible organic solvent, converting the solution obtained into a spray, and igniting the spray whereby an oxide powder is obtained. The oxide powder is then slurried in an aqueous soiution of a substance which is adsorbable by said oxides, dspersed in a colloid mill whereby a suspension is obtained, and electrodialyzed until a low spectiic conductance is reached.

48 CFR 1222.406-9 - Withholding from or suspension of contract payments.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Labor Standards for Contracts Involving Construction 1222.406-9 Withholding from or suspension of... 4220.7, Employee Claim for Wage Restitution, is obtained from each employee claiming restitution under the contract. The Comptroller General (Claims Division) must receive this form with a completed...

48 CFR 1222.406-9 - Withholding from or suspension of contract payments.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Labor Standards for Contracts Involving Construction 1222.406-9 Withholding from or suspension of... 4220.7, Employee Claim for Wage Restitution, is obtained from each employee claiming restitution under the contract. The Comptroller General (Claims Division) must receive this form with a completed...

48 CFR 1222.406-9 - Withholding from or suspension of contract payments.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Labor Standards for Contracts Involving Construction 1222.406-9 Withholding from or suspension of... 4220.7, Employee Claim for Wage Restitution, is obtained from each employee claiming restitution under the contract. The Comptroller General (Claims Division) must receive this form with a completed...

48 CFR 1222.406-9 - Withholding from or suspension of contract payments.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Labor Standards for Contracts Involving Construction 1222.406-9 Withholding from or suspension of... 4220.7, Employee Claim for Wage Restitution, is obtained from each employee claiming restitution under the contract. The Comptroller General (Claims Division) must receive this form with a completed...

48 CFR 1222.406-9 - Withholding from or suspension of contract payments.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Labor Standards for Contracts Involving Construction 1222.406-9 Withholding from or suspension of... 4220.7, Employee Claim for Wage Restitution, is obtained from each employee claiming restitution under the contract. The Comptroller General (Claims Division) must receive this form with a completed...

Optical tweezers for measuring the interaction of the two single red blood cells in flow condition

NASA Astrophysics Data System (ADS)

Lee, Kisung; Muravyov, Alexei; Semenov, Alexei; Wagner, Christian; Priezzhev, Alexander

2017-03-01

Aggregation of red blood cells (RBCs) is an intrinsic property of blood, which has direct effect on the blood viscosity and therefore affects overall the blood circulation throughout the body. It is attracting interest for the research in both fundamental science and clinical application. Despite of the intensive research, the aggregation mechanism is remaining not fully clear. Recent advances in methods allowed measuring the interaction between single RBCs in a well-defined configuration leading the better understanding of the mechanism of the process. However the most of the studies were made on the static cells. Thus, the measurements in flow mimicking conditions are missing. In this work, we aim to study the interaction of two RBCs in the flow conditions. We demonstrate the characterization of the cells interaction strength (or flow tolerance) by measuring the flow velocity to be applied to separate two aggregated cells trapped by double channel optical tweezers in a desired configuration. The age-separated cells were used for this study. The obtained values for the minimum flow velocities needed to separate the two cells were found to be 78.9 +/- 6.1 μm/s and 110 +/- 13 μm/s for old and young cells respectively. The data obtained is in agreement with the observations reported by other authors. The significance of our results is in ability for obtaining a comprehensible and absolute physical value characterizing the cells interaction in flow conditions (not like the Aggregation Index measured in whole blood suspensions by other techniques, which is some abstract parameter)

Mechanochemistry of Chitosan-Coated Zinc Sulfide (ZnS) Nanocrystals for Bio-imaging Applications.

PubMed

Bujňáková, Zdenka; Dutková, Erika; Kello, Martin; Mojžiš, Ján; Baláž, Matej; Baláž, Peter; Shpotyuk, Oleh

2017-12-01

The ZnS nanocrystals were prepared in chitosan solution (0.1 wt.%) using a wet ultra-fine milling. The obtained suspension was stable and reached high value of zeta potential (+57 mV). The changes in FTIR spectrum confirmed the successful surface coating of ZnS nanoparticles by chitosan. The prepared ZnS nanocrystals possessed interesting optical properties verified in vitro. Four cancer cells were selected (CaCo-2, HCT116, HeLa, and MCF-7), and after their treatment with the nanosuspension, the distribution of ZnS in the cells was studied using a fluorescence microscope. The particles were clearly seen; they passed through the cell membrane and accumulated in cytosol. The biological activity of the cells was not influenced by nanoparticles, they did not cause cell death, and only the granularity of cells was increased as a consequence of cellular uptake. These results confirm the potential of ZnS nanocrystals using in bio-imaging applications.

Scanning electron microscopy study of adhesion in sea urchin blastulae. M.S. Thesis

NASA Technical Reports Server (NTRS)

Crowther, Susan D.

1988-01-01

The dissociation supernatant (DS) isolated by disaggregating Strongylocentrotus purpuratus blastulae in calcium- and magnesium-free seawater specifically promotes reaggregation of S. purpuratus blastula cells. The purpose of this study was to use scanning electron microscopy to examine the gross morphology of aggregates formed in the presence of DS to see if it resembles adhesion in partially dissociated blastulae. A new reaggregation procedure developed here, using large volumes of cell suspension and a large diameter of rotation, was utilized to obtain sufficient quantities of aggregates for scanning electron microscopy. The results indicate that aggregates formed in the presence of DS resemble partially dissociated intact embryos in terms of the direct cell-cell adhesion observed. DS did not cause aggregation to form as a result of the entrapment of cells in masses of extracellular material. These studies provide the groundwork for further studies using transmission electron microscopy to more precisely define the adhesive contacts made by cells in the presence of the putative adhesion molecules present in DS.

