Sample records for cell culture assay

  1. Defining cell culture conditions to improve human norovirus infectivity assays

    SciTech Connect

    Straub, Tim M.; Hutchison, Janine R.; Bartholomew, Rachel A.; Valdez, Catherine O.; Valentine, Nancy B.; Dohnalkova, Alice; Ozanich, Richard M.; Bruckner-Lea, Cindy J.

    2013-01-10

    Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that leads to more reproducible hNoV infectivity in vitro requires that the cell line be 1) of human gastrointestinal origin, 2) expresses apical microvilli, and 3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both reverse transcription quantitative PCR (qRT-PCR) and microscopy. Using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using quantitative reverse transcription PCR (qRT-PCR) that measures all RNA vs. plaque assays that measure infectious virus.

  2. A cell culture assay for the detection of cardiotoxicity

    SciTech Connect

    Loew-Friedrich, Iv.; von Bredow, F.; Schoeppe, W. (Department of Nephrology, Hospital of the Johann Wolfgang Goethe-University, Frankfurt am Main (Germany))

    1991-04-01

    An important step in minimizing the number of animal experiments in medical research is the study of in vitro model systems. The authors propose the use of shock protein formation, which is a cellular response to cell-damaging stress as an assay to monitor cardiotoxicity. Isolated and cultured cardiac myocytes were prepared by a trypsin digestion method from 18-day-old fetal mice. These cells respond to typical substances inducing shock protein formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of shock proteins. Pharmaceuticals relevant in transplant medicine were tested for possible cardiotoxic effects: Cyclosporine A evokes shock protein formation at subtherapeutic concentrations. Azathioprine and methyl-prednisolone exert the same effect but at concentration ranges highly above the therapeutic level. The ability to induce shock protein synthesis obviously seems to be restricted to toxic drugs. The data presented demonstrate that the proposed in vitro model system for cardiotoxicity is animal saving and sensitive.

  3. Luciferase reporter assay in Drosophila and mammalian tissue culture cells

    PubMed Central

    Yun, Chi

    2014-01-01

    Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell-based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA /dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions. PMID:24652620

  4. In vitro cell culture infectivity assay for human noroviruses.

    PubMed

    Straub, Timothy M; Höner zu Bentrup, Kerstin; Orosz-Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K; Bartholomew, Rachel A; Valdez, Catherine O; Bruckner-Lea, Cynthia J; Gerba, Charles P; Abbaszadegan, Morteza; Nickerson, Cheryl A

    2007-03-01

    Human noroviruses cause severe, self-limiting gastroenteritis that typically lasts 24-48 hours. Because of the lack of suitable tissue culture or animal models, the true nature of norovirus pathogenesis remains unknown. We show, for the first time, that noroviruses can infect and replicate in a physiologically relevant 3-dimensional (3-D), organoid model of human small intestinal epithelium. This level of cellular differentiation was achieved by growing the cells on porous collagen-I coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in situ hybridization provided evidence of norovirus infection. Cytopathic effect and norovirus RNA were detected at each of the 5 cell passages for genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts that used differentiated monolayer cultures failed. PMID:17552092

  5. Heat-Transfer-Method-Based Cell Culture Quality Assay through Cell Detection by Surface Imprinted Polymers.

    PubMed

    Eersels, Kasper; van Grinsven, Bart; Khorshid, Mehran; Somers, Veerle; Püttmann, Christiane; Stein, Christoph; Barth, Stefan; Diliën, Hanne; Bos, Gerard M J; Germeraad, Wilfred T V; Cleij, Thomas J; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick

    2015-02-17

    Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time. PMID:25654744

  6. CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures.

    PubMed

    Trehan, Ashutosh; Rotgers, Emmi; Coffey, Eleanor T; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

    2014-11-01

    BackgroundG protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to G¿S stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in studies of GPCR signaling. Primary cell cultures represent a more robust model than cell lines to study GPCR signaling since they physiologically resemble the parent tissue. Current cAMP assays have two fundamental limitations: 1) absence of cAMP kinetics as competition-based assays require cell lysis and measure only a single time-point, and 2) high variation with separate samples needed to measure consecutive time points. The utility of real-time cAMP biosensors is also limited in primary cell cultures due to their poor transfection efficiency, variable expression levels and inability to select stable clones. We therefore, decided to develop an assay that can measure cAMP not only at a single time-point but the entire cAMP kinetics after GPCR activation in untransfected primary cells.ResultsCANDLES (Cyclic AMP iNdirect Detection by Light Emission from Sensor cells) assay for monitoring cAMP kinetics in cell cultures, particularly in primary cultures was developed. The assay requires co-culturing of primary cells with sensor cells that stably express a luminescent cAMP sensor. Upon GPCR activation in primary cells, cAMP is transferred to sensor cells via gap junction channels, thereby evoking a luminescent read-out. GPCR activation using primary cultures of rat cortical neurons and mouse granulosa cells was measured. Kinetic responses of different agonists to adrenergic receptors were also compared using rat cortical neurons. The assay optimization was done by varying sensor-test cell ratio, using phosphodiesterase inhibitors and testing cell-cell contact requirement.ConclusionsHere we present CANDLES assay based on co-culturing test cells with cAMP-detecting sensor cells. This co-culture setup allows kinetic measurements, eliminates primary cell transfections and reduces variability. A variety of cell types (rat cortical neurons, mouse granulosa cells and established cell lines) and receptors (adrenergic, follicle stimulating hormone and luteinizing hormone/chorionic gonadotropin receptors) were tested for use with CANDLES. The assay is best applied while comparing cAMP generation curves upon different drug treatments to untransfected primary cells. PMID:25366423