Mechanochemistry of Chitosan-Coated Zinc Sulfide (ZnS) Nanocrystals for Bio-imaging Applications

NASA Astrophysics Data System (ADS)

Bujňáková, Zdenka; Dutková, Erika; Kello, Martin; Mojžiš, Ján; Baláž, Matej; Baláž, Peter; Shpotyuk, Oleh

2017-05-01

The ZnS nanocrystals were prepared in chitosan solution (0.1 wt.%) using a wet ultra-fine milling. The obtained suspension was stable and reached high value of zeta potential (+57 mV). The changes in FTIR spectrum confirmed the successful surface coating of ZnS nanoparticles by chitosan. The prepared ZnS nanocrystals possessed interesting optical properties verified in vitro. Four cancer cells were selected (CaCo-2, HCT116, HeLa, and MCF-7), and after their treatment with the nanosuspension, the distribution of ZnS in the cells was studied using a fluorescence microscope. The particles were clearly seen; they passed through the cell membrane and accumulated in cytosol. The biological activity of the cells was not influenced by nanoparticles, they did not cause cell death, and only the granularity of cells was increased as a consequence of cellular uptake. These results confirm the potential of ZnS nanocrystals using in bio-imaging applications.

Induction of phytic acid synthesis by abscisic acid in suspension-cultured cells of rice.

PubMed

Matsuno, Koya; Fujimura, Tatsuhito

2014-03-01

A pathway of phytic acid (PA) synthesis in plants has been revealed via investigations of low phytic acid mutants. However, the regulation of this pathway is not well understood because it is difficult to control the environments of cells in the seeds, where PA is mainly synthesized. We modified a rice suspension culture system in order to study the regulation of PA synthesis. Rice cells cultured with abscisic acid (ABA) accumulate PA at higher levels than cells cultured without ABA, and PA accumulation levels increase with ABA concentration. On the other hand, higher concentrations of sucrose or inorganic phosphorus do not affect PA accumulation. Mutations in the genes RINO1, OsMIK, OsIPK1 and OsLPA1 have each been reported to confer low phytic acid phenotypes in seeds. Each of these genes is upregulated in cells cultured with ABA. OsITPK4 and OsITPK6 are upregulated in cells cultured with ABA and in developing seeds. These results suggest that the regulation of PA synthesis is similar between developing seeds and cells in this suspension culture system. This system will be a powerful tool for elucidating the regulation of PA synthesis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

An injectable spheroid system with genetic modification for cell transplantation therapy.

PubMed

Uchida, Satoshi; Itaka, Keiji; Nomoto, Takahiro; Endo, Taisuke; Matsumoto, Yu; Ishii, Takehiko; Kataoka, Kazunori

2014-03-01

The new methodology to increase a therapeutic potential of cell transplantation was developed here by the use of three-dimensional spheroids of transplanting cells subsequent to the genetic modification with non-viral DNA vectors, polyplex nanomicelles. Particularly, spheroids in regulated size of 100-μm of primary hepatocytes transfected with luciferase gene were formed on the micropatterned culture plates coated with thermosensitive polymer, and were recovered in the form of injectable liquid suspension simply by cooling the plates. After subcutaneously transplanting these hepatocyte spheroids, efficient transgene expression was observed in host tissue for more than a month, whereas transplantation of a single-cell suspension from a monolayer culture resulted in an only transient expression. The spheroid system contributed to the preservation of innate functions of transplanted hepatocytes in the host tissue, such as albumin expression, thereby possessing high potential for expressing transgene. Intravital observation of transplanted cells showed that those from spheroid cultures had a tendency to localize in the vicinity of blood vessels, making a favorable microenvironment for preserving cell functionality. Furthermore, spheroids transfected with erythropoietin-expressing DNA showed a significantly higher hematopoietic effect than that of cell suspensions from monolayer cultures, demonstrating high potential of this genetically-modified spheroid transplantation system for therapeutic applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

Optical sensing of concentration and refractive index of pigments in a suspension.

PubMed

Niskanen, Ilpo; Räty, Jukka; Peiponen, Kai-Erik

2010-06-10

We describe an immersion liquid and data analysis method for the simultaneous determination of the refractive index and concentration of pigments by measurement of light transmission of suspensions. A new innovation is that, in the event that two different pigments are simultaneously present in a suspension, it is possible to detect the refractive index of an unknown pigment with the aid of half-width of transmittance and, furthermore, to obtain the concentration of the unknown pigment.

The effect of space microgravity on the physiological activity of mammalian resident cardiac stem cells

NASA Astrophysics Data System (ADS)

Belostotskaya, Galina; Zakharov, Eugeny

Prolonged exposure to weightlessness during space flights is known to cause depression of heart function in mammals. The decrease in heart weight and its remodeling under the influence of prolonged weightlessness (or space microgravity) is assumed to be due to both morphological changes of working cardiomyocytes and their progressive loss, as well as to possible depletion of resident cardiac stem cells (CSCs) population, or their inability to self-renewal and regeneration of muscle tissue under conditions of weightlessness. We have previously shown that the presence of different maturity clones formed by resident CSCs not only in culture but also in the mammalian myocardium can be used as an indicator of the regenerative activity of myocardial cells [Belostotskaya, et al., 2013: 2014]. In this study, we were interested to investigate whether the 30-day near-Earth space flight on the spacecraft BION-M1 affects the regenerative potential of resident CSCs. Immediately after landing of the spacecraft, we had examined the presence of resident c-kit+, Sca-1+ and Isl1+ CSCs and their development in suspension of freshly isolated myocardial cells of C57BL mice in comparison to controls. Cardiac cell suspension was obtained by enzymatic digestion of the heart [Belostotskaya and Golovanova, 2014]. Immunocytochemically stained preparations of fixed cells were analyzed with confocal microscope Leica TCS SP5 (Germany) in the Resource Center of St-Petersburg State University. CSCs were labeled with appropriate antibodies. CSCs differentiation into mature cardiomyocytes was verified using antibodies to Sarcomeric α-Actinin and Cardiac Troponin T. Antibodies to Connexin43 were used to detect cell-cell contacts. All antibodies were conjugated with Alexa fluorochromes (488, 532, 546, 568, 594 and/or 647 nm), according to Zenon-technology (Invitrogen). It has been shown that, under identical conditions of cell isolation, more complete digestion of heart muscle was observed in weightlessness-treated samples vs. controls. These findings correlated with reduced expression of Connexin43. Typical elongated cardiomyocytes, presenting as both individual cells and conglomerates, were present in the control samples, whereas the shortened and thickened individual cardiac myocytes prevailed in the samples subjected to space microgravity. Both control samples and microgravity-treated samples contained resident CSCs of all subtypes. Both individual CSCs and CSC-derived clones were present in the suspension of myocardial cells. However, the number of CSC-formed clones of different maturity was significantly higher in the samples subjected to space microgravity. Some clones comprised only small undifferentiated cells of one CSCs subtype, while the cells of the other clones expressed some of the specific cardiac antigens (α-Actinin and Troponin T) at varying rate. In addition, large α-actinin- and troponin T-positive individual cardiomyocytes with readily discernible sarcomeric structure still expressing the original CSC antigens were also identified. The data obtained suggest that prolonged space microgravity exposure during space flight causes significant structural changes in the mammalian myocardium which may affect cardiac contractile function. Weightlessness-induced loss in heart muscle weight is assumed to be compensated by an increase in the activity of resident CSCs, which form new cardiomyocytes proliferating and differentiating inside the clones. The authors express their gratitude to the staff of Institute of Biomedical Problems of the Russian Academy of Sciences and Company "Progress" for the preparation of experimental animals for the biosatellite flight. The study was in part supported by grants from BION-M1 Project and Program of Presidium of Russian Academy of Sciences “Fundamental Sciences for Medicine” (2013).

Surfactant-assisted dispersion of carbon nanotubes: mechanism of stabilization and biocompatibility of the surfactant

NASA Astrophysics Data System (ADS)

Singh, Raman Preet; Jain, Sanyog; Ramarao, Poduri

2013-10-01

Nanoparticles (NPs) are thermodynamically unstable system and tend to aggregate to reduce free energy. The aggregation property of NPs results in inhomogeneous exposure of cells to NPs resulting in variable cellular responses. Several types of surfactants are used to stabilize NP dispersions and obtain homogenous dispersions. However, the effects of these surfactants, per se, on cellular responses are not completely known. The present study investigated the application of Pluronic F68 (PF68) for obtaining stable dispersion of NPs using carbon nanotubes as model NPs. PF68-stabilized NP suspensions are stable for long durations and do not show signs of aggregation or settling during storage or after autoclaving. The polyethylene oxide blocks in PF68 provide steric hindrance between adjacent NPs leading to stable NP dispersions. Further, PF68 is biocompatible in nature and does not affect integrity of mitochondria, lysosomes, DNA, and nuclei. Also, PF68 neither induce free radical or cytokine production nor does it interfere with cellular uptake mechanisms. The results of the present study suggest that PF68-assisted dispersion of NPs produced suspensions, which are stable after autoclaving. Further, PF68 does not interfere with normal physiological functions suggesting its application in nanomedicine and nanotoxicity evaluation.

Effect of Different Carbon Sources on Relative Growth Rate, Internal Carbohydrates, and Mannitol 1-Oxidoreductase Activity in Celery Suspension Cultures.

PubMed Central

Stoop, JMH.; Pharr, D. M.

1993-01-01

Little information exists concerning the biochemical route of mannitol catabolism in higher plant cells. In this study, the role of a recently discovered mannitol 1-oxidoreductase (MDH) in mannitol catabolism was investigated. Suspension cultures of celery (Apium graveolens L. var dulce [Mill.] Pers.) were successfully grown on nutrient media with either mannitol, mannose, or sucrose as the sole carbon source. Cell cultures grown on any of the three carbon sources did not differ in relative growth rate, as measured by packed cell volume, but differed drastically in internal carbohydrate concentration. Mannitol-grown cells contained high concentrations of mannitol and extremely low concentrations of sucrose, fructose, glucose, and mannose. Sucrose-grown cells had high concentrations of sucrose early in the growth cycle and contained a substantial hexose pool. Mannose-grown cells had a high mannose concentration early in the cycle, which decreased during the growth cycle, whereas their internal sucrose concentrations remained relatively constant during the entire growth cycle. Celery suspension cultures on all three carbon substrates contained an NAD-dependent MDH. Throughout the growth cycle, MDH activity was 2- to 4-fold higher in mannitol-grown cells compared with sucrose- or mannose-grown cells, which did not contain detectable levels of mannitol, indicating that MDH functions pre-dominantly in an oxidative capacity in situ. The MDH activity observed in celery cells was 3-fold higher than the minimum amount required to account for the observed rate of mannitol utilization from the media. Cultures transferred from mannitol to mannose underwent a decrease in MDH activity over a period of days, and transfer from mannose to mannitol resulted in an increase in MDH activity. These data provide strong evidence that MDH plays an important role in mannitol utilization in celery suspension cultures. PMID:12231996