  7. Gonococcal and meningococcal pathogenesis as defined by human cell, cell culture, and organ culture assays.

    PubMed Central

    Stephens, D S

    1989-01-01

    Human cells, cell cultures, and organ cultures have been extremely useful for studying the events that occur when gonococci and meningococci encounter human mucosal surfaces. The specificity and selectivity of these events for human cells are striking and correlate with the adaptation of these pathogens for survival on human mucous membranes. To colonize these sites, meningococci and gonococci have developed mechanisms to damage local host defenses such as the mucociliary blanket, to attach to epithelial cells, and to invade these cells. Attachment to epithelial cells mediated by pili, and to some types of cells mediated by PIIs, serves to anchor the organism close to sources of nutrition and allows multiplication. Intracellular invasion, possibly initiated by the major porin protein, may provide additional nutritional support and protection from host defenses. Mucosal invasion may also result in access of gonococci and meningococci to the bloodstream, leading to dissemination. Images PMID:2497953

  8. In Vitro Cell Culture Infectivity Assay for Human Noroviruses

    SciTech Connect

    Straub, Tim M.; Honer Zu Bentrup, Kerstin A.; Orosz Coghlan, Patricia A.; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza; Nickerson, Cheryl A.

    2007-01-30

    Human noroviruses (NoV) cause severe, self-limiting gastroenteritis that typically lasts 24 - 48 hours. The true nature of NoV pathogenesis remains unknown due to the lack of suitable tissue culture or animal models. Here we show, for the first time, that NoV can infect and replicate in an organoid, three-dimensional (3-D) model of human small intestinal epithelium (INT-407). Cellular differentiation for this model was achieved by growing the cells in 3-D on porous collagen I-coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in-situ hybridization were employed to provide evidence of NoV infection. CPE and norovirus RNA was detected at each of the five cell passages for both genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts using differentiated monolayer cultures failed.

  9. Biochemical assays of cultured cells. [space shuttle oft-3

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1981-01-01

    Assay systems were developed for use in interpreting samples to be returned on the space shuttle OFT-3 flights. Samples from electrophoretic separation were used to evaluate the techniques. All assays were determinable on the growth media. Approaches are described for assaying: (1) the human granulocyte conditioning factor; (2) urokinase activity; (3) erythropoietin; (4) the molecular form of urokinase; and (5) protein distribution. Other studies are planned to validate that the activity observed is urokinase and not that of other activators or proteases.

  10. TOTAL CULTURABLE VIRUS QUANTAL ASSAY

    EPA Science Inventory

    This chapter describes a quantal method for assaying culturable human enteric viruses from water matrices. The assay differs from the plaque assay described in Chapter 10 (December 1987 Revision) in that it is based upon the direct microscopic viewing of cells for virus-induced ...

  11. The Effect of Primary Cancer Cell Culture Models on the Results of Drug Chemosensitivity Assays: The Application of Perfusion Microbioreactor System as Cell Culture Vessel

    PubMed Central

    Chen, Yi-Dao; Huang, Shiang-Fu; Wang, Hung-Ming

    2015-01-01

    To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that minor environmental pH change could significantly affect the metabolic activity of cells, demonstrating the importance of stable culture condition for such assays. Moreover, the culture models could also significantly influence the metabolic activity and proliferation of cells. Furthermore, the choice of culture models might lead to different outcomes of chemosensitivity assays. Compared with the similar test based on tumor-level assays, the spheroid model could overestimate the drug resistance of cells to cisplatin, whereas the 2D and 3D culture models might overestimate the chemosensitivity of cells to such anticancer drug. In this study, the 3D culture models with same cell density as that in tumor samples showed comparable chemosensitivity assay results as the tumor-level assays. Overall, this study has provided some fundamental information for establishing a precise and faithful drug chemosensitivity assay. PMID:25654105

  12. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture.