Interactions in charged colloidal suspensions: A molecular dynamics simulation study

NASA Astrophysics Data System (ADS)

Padidela, Uday Kumar; Behera, Raghu Nath

2017-07-01

Colloidal suspensions are extensively used in everyday life and find several applications in the pharmaceutical, chemical, food industries, etc. We present the classical molecular dynamics simulation results of the structural and transport properties of charged colloidal suspensions as a function of its size, charge and concentration. The system is viewed as a two-component (colloids and counterions) primitive model consisting of spherical colloid particle (macroion) and the counterions (micro-particles), which are treated explicitly. The solvent is treated as dielectric continuum. A systematic trend in the radial distribution functions g(r), potential of mean force W(r), different thermodynamic properties and diffusion coefficients is obtained as a function of colloid charge, size and concentration. An attractive minimum in W(r) is obtained at short interparticle distance.

In situ distributed diagnostics of flowable electrode systems: resolving spatial and temporal limitations.

PubMed

Dennison, C R; Gogotsi, Y; Kumbur, E C

2014-09-14

In this study, we have developed an in situ distributed diagnostics tool to investigate spatial and temporal effects in electrochemical systems based on flowable electrodes. Specifically, an experimental approach was developed that enables spatially-resolved voltage measurements to be obtained in situ, in real-time. To extract additional data from these distributed measurements, an experimentally-parameterized equivalent circuit model with a new 'flow capacitor' circuit element was developed to predict the distributions of various system parameters during operation. As a case study, this approach was applied to investigate the behavior of the suspension electrodes used in an electrochemical flow capacitor under flowing and static conditions. The volumetric capacitance is reduced from 15.6 F ml(-1) to 1.1 F ml(-1) under flowing conditions. Results indicate that the majority of the charging in suspension electrodes occurs within ∼750 μm of the current collectors during flow, which gives rise to significant state-of-charge gradients across the cell, as well as underutilization of the available active material. The underlying cause of this observation is attributed to the relatively high electrical resistance of the slurry coupled with a stratified charging regime and insufficient residence time. The observations highlight the need to develop more conductive slurries and to design cells with reduced charge transport lengths.

Early detection of disease program: Evaluation of the cellular immune response

NASA Technical Reports Server (NTRS)

Criswell, B. S.; Knight, V.; Martin, R. R.; Kasel, J. A.

1975-01-01

Surfaces of normal, cultured, and mitogen-stimulated mouse lymphoid cells were examined by scanning electron microscopy (SEM). Lymphocytes with smooth, highly villous and intermediate surfaces were observed in cell suspensions from both spleens and thymuses of normal mice and from spleens of congenitally athymic (nude) mice. Several strain-specific surface features were noted, including the spine-like appearance of microvilli on C57B1/6 lymphocytes. Although thymus cell suspensions contained somewhat more smooth cells than did spleen cell preparations, lymphocyte derivation could not be inferred from SEM examination. Studies of cells stimulated with mitogenic agents for thymus-derived lymphocytes (concanavalin A) or for bone marrow-derived lymphocytes (lipopolysaccharide) suggested that, in the mouse, development of a complex villous surface is a general concomitant of lymphocyte activation and transformation.

Screening and selection of high carotenoid producing in vitro tomato cell culture lines for [13C]-carotenoid production.

PubMed

Engelmann, Nancy J; Campbell, Jessica K; Rogers, Randy B; Rupassara, S Indumathie; Garlick, Peter J; Lila, Mary Ann; Erdman, John W

2010-09-22

Isotopically labeled tomato carotenoids, phytoene, phytofluene, and lycopene, are needed for mammalian bioavailability and metabolism research but are currently commercially unavailable. The goals of this work were to establish and screen multiple in vitro tomato cell lines for carotenoid production, test the best producers with or without the bleaching herbicides, norflurazon and 2-(4-chlorophenyl-thio)triethylamine (CPTA), and to use the greatest carotenoid accumulator for in vitro 13C-labeling. Different Solanum lycopersicum allelic variants for high lycopene and varying herbicide treatments were compared for carotenoid accumulation in callus and suspension culture, and cell suspension cultures of the hp-1 line were chosen for isotopic labeling. When grown with [U]-13C-glucose and treated with CPTA, hp-1 suspensions yielded highly enriched 13C-lycopene with 45% of lycopene in the M+40 form and 88% in the M+35 to M+40 isotopomer range. To the authors' knowledge this is the first report of highly enriched 13C-carotenoid production from in vitro plant cell culture.

Automated catalyst processing for cloud electrode fabrication for fuel cells

DOEpatents

Goller, Glen J.; Breault, Richard D.

1980-01-01

A process for making dry carbon/polytetrafluoroethylene floc material, particularly useful in the manufacture of fuel cell electrodes, comprises of the steps of floccing a co-suspension of carbon particles and polytetrafluoroethylene particles, filtering excess liquids from the co-suspension, molding pellet shapes from the remaining wet floc solids without using significant pressure during the molding, drying the wet floc pellet shapes within the mold at temperatures no greater than about 150.degree. F., and removing the dry pellets from the mold.

Physical and Chemical Stability of Mycophenolate Mofetil (MMF) Suspension Prepared at the Hospital

PubMed Central

Fahimi, Fanak; Baniasadi, Shadi; Mortazavi, Seyed Alireza; Dehghan, Hanie; Zarghi, Afshin

2012-01-01

To evaluate the physical and chemical stability of a suspension of mycophenolate mofetil (MMF) prepared in the hospital from commercially available MMF capsules and tablets. Extemporaneous pharmacy was used as a feasible method in this experimental study to prepare suspension form of MMF. Suspension formulations were prepared from both tablets and capsules forms of MMF. Thereafter the stability parameters such as pH, microbial control, thermal and physical stability and particle sizes were evaluated. The amount of MMF, in the suspension was measured at various time points by HPLC. The HPLC method showed that concentration of suspensions prepared from tablets and capsules were 49 mg/mL and 50 mg/mL at time 0, respectively. The effective amount of suspensions prepared from capsules was 101% at time 0, 100% after 7 days, 98% after 14 days, and less than 70% after 28 days. According to the obtained results in this study, capsule-based suspension was stable for as long as 14 days at 5°C. This formulation appears to be clinically acceptable and provides a convenient dosage form for pediatric patients and for adults during the early postoperative period. PMID:24250439

SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture.

PubMed

Arhoma, A; Chantry, A D; Haywood-Small, S L; Cross, N A

2017-11-15

Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL-induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate-based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter-Glo luminescence assay, Caspase-8 and -9 activities were measured by Caspase-Glo™ assay kit. TRAIL-resistant cells were generated by culture of RPMI 8226 and NCI-H929 by acute exposure to TRAIL followed by selection of TRAIL-resistant cells. TRAIL significantly induced apoptosis in a dose-dependent manner in OPM-2, RPMI 8226, NCI-H929, U266, JJN-3 MM cell lines and ADC-1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM-2, and enhanced TRAIL responses were both via Caspase-8 and -9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co-treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI-H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL-sensitive cell populations that had been selected for TRAIL-resistance from initially TRAIL-sensitive populations. SAHA may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Improved design of dynamic vibration absorber by using the inerter and its application in vehicle suspension

NASA Astrophysics Data System (ADS)

Shen, Yujie; Chen, Long; Yang, Xiaofeng; Shi, Dehua; Yang, Jun

2016-01-01

Inerter is a recently proposed mechanical element with two terminals. The novelty of this paper is to present the improved design which aims to add traditional dynamic vibration absorber to the vehicle body by using the inerter. Based on this background, a new vehicle suspension structure called ISD suspension, including the inerter, spring and damper has been created. A dual-mass vibration model including the ISD suspension is considered in this study. Parameters are obtained by using the genetic optimizing algorithm. The frequency-domain simulation confirms that the ISD suspension can effectively improve the damping performance of the suspension system, especially at the offset frequency of the vehicle body, which is consistent with the feature of the dynamic vibration absorber added to the vehicle body mass. At last, a prototype ball screw inerter has been designed and the bench test of a quarter-car model has been undertaken. Under the conditions of the random road input, the vehicle ride comfort evaluation of body acceleration RMS value decreases by 4% at most, the suspension deflection RMS value decreases by 16% at most, the tire dynamic load RMS value decreases by 6% at most. Power spectral density results also indicate that the ISD suspension has superior damping performance than passive suspension which proves that the proposed ISD suspension is deemed effective.

Phenylalanine ammonia-lyase activity in suspension cultures of Ulmus pumila and U. campestris treated with spores of Ceratocystis ulmi.

PubMed

Corchete, M P; Diez, J J; Valle, T

1993-12-01

Cell suspension cultures of a Ceratocystis ulmi-resistant (Ulmus pumila) and a -susceptible elm (U.campestris) were established from leaf callus tissue. Treatment of cultures with spores of C.ulmi induced a large increase in the activity of phenylalanine ammonialyase, only in the cells of the resistant species U.pumila with a maximum after 24 h. Inoculated U.pumila cells also excreted a red unidentified chemical into the culture medium. Neither responses were induced in inoculated U.campestris cultures. The results are discussed in relation to the development of the elm cell culture system as a model for studying the differential biochemical mechanisms of disease resistance in elms.

Hybrid anodes for redox flow batteries

DOEpatents

Wang, Wei; Xiao, Jie; Wei, Xiaoliang; Liu, Jun; Sprenkle, Vincent L.

2015-12-15

RFBs having solid hybrid electrodes can address at least the problems of active material consumption, electrode passivation, and metal electrode dendrite growth that can be characteristic of traditional batteries, especially those operating at high current densities. The RFBs each have a first half cell containing a first redox couple dissolved in a solution or contained in a suspension. The solution or suspension can flow from a reservoir to the first half cell. A second half cell contains the solid hybrid electrode, which has a first electrode connected to a second electrode, thereby resulting in an equipotential between the first and second electrodes. The first and second half cells are separated by a separator or membrane.

Handling, storage, and preparation of human tissues.

PubMed

Dressler, L G; Visscher, D

2001-05-01

Human tissue for flow cytometry must be prepared as an adequate single-cell suspension. The appropriate methods for tissue collection, transport, storage, and dissociation depend on the cell parameters being measured and the localization of the markers. This unit includes a general method for collecting and transporting human tissue and preparing a tissue imprint. Protocols are supplied for tissue disaggregation by either mechanical or enzymatic means and for preparation of single-cell suspensions of whole cells from fine-needle aspirates, pleural effusions, abdominal fluids, or other body fluids. Other protocols detail preparation of intact nuclei from fresh, frozen, or paraffin-embedded tissue. Support protocols cover fixation, cryospin preparation, cryopreservation, and removal of debris.