    PubMed

    Cory, A H; Owen, T C; Barltrop, J A; Cory, J G

    1991-07-01

    A new tetrazolium analog of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was evaluated as a substitute for MTT in the microculture screening assay for in vitro cell growth. This new tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), in the presence of phenazine methosulfate (PMS), gave a water-soluble formazan product that had an absorbance maximum at 490-500 nm in phosphate-buffered saline. The amount of colored product formed was proportional to the number of cells and the time of incubation of the cells with MTS/PMS. MTS/PMS was reactive in all the cell lines tested which included mouse leukemia L1210 cells, mouse Ehrlich tumor cells, mouse 3T3 fibroblasts, and human colon tumor cells (HT-29). HT-29 and 3T3 fibroblasts reduced MTS/PMS more efficiently than they reduced MTT. Comparable to the amount of product formed from MTT, MTS/PMS gave excellent product formation. The IC50 value for pyrazoloimidazole obtained using MTS/PMS was 200 microM; for 5-fluoro-2'-deoxyuridine, the IC50 value was 0.9 nM. These values compared very favorably with the IC50 values obtained by direct cell counts. Further, the same IC50 values were obtained when the absorbances of the formazan product in the 96-well plates were determined after different times of incubation. PMID:1867954

  13. Development of a novel perfusion microfluidic cell culture device for cell-based assays

    Microsoft Academic Search

    Yunhuan Zheng; Jianzhang Wu; Jianbo Shao; Qinghui Jin; Jianlong Zhao

    2009-01-01

    This study reports on the development of a novel perfusion microfluidic cell culture device integrated drug delivering, fluid controlling and cell culturing functions on a two- layer PDMS chip. The device consists of an array of 6 X 6 cell culture chambers, a drug gradient generator and two control valves. Cells are physically trapped and cultured in the center of

  14. In Vitro Cell Culture Infectivity Assay for Human Noroviruses

    Microsoft Academic Search

    Timothy M. Straub; Kerstin A. Honer Zu Bentrup; Patricia A. Orosz Coghlan; Alice Dohnalkova; Brooke K. Mayer; Rachel A. Bartholomew; Catherine O. Valdez; Cindy J. Bruckner-Lea; Charles P. Gerba; Morteza Abbaszadegan; Cheryl A. Nickerson

    2007-01-01

    Human noroviruses (NoV) cause severe, self-limiting gastroenteritis that typically lasts 24 - 48 hours. The true nature of NoV pathogenesis remains unknown due to the lack of suitable tissue culture or animal models. Here we show, for the first time, that NoV can infect and replicate in an organoid, three-dimensional (3-D) model of human small intestinal epithelium (INT-407). Cellular differentiation

  15. COMPARATIVE TOXICITIES OF DIFFERENT FORMS OF ASBESTOS IN A CELL CULTURE ASSAY

    EPA Science Inventory

    Three forms of Union Internationale Contre le Cancer (UICC) asbestos, amosite, crocidolite, and chrysotile, were assayed for their cytotoxicity (inhibition of colony formation) in cell culture. Using embryonic human intestine-derived (I-407) and adult rat liver-derived (ARL-6) ep...

  16. Nine surface plasmon resonance assays for specific protein quantitation during cell culture and process development.

    PubMed

    Frostell, Sa; Mattsson, Anna; Eriksson, Sa; Wallby, Elisabeth; Kärnhall, Johan; Illarionova, Nina B; Estmer Nilsson, Camilla

    2015-05-15

    Quantitation of protein is essential during pharmaceutical development, and a variety of methods and technologies for determination of total and specific protein concentration are available. Here we describe the development of a streamlined assay platform for specific quantitation assays using surface plasmon resonance (SPR) technology. A total of nine different assays were developed using similar conditions, of which eight assays were for quantitation of different human blood plasma proteins (IgG, IgG1-4 subclasses, IgA, transferrin, and albumin) from a chromatography-based IgG plasma process. Lastly, an assay for monitoring the concentration of a recombinant monoclonal antibody during 13days of CHO cell culturing was developed. Assay performances were compared with enzyme-linked immunosorbent assay (ELISA), nephelometry, ARCHITECT, and Cobas c501. SPR assays were shown to have higher sensitivity than analysis using nephelometry, ARCHITECT, and Cobas and to have significantly lower analysis and hands-on time compared with ELISA. Furthermore, the SPR assays were robust enough to be used for up to 12days, allowing specific protein concentration measurement of a sample to be completed at line within 10min. Using the same platform with only few varied parameters between different assays has saved time in the lab as well as for evaluation and presentation of results. PMID:25700863

  17. Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

    PubMed Central

    Azari, Hassan; Sharififar, Sharareh; Rahman, Maryam; Ansari, Saeed; Reynolds, Brent A.

    2011-01-01

    In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum?free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments. PMID:21248704

  18. Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay

    PubMed Central

    Nickson, Catherine M.; Parsons, Jason L.