A study of cell electrophoresis as a means of purifying growth hormone secreting cells

NASA Technical Reports Server (NTRS)

Plank, Lindsay D.; Hymer, W. C.; Kunze, M. Elaine; Marks, Gary M.; Lanham, J. Wayne

1983-01-01

Growth hormone secreting cells of the rat anterior pituitary are heavily laden with granules of growth hormone and can be partialy purified on the basis of their resulting high density. Two methods of preparative cell electrophoresis were investigated as methods of enhancing the purification of growth hormone producing cells: density gradient electrophoresis and continuous flow electrophoresis. Both methods provided a two- to four-fold enrichment in growth hormone production per cell relative to that achieved by previous methods. Measurements of electrophoretic mobilities by two analytical methods, microscopic electrophoresis and laser-tracking electrophoresis, revealed very little distinction between unpurified anterior pituitary cell suspensions and somatotroph-enriched cell suspensions. Predictions calculated on the basis of analytical electrophoretic data are consistent with the hypothesis that sedimentation plays a significant role in both types of preparative electrophoresis and the electrophoretic mobility of the growth hormone secreting subpopulation of cells remains unknown.

STUDIES OF ANTIGENIC DIFFERENCES AMONG STRAINS OF INFLUENZA A BY MEANS OF RED CELL AGGLUTINATION

PubMed Central

Hirst, George K.

1943-01-01

A study of cross inhibition tests among strains of influenza A virus and their antisera showed that the results obtained were subject to a certain amount of variation due to the red cells, the virus suspensions, and the ferret antisera employed. Methods have been demonstrated for handling the data obtained from such tests, so that these variables were corrected or avoided, making it possible to use the agglutination technique for antigenic comparisons. The antigenic pattern of eighteen strains of influenza A virus, obtained from the 1940–41 epidemic in the United States, has been compared by means of agglutination inhibition tests with ferret antisera. No significant antigenic differences were found among sixteen of these strains (all isolated from throat washings by the inoculation of chick embryos) although they were obtained from individuals in widely separated regions of the country. Two strains, from cases occurring early in the epidemic and isolated from throat washings by ferret and mouse passage, showed a slight but significant strain difference from the other strains and from each other. One of the 1940–41 strains on cross test resembled the PR8 strain more closely than any other stock strain tested. PMID:19871338

Detection of early changes in lung cell cytology by flow-systems analysis techniques, July 1--December 31, 1975

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

Steinkamp, J.A.; Ingram, M.; Hansen, K.M.

1976-03-01

This report summarizes results of preliminary experiments to demonstrate the feasibility of using automated flow-systems analysis in detecting early changes of respiratory epithelium exposed to physical and chemical agents associated with the by-products of nonnuclear energy production. The Syrian hamster was selected as the experimental test animal to begin investigation of the effects of toxic agents to cells of the respiratory tract. Since initiation of the program approximately six months ago, the goals have been acquisition of adequate numbers of exfoliated cells from the lung; adaptation of cytological techniques developed on human exfoliated gynecological samples to hamster lung epithelium formore » obtaining single-cell suspensions; utilization of existing cell staining methods to measure DNA content in lung cells; and analysis of DNA content and cell size. As the flow-system cell analysis technology is adapted to the measurement of exfoliated lung cells, rapid and quantitative determination of early changes in the physical and biochemical cellular properties will be attempted as a function of exposure to the toxic agents. (auth)« less

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.

Theoretical and Practical Issues That Are Relevant When Scaling Up hMSC Microcarrier Production Processes

PubMed Central

Jossen, Valentin; Schirmer, Cedric; Mostafa Sindi, Dolman; Eibl, Regine; Kraume, Matthias; Pörtner, Ralf; Eibl, Dieter

2016-01-01

The potential of human mesenchymal stem cells (hMSCs) for allogeneic cell therapies has created a large amount of interest. However, this presupposes the availability of efficient scale-up procedures. Promising results have been reported for stirred bioreactors that operate with microcarriers. Recent publications focusing on microcarrier-based stirred bioreactors have demonstrated the successful use of Computational Fluid Dynamics (CFD) and suspension criteria (N S1u, N S1) for rapidly scaling up hMSC expansions from mL- to pilot scale. Nevertheless, one obstacle may be the formation of large microcarrier-cell-aggregates, which may result in mass transfer limitations and inhomogeneous distributions of stem cells in the culture broth. The dependence of microcarrier-cell-aggregate formation on impeller speed and shear stress levels was investigated for human adipose derived stromal/stem cells (hASCs) at the spinner scale by recording the Sauter mean diameter (d 32) versus time. Cultivation at the suspension criteria provided d 32 values between 0.2 and 0.7 mm, the highest cell densities (1.25 × 106 cells mL−1 hASCs), and the highest expansion factors (117.0 ± 4.7 on day 7), while maintaining the expression of specific surface markers. Furthermore, suitability of the suspension criterion N S1u was investigated for scaling up microcarrier-based processes in wave-mixed bioreactors for the first time. PMID:26981131

Biochemical precursor effects on the fatty acid production in cell suspension cultures of Theobroma cacao L.

PubMed

Parra, O; Gallego, A M; Urrea, A; Rojas, L F; Correa, C; Atehortúa, L

2017-02-01

Cocoa butter (CB) is composed of 96% palmitic, stearic, oleic, linoleic and linolenic fatty acids that are responsible for the hardness, texture and fusion properties of chocolate. Through in vitro plant cell culture it is possible to modify CB lipid profiles and to study the fatty acid biosynthesis pathway on a subcellular level, evaluating fundamental aspects to enhance in vitro fatty acid production in a specific and controlled way. In this research, culture media was supplemented with acetate, biotin, pyruvate, bicarbonate and glycerol at three different concentrations and the effects on the biomass production (g/L), cell viability, and fatty acids profile and production was evaluated in in vitro cell suspensions culture. It was found that biotin stimulated fatty acid synthesis without altering cell viability and cell growth. It was also evident a change in the lipid profile of cell suspensions, increasing middle and long chain fatty acids proportion, which are unusual to those reported in seeds; thus implying that it is possible to modify lipid profiles according to the treatment used. According to the results of sucrose gradients and enzyme assays performed, it is proposed that cacao cells probably use the pentose phosphate pathway, mitochondria being the key organelle in the carbon flux for the synthesis of reductant power and fatty acid precursors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Transient maintenance in bioreactor improves health of neuronal cells.

PubMed

Di Loreto, Silvia; Sebastiani, Pierluigi; Benedetti, Elisabetta; Zimmitti, Vincenzo; Caracciolo, Valentina; Amicarelli, Fernanda; Cimini, Annamaria; Adorno, Domenico

2006-01-01

To examine whether a neuronal cell suspension can be held in vitro for a relatively short period without compromising survival rates and functionality, we have set up an experimental protocol planning 24 h of suspension culture in a rotary wall vessel (RWV) bioreactor before plating in a conventional adherent system. Apoptosis measurement and activated caspase-8, -9, and -3 detection have demonstrated that survey of the cells was not affected. The activity of major antioxidant enzymes (AOE), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), was significantly decreased in RWV-maintained cells. A significant decrease of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) is coupled with a level of activated nuclear factor-kappaB (NF-kappaB) protein significantly lower in RVW cells than in the control. On the contrary, the level of IL-6 expression did not change between the test and the control. A significant up-regulation of growth-associated protein-43 (GAP-43), peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta), and acyl-CoA synthetase 2 (ACS2) in RWV cells has been detected. We provide the evidence that primary neuronal cells, at an early stage of development, can be maintained in a suspension condition before adherent plating. This experimental environment does not induce detrimental effects but may have an activator role, leading cells to development and maturation in a tridimensional state.

Design and Analysis of the Warm-To Suspension Links for Jefferson Lab's 11 Gev/c Super High Momentum Spectrometer

NASA Astrophysics Data System (ADS)

Sun, E.; Brindza, P.; Lassiter, S.; Fowler, M.

2010-04-01

This paper describes design and analysis performed for the warm-to-cold suspension links of the warm iron yoke superconducting quadrupole magnets, and superconducting dipole magnet. The results of investigation of titanium Ti-6Al-4V and Nitronic 50 stainless steel for the suspension links to support the cold mass, preloads, forces due to cryogenic temperature, and imbalanced magnetic forces from misalignments are presented. Allowable stresses at normal-case scenarios and worst-case scenarios, space constraints, and heat leak considerations are discussed. Principles of the ASME Pressure Vessel Code were used to determine allowable stresses. Optimal angles of the suspension links were obtained by calculation and finite element methods. The stress levels of suspension links at multiple scenarios are presented, discussed, and compared with the allowable stresses.

Technique for obtaining highly enriched, quiescent immature Langerhans cells suitable for ex vivo assays.

PubMed

Tchou, Isabelle; Sabido, Odile; Lambert, Claude; Misery, Laurent; Garraud, Olivier; Genin, Christian

2003-03-03

Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a. These cells can capture a pathogen and then migrate and differentiate to a more mature stage. During this maturation process, dentritic cells express surface markers differentially. In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells. For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery. This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage. After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process. Both systems were equally efficient in terms of purification and yield. By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a(+) LCs, contrary to the repeated Ficoll floatation. This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells. Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.

Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.

PubMed

Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc

2016-01-01

Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.

Unexpected features of exponentially growing Tobacco Bright Yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition.

PubMed

Issawi, Mohammad; Muhieddine, Mohammad; Girard, Celine; Sol, Vincent; Riou, Catherine

2017-10-01

This article presents a new insight about TBY-2 cells; from extracellular polysaccharides secretion to cell wall composition during cell suspension culture. In the medium of cells taken 2 days after dilution (end of lag phase), a two unit pH decrease from 5.38 to 3.45 was observed and linked to a high uronic acid (UA) amount secretion (47.8%) while, in 4 and 7 day-old spent media, pH increased and UA amounts decreased 35.6 and 42.3% UA, respectively. To attain deeper knowledge of the putative link between extracellular polysaccharide excretion and cell wall composition, we determined cell wall UA and neutral sugar composition of cells from D2 to D12 cultures. While cell walls from D2 and D3 cells contained a large amount of uronic acid (twice as much as the other analysed cell walls), similar amounts of neutral sugar were detected in cells from lag to end of exponential phase cells suggesting an enriched pectin network in young cultures. Indeed, monosaccharide composition analysis leads to an estimated percentage of pectins of 56% for D3 cell wall against 45% D7 cell walls indicating that the cells at the mid-exponential growth phase re-organized their cell wall linked to a decrease in secreted UA that finally led to a stabilization of the spent medium pH to 5.4. In conclusion, TBY-2 cell suspension from lag to stationary phase showed cell wall remodeling that could be of interest in drug interaction and internalization study.