    2014-01-01

    Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (?20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase ?, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. PMID:25076968

  19. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

    PubMed Central

    Shin, Yoojin; Han, Sewoon; Jeon, Jessie S.; Yamamoto, Kyoko; Zervantonakis, Ioannis K.; Sudo, Ryo; Kamm, Roger D.; Chung, Seok

    2014-01-01

    This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel incorporating chambers between surface-accessible microchannels. Using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flows and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 days to fabricate the system and experiments can run for up to several weeks. PMID:22678430

  20. Detection of Hepatitis A Virus by Using a Combined Cell Culture-Molecular Beacon Assay?

    PubMed Central

    Yeh, Hsiao-Yun; Hwang, Yu-Chen; Yates, Marylynn V.; Mulchandani, Ashok; Chen, Wilfred

    2008-01-01

    Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5? noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays. PMID:18263747

  1. An improved system for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay

    Microsoft Academic Search

    Masumi Asakura; Toshiaki Sasaki; Toshie Sugiyama; Heihachiro Arito; Shoji Fukushima; Taijiro Matsushima

    2008-01-01

    A gas exposure system using rotating vessels was improved for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay. This system was composed of 12 square culture vessels, a device for preparation of air containing test gas, and positive and negative control gases at target concentrations and for supplying these gases to the culture vessels, and

  2. Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models.

    PubMed

    Bonnier, F; Keating, M E; Wróbel, T P; Majzner, K; Baranska, M; Garcia-Munoz, A; Blanco, A; Byrne, H J

    2015-02-01

    Comparisons of 2D and 3D cell culture models in literature have indicated differences in cellular morphology and metabolism, commonly attributed the better representation of in vivo conditions of the latter cell culture environment. Thus, interest in the use of 3D collagen gels for in vitro analysis has been growing. Although comparative studies to date have indicated an enhanced resistance of cells on collagen matrices against different toxicants, in the present study it is demonstrated that non-adapted protocols can lead to misinterpretation of results obtained from classical colorimetric dye-based cytotoxic assays. Using the well established Alamar blue assay, the study demonstrates how the transfer from 2D substrates to 3D collagen matrices can affect the uptake of the resazurin itself, affecting the outcome of the assay. Using flow cytometry, it is demonstrated that the cell viability is unaffected when cells are grown on collagen matrices, thus the difference seen in the fluorescence is a result of a dilution of the resazurin dye in the collagen matrix, and an increased uptake rate due to the larger cell surface exposed to the surrounding environment, facilitating more effective diffusion through the cellular membrane. The results are supported by a rate equation based simulation, verifying that differing uptake kinetics can result in apparently different cell viability. Finally, this work highlights the feasibility to apply classical dye-based assays on collagen based 3D cell culture models. However, the diffusion and bioavailability of test substances in 3D matrices used in in vitro toxicological assays must be considered and adaption of the protocols is necessary for direct comparison with the traditional 2D models. Moreover, the observations made based on the resazurin dye can be applied to drugs or nanoparticles which freely diffuse through the collagen matrices, thus affecting the effective concentration exposed to the cells. PMID:25300790

  3. Comparison of In Vitro Cell Culture and a Mouse Assay for Measuring Infectivity of Cryptosporidium parvum

    PubMed Central

    Rochelle, Paul A.; Marshall, Marilyn M.; Mead, Jan R.; Johnson, Anne M.; Korich, Dick G.; Rosen, Jeffrey S.; De Leon, Ricardo

    2002-01-01

    In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the “gold standard,” mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all assays indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences. PMID:12147476

  4. Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay.

    PubMed

    Farkash, Evan A; Kao, Gary D; Horman, Shane R; Prak, Eline T Luning

    2006-01-01

    Long Interspersed Elements (LINE-1s, L1s) are the most active mobile elements in the human genome and account for a significant fraction of its mass. The propagation of L1 in the human genome requires disruption and repair of DNA at the site of integration. As Barbara McClintock first hypothesized, genotoxic stress may contribute to the mobilization of transposable elements, and conversely, element mobility may contribute to genotoxic stress. We tested the ability of genotoxic agents to increase L1 retrotransposition in a cultured cell assay. We observed that cells exposed to gamma radiation exhibited increased levels of L1 retrotransposition. The L1 retrotransposition frequency was proportional to the number of phosphorylated H2AX foci, an indicator of genotoxic stress. To explore the role of the L1 endonuclease in this context, endonuclease-deficient tagged L1 constructs were produced and tested for their activity in irradiated cells. The activity of the endonuclease-deficient L1 was very low in irradiated cells, suggesting that most L1 insertions in irradiated cells still use the L1 endonuclease. Consistent with this interpretation, DNA sequences that flank L1 insertions in irradiated cells harbored target site duplications. These results suggest that increased L1 retrotransposition in irradiated cells is endonuclease dependent. The mobilization of L1 in irradiated cells potentially contributes to genomic instability and could be a driving force for secondary mutations in patients undergoing radiation therapy. PMID:16507671

  5. Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment.

    PubMed Central

    Dahling, D R; Wright, B A

    1986-01-01

    An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures. Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents. Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas. The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses. A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system. PMID:3010860

  6. Clonogenic Assay: Adherent Cells

    PubMed Central

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation. PMID:21445039

  7. Clonogenic assay: adherent cells.

    PubMed

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation. PMID:21445039

  8. A microtiter trypan blue absorbance assay for the quantitative determination of excitotoxic neuronal injury in cell culture.

    PubMed

    Uliasz, T F; Hewett, S J

    2000-07-31

    An automated method for the determination of neuronal cell death using trypan blue is described. Following various excitotoxic insults, murine mixed cortical cell cultures are stained with trypan blue (0.05%; 15 min), followed by SDS (1%) lysis. The absorbance of the dye is measured spectrophotometrically at 590 nm using a microtiter plate reader. When compared to the biochemical lactate dehydrogenase assay, no statistical difference in the calculated levels of excitotoxic neuronal cell death was noted between the assays in any given paradigm. This method is fast and reliable. It eliminates the need for cell counting, thus allowing for high volume sample analysis with a minimum of sample error. Utility of this trypan blue absorbance spectrophotometric assay is likely to extend beyond the study of excitotoxic neuronal injury and should complement existing methods for measuring neuronal viability and cytotoxicity in cell culture. PMID:11040379

  9. Accurate non-invasive image-based cytotoxicity assays for cultured cells

    Microsoft Academic Search

    Patricia Marqués-Gallego; Hans den Dulk; Claude Backendorf; Jaap Brouwer; Jan Reedijk; Julian F Burke

    2010-01-01

    BACKGROUND: The CloneSelect™ Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay. RESULTS: Here we show that both the CloneSelect Imager and the MTT approach result in comparable EC50 values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. However, the image-based technique was found

  10. Detection of Hepatitis A Virus by Using a Combined Cell Culture-Molecular Beacon Assay

    Microsoft Academic Search

    Hsiao-Yun Yeh; Yu-Chen Hwang; Marylynn V. Yates; Ashok Mulchandani; Wilfred Chen

    2008-01-01

    Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow.

  11. Improved chicken embryo cell culture plaque assay for scrub typhus rickettsiae.

    PubMed Central

    Woodman, D R; Grays, R; Weiss, E

    1977-01-01

    The plaque technique for three strains of Rickettsia tsutsugamushi in chicken embryo cell cultures was greatly improved by modifying the trypsinizing procedure and employing homologous chicken serum in the overlay medium. Images PMID:412863

  12. Microscale 3D Collagen Cell Culture Assays in Conventional Flat-Bottom 384-Well Plates.

    PubMed

    Leung, Brendan M; Moraes, Christopher; Cavnar, Stephen P; Luker, Kathryn E; Luker, Gary D; Takayama, Shuichi

    2015-04-01

    Three-dimensional (3D) culture systems such as cell-laden hydrogels are superior to standard two-dimensional (2D) monolayer cultures for many drug-screening applications. However, their adoption into high-throughput screening (HTS) has been lagging, in part because of the difficulty of incorporating these culture formats into existing robotic liquid handling and imaging infrastructures. Dispensing cell-laden prepolymer solutions into 2D well plates is a potential solution but typically requires large volumes of reagents to avoid evaporation during polymerization, which (1) increases costs, (2) makes drug penetration variable and (3) complicates imaging. Here we describe a technique to efficiently produce 3D microgels using automated liquid-handling systems and standard, nonpatterned, flat-bottomed, 384-well plates. Sub-millimeter-diameter, cell-laden collagen gels are deposited on the bottom of a ~2.5 mm diameter microwell with no concerns about evaporation or meniscus effects at the edges of wells, using aqueous two-phase system patterning. The microscale cell-laden collagen-gel constructs are readily imaged and readily penetrated by drugs. The cytotoxicity of chemotherapeutics was monitored by bioluminescence and demonstrated that 3D cultures confer chemoresistance as compared with similar 2D cultures. Hence, these data demonstrate the importance of culturing cells in 3D to obtain realistic cellular responses. Overall, this system provides a simple and inexpensive method for integrating 3D culture capability into existing HTS infrastructure. PMID:25510473

  13. Multilayer and monolayer cell cultures in a cytotoxicity assay of root canal sealers.

    PubMed

    Vajrabhaya, L; Sithisarn, P

    1997-03-01

    The aim of this study was to evaluate the response of a multilayer compared with a monolayer cell culture using six root canal sealers. Both monolayer and multilayer of MU-mu-1 (Mahidol University mouse cell line 1) were cultured in separate 96-well plates. Following incubation at 37 degrees C in 5% CO2 for 4 h in the presence of each sealer, cells were stained with 0.4% Sulphorhodamine-B, viable dye staining and the absorbance at 540 nm determined and calculated as cell viability. There was no statistical difference in the percentage viability for each sealer in both the multilayer and the monolayer cell culture (P > 0.01). Apexit and AH-26 were less toxic (P < 0.01) than MU (Mahidol University) sealer, ROCANAL 3, ROCANAL 2, and Endomethasone which demonstrated the same toxicity (P > 0.01). PMID:10332248

  14. A PCR-based Rapid Neutralization Assay for GII.4 Norovirus Infection in HIEC6 Cell Culture.

    PubMed

    Fan, Yi Sun; Liu, Cheng; Zhu, Hui Juan; Ding, Yi; Zeng, Wan Jie; Yin, Xu Fang; Ding, Shuang Shuang; Zhang, Jun

    2015-03-01

    Because of limited viral replication and lack of cytopathic effect in cell culture, a new PCR-based rapid seroneutralization assay for detection of GII.4 norovirus neutralized antibodies was developed with serum samples from acute-phase patients, convalescent-phase patients and healthy controls. According to this study, neutralizing antibodies were detected in 100% of convalescent-phase sera, and in 2.5% of healthy controls sera. However, all of the acute-phase serum samples could not neutralize virus efficiently. Compared to the results from ELISA (96.2% at sensitivity and 80% at specificity), the present in vitro neutralization assay is more specific and more sensitive. PMID:25800447

  15. Validation of a Cell-Free Translation Assay for Detecting Shiga Toxin 2 in Bacterial Culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have validated a cell-free translation (CFT) assay for detecting Shiga toxin (Stx). The limit of detection (LOD) for pure Stx2 (PStx2) and partially pure Stx2 (PPStx2) in water reached 20 pg/µl and 3.5 pg/µL respectively without the artificial process of proteolytic activation and reduction of th...

  16. Preliminary evaluation of in vitro prescreen assays for developmental toxicants based on cultured murine preimplantation embryos and a cell line developed from a bovine preimplantation embryo

    Microsoft Academic Search

    B. W. Kemppainen; P. Terse; O. Zurovac; D. Stringfellow

    1996-01-01

    The purpose of this research was to evaluate in vitro assays and compare their efficiency in accurate prediction of the potential of chemicals to cause abnormal embryonic\\/foetal development. In vitro assays were based on cultured murine preimplantation embryos and a continuous cell line derived from a bovine preimplantation embryo. Preimplantation embryos collected from superovulated mice were cultured for 72 hr

  17. GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells

    PubMed Central

    Santillan, Beatriz A.; Moye, Christopher; Mittelman, David; Wilson, John H.

    2014-01-01

    Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries. PMID:25423602

  18. Metabolic response of environmentally isolated microorganisms to industrial effluents: Use of a newly described cell culture assay

    NASA Technical Reports Server (NTRS)

    Ferebee, Robert N.

    1992-01-01

    An environmental application using a microtiter culture assay to measure the metabolic sensitivity of microorganisms to petrochemical effluents will be tested. The Biomedical Operations and Research Branch at NASA JSC has recently developed a rapid and nondestructive method to measure cell growth and metabolism. Using a colorimetric procedure the uniquely modified assay allows the metabolic kinetics of prokaryotic and eukaryotic cells to be measured. Use of such an assay if adapted for the routine monitoring of waste products, process effluents, and environmentally hazardous substances may prove to be invaluable to the industrial community. The microtiter method as described will be tested using microorganisms isolated from the Galveston Bay aquatic habitat. The microbial isolates will be identified prior to testing using the automated systems available at JSC. Sodium dodecyl sulfate (SDS), cadmium, and lead will provide control toxic chemicals. The toxicity of industrial effluent from two industrial sites will be tested. An effort will be made to test the efficacy of this assay for measuring toxicity in a mixed culture community.

  19. Polymer-Based Mesh as Supports for Multi-layered 3D Cell Culture and Assays

    PubMed Central

    Simon, Karen A.; Park, Kyeng Min; Mosadegh, Bobak; Subramaniam, Anand Bala; Mazzeo, Aaron; Ngo, Phil M.; Whitesides, George M.

    2013-01-01

    Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system – Cells-in-Gels-in-Mesh (CiGiM) – that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells—layer-by-layer—within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis—(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format. PMID:24095253

  20. Comparison of a Commercial Real-Time PCR Assay for tcdB Detection to a Cell Culture Cytotoxicity Assay and Toxigenic Culture for Direct Detection of Toxin-Producing Clostridium difficile in Clinical Samples?

    PubMed Central

    Stamper, Paul D.; Alcabasa, Romina; Aird, Deborah; Babiker, Wisal; Wehrlin, Jennifer; Ikpeama, Ijeoma; Carroll, Karen C.

    2009-01-01

    Rapid detection of toxin-producing strains of Clostridium difficile is essential for optimal management of patients with C. difficile infection. The BD GeneOhm (San Diego, CA) Cdiff assay, a real-time PCR assay that amplifies tcdB, was compared to a cell culture neutralization assay (Wampole C. difficile Toxin B [TOX-B] test; TechLab, Blacksburg, VA) and to toxigenic culture. Using liquid (n = 273) and soft (n = 131) stool specimens from 377 symptomatic patients, all testing was performed on the same day by independent laboratory staff according to the manufacturers' protocols. Toxigenic bacterial culture was performed as follows. A 0.5-ml aliquot of stool was heated to 80°C for 10 min, followed by inoculation onto modified cycloserine cefoxitin fructose agar with and without horse blood (Remel, Lenexa, KS) and into prereduced chopped-meat broth. Of the 404 stool specimens tested, 340 were negative and 40 were positive (10.0% prevalence) both by PCR for tcdB and by cytotoxin production. The overall agreement between the BD GeneOhm Cdiff assay and the TOX-B test was 94.8% (380/401). When the TOX-B test was used as the reference method, the initial sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 90.9% (40/44), 95.2% (340/357), 70.2% (40/57), and 98.8% (340/344), respectively. When toxigenic culture was used as the “gold standard,” the sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 83.6%, 98.2%, 89.5%, and 97.1%, respectively, and those of the TOX-B test were 67.2%, 99.1%, 93.2%, and 94.4%, respectively. PCRs for three samples were inhibited upon initial testing; one sample was resolved upon retesting. One sample produced nonspecific cytotoxin results. The BD GeneOhm Cdiff assay performed well compared to a standard cell culture neutralization assay and to toxigenic culture for the detection of toxigenic C. difficile directly from fecal specimens. PMID:19073875

  1. Comparison of BGM and PLC/PRC/5 cell lines for total culturable viral assay of treated sewage.

    PubMed

    Rodríguez, Roberto A; Gundy, Patricia M; Gerba, Charles P

    2008-05-01

    The objective of this study was to compare PLC/PRF/5 and BGM cell lines for use in a total culturable viral assay (TCVA) of treated sewage effluents. Samples were collected before and after chlorination from an activated sludge wastewater treatment plant and from the effluent of a high-rate enhanced flocculation system, followed by UV light disinfection. Cell monolayers were observed for cytopathic effect (CPE) after two passages of 14 days each. Monolayers exhibiting viral CPE were tested for the presence of adenoviruses and enteroviruses by PCR or reverse transcription-PCR. Eight percent of the samples exhibited CPE on BGM cells, and 57% showed CPE on PLC/PRF/5 cells. Only enteroviruses were detected on the BGM cells, while 30% and 52% of the samples were positive for enteroviruses and adenoviruses, respectively, on the PLC/PRF/5 cells. Thirty percent of the samples were positive for both adenoviruses and enteroviruses in chlorinated activated sludge effluent. Thirty percent of the samples were positive for adenoviruses in the UV treatment effluent, but no enteroviruses were detected. In conclusion, the PLC/PRF/5 cells were more susceptible than BGM cells to viruses found in treated sewage. The use of BGM cells for TCVA may underestimate viral concentration in sewage effluent samples. The PLC/PRF/5 cells were more susceptible to adenoviruses, which is important in the evaluation of UV disinfection systems because adenoviruses are highly resistant to UV inactivation. PMID:18326686

  2. A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR.

    PubMed

    Schalk, Johanna A C; de Vries, Claudette G J C A; Orzechowski, Tom J H; Rots, Marianne G

    2007-11-01

    The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by E1-specific quantitative PCR. The detection limit of this assay is 3 RCAs in 1 x 10(10) vector particles per 70 ml of microcarrier cell culture. The main advantage of the combination of cell culture and PCR detection is that replicated virus can be detected long before cytopathic effects become visible and therefore, it is much faster than conventional cell culture-based assays. This assay was validated by spiking replication-incompetent adenoviral vectors with wild-type adenovirus serotype 5 (wt Ad5) as a positive control for RCA. It was found that the replication of wt Ad5 is hampered above a vector particle per cell ratio of 50. However, if microcarrier beads are used, many cells can be grown in a small suspension culture and consequently a large number of vector particles can be tested for contamination with RCA. PMID:17588680

  3. Evaluation of a Soluble Tetrazolium\\/Formazan Assay for Cell Growth and Drug Sensitivity in Culture Using Human and Other Tumor Cell Lines1

    Microsoft Academic Search

    Dominic A. Scudiere; Robert H. Shoemaker; Kenneth D. Paul; Anne Monks; Siobhan Tierney; Thomas H. Nofziger; Michael J. Currens; Donna Seniff; Michael R. Boyd

    We have previously described the application of an automated micro- culture tetrazolium assay (MTA) involvingdimethyl sulfoxide solubiliza- tion of cellular-generated 3-{4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide (MTTHormazan to the in vitro assessment of drug effects on cell growth (M. C. Alley et al., Proc. Am. Assen. Cancer Res., 27:389,1986; M. C. Alley et al.. Cancer Res. 48:589-601,1988). There are several inherent disadvantages of

  4. Microfluidic Cell Culture and Its Application in High Throughput Drug Screening: Cardiotoxicity Assay for hERG Channels

    PubMed Central

    Su, Xiaojing; Young, Edmond W.K.; Underkofler, Heather A. S.; Kamp, Timothy J.; January, Craig T.; Beebe, David J.

    2011-01-01

    Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval, and hence risk for life threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, we demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. We validate our microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP) and poly(dimethylsiloxane) (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG), and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Our results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. We demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. We further showed that experimental procedures can be streamlined by using direct in-channel immunofluorescent staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with five different drugs suggested that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules. PMID:21131594

  5. An improved filter elution and cell culture assay procedure for evaluating public groundwater systems for culturable enteroviruses.

    PubMed

    Dahling, Daniel R

    2002-01-01

    Large-scale virus studies of groundwater systems require practical and sensitive procedures for both sample processing and viral assay. Filter adsorption-elution procedures have traditionally been used to process large-volume water samples for viruses. In this study, five filter elution procedures using cartridge filters were evaluated for their effectiveness in processing samples. Of the five procedures tested, the third method, which incorporated two separate beef extract elutions (one being an overnight filter immersion in beef extract), recovered 95% of seeded poliovirus compared with recoveries of 36 to 70% for the other methods. For viral enumeration, an expanded roller bottle quantal assay was evaluated using seeded poliovirus. This cytopathic-based method was considerably more sensitive than the standard plaque assay method. The roller bottle system was more economical than the plaque assay for the evaluation of comparable samples. Using roller bottles required less time and manipulation than the plaque procedure and greatly facilitated the examination of large numbers of samples. The combination of the improved filter elution procedure and the roller bottle assay for viral analysis makes large-scale virus studies of groundwater systems practical. This procedure was subsequently field tested during a groundwater study in which large-volume samples (exceeding 800 L) were processed through the filters. PMID:12540097

  6. Detection of Infectious Cryptosporidium Oocysts by Cell Culture Immunofluorescence Assay: Applicability to Environmental Samples

    Microsoft Academic Search

    F. M. Schets; G. B. Engels; A. M. de Roda Husman

    2005-01-01

    In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Crypto- sporidium this information is required. For environmental samples the oocyst counts

  7. Selective extraction, concentration, and assay of orthophosphate from microliter quantities of cultured mammalian cells.

    PubMed

    Bevington, A; Angier, C M; Kemp, G J; Russell, R G

    1989-08-15

    A colorimetric procedure is described for determination of orthophosphate (0.2-2.5 nmol) in sample volumes up to 400 microliters. Orthophosphate is selectively extracted (in the form of phosphomolybdate) into an organic solvent mixture (2-methylpropan-1-ol and petroleum spirit) leaving interfering substances, such as labile organic phosphates, in the aqueous phase. Orthophosphate is then back-extracted into a small volume of aqueous sodium hydroxide. By keeping this volume small, orthophosphate from large dilute samples can be concentrated into small volumes and assayed colorimetrically in microcuvettes using the dye malachite green. The procedure is highly reproducible and insensitive to interfering substances, as shown by comparison with a conventional malachite green assay without the solvent extraction. PMID:2817373

  8. Basics of Cell Culture

    NSDL National Science Digital Library

    Afshar, Golnar

    These manuals are used in the Stem Cell Culture Course at City College of San Francisco. This course is about general mammalian cell culture techniques but includes a laboratory exercise using stem cells (takes 3 weeks to complete). The course is taught to high school students but the materials are also used for college students. Laboratory exercises provide instruction in basic techniques of routine cell culture using common cell lines before progressing to differentiation of mouse embryonic stem cells. Photographs and explanations of common equipment (laminar flow hood, inverted microscope, etc.) and reagents are provided. Laboratory exercises include the following: Basic Aseptic Technique; Media Preparation; Plating cells from frozen stock; Cell counting and plating; Survival assay (UV); Live Cell Identification; Transfection; Freezing cells; Stem cell differentiation. A student lab manual and an instructor manual are provided.

  9. Detection of Epstein–Barr virus-specific memory CD4+ T cells using a peptide-based cultured enzyme-linked