The microwave properties of composites including lightweight core-shell ellipsoids

NASA Astrophysics Data System (ADS)

Yuan, Liming; Xu, Yonggang; Dai, Fei; Liao, Yi; Zhang, Deyuan

2016-12-01

In order to study the microwave properties of suspensions including lightweight core-shell ellipsoids, the calculation formula was obtained by substituting an equivalent ellipsoid for the original core-shell ellipsoid. Simulations for Fe-coated diatomite/paraffin suspensions were performed. Results reveal that the calculated results fitted the measured results very well when the inclusion concentration was no more than 15 vol%, but there was an obvious deviation when the inclusion concentration reached 24 vol%. By comparisons, the formula for less diluted suspensions was more suitable for calculating the electromagnetic parameter of suspensions especially when the ratio was smaller between the electromagnetic parameter of the inclusion and that of the host medium.

Pulsed near-infrared photoacoustic spectroscopy of blood

NASA Astrophysics Data System (ADS)

Laufer, Jan G.; Elwell, Clare E.; Delpy, Dave T.; Beard, Paul C.

2004-07-01

The aim of this study was to use pulsed near infrared photoacoustic spectroscopy to determine the oxygen saturation (SO2) of a saline suspension of red blood cells in vitro. The photoacoustic measurements were made in a cuvette which formed part of a larger circuit through which the red blood cell suspension was circulated. Oxygen saturation of the red blood cell suspension was altered between 2-3% to 100% in step increments using a membrane oxygenator and at each increment an independent measurement of oxygen saturation was made using a co-oximeter. An optical parametric oscillator laser system provided nanosecond excitation pulses at a number of wavelengths in the near-infrared spectrum (740-1040nm) which were incident on the cuvette. The resulting acoustic signals were detected using a broadband (15MHz) Fabry-Perot polymer film transducer. The optical transport coefficient and amplitude were determined from the acoustic signals as a function of wavelength. These data were then used to calculate the relative concentrations of oxy- and deoxyhaemoglobin, using their known specific absorption coefficients and an empirically determined wavelength dependence of optical scattering over the wavelength range investigated. From this, the oxygen saturation of the suspension was derived with an accuracy of +/-5% compared to the co-oximeter SO2 measurements.

Absorption Spectra of Gold Nanoparticle Suspensions

NASA Astrophysics Data System (ADS)

Anan'eva, M. V.; Nurmukhametov, D. R.; Zverev, A. S.; Nelyubina, N. V.; Zvekov, A. A.; Russakov, D. M.; Kalenskii, A. V.; Eremenko, A. N.

2018-02-01

Three gold nanoparticle suspensions are obtained, and mean radii in distributions - (6.1 ± 0.2), (11.9 ± 0.3), and (17.3 ± 0.7) nm - are determined by the transmission electron microscopy method. The optical absorption spectra of suspensions are obtained and studied. Calculation of spectral dependences of the absorption index of suspensions at values of the gold complex refractive index taken from the literature showed a significant deviation of experimental and calculated data in the region of 450-800 nm. Spectral dependences of the absorption of suspensions are simulated within the framework of the Mie-Drude theory taking into account the interband absorption in the form of an additional term in the imaginary part of the dielectric permittivity of the Gaussian type. It is shown that to quantify the spectral dependences in the region of the plasmon absorption band of nanoparticles, correction of the parameters of the interband absorption is necessary in addition to the increase of the relaxation parameter of the Drude theory. Spectral dependences of the dielectric permittivity of gold in nanodimensional state are refined from the solution of the inverse problem. The results of the present work are important for predicting the special features of operation of photonic devices and optical detonators based on gold nanoparticles.

Activity of lysozyme on Lactobacillus hilgardii strains isolated from Port wine.

PubMed

Dias, Rita; Vilas-Boas, Eduardo; Campos, Francisco M; Hogg, Tim; Couto, José António

2015-08-01

This work evaluated the effect of lysozyme on lactobacilli isolated from Port wine. Bacterial growth experiments were conducted in MRS/TJ medium and inactivation studies were performed in phosphate buffer (KH2PO4), distilled water and wine supplemented with different concentrations of lysozyme. The response of bacteria to lysozyme was found to be highly strain dependent. Some strains of Lactobacillus hilgardii together with Lactobacillus collinoides and Lactobacillus fructivorans were found to be resistant to concentrations of lysozyme as high as 2000 mg/L. It was observed that among the L. hilgardii taxon the resistant strains possess an S-layer coat. Apparently, the strains of L. collinoides and L. fructivorans studied are also S-layer producers as suggested by the total protein profile obtained by SDS-PAGE. Thus, the hypothetical protective role of the S-layer against the action of lysozyme was investigated. From the various treatments used to remove the protein from the surface of the cells, the one employing LiCl (5 M) was the most effective. LiCl pre-treated cells exposed to lysozyme (2000 mg/L) in KH2PO4 buffer maintained its resistance. However, when cells were suspended in distilled water an increased sensitivity to lysozyme was observed. Moreover, it was found that the addition of ethanol (20% v/v) to the suspension medium (distilled water) triggered a strong inactivation effect especially on cells previously treated with LiCl (reduction of >6 CFU log cycles). The results suggest that the S-layer exerts a protective effect against lysozyme and that the cell suspension medium influences the bacteriolysis efficiency. It was also noted that ethanol enhances the inactivation effect of lysozyme. Copyright © 2015 Elsevier Ltd. All rights reserved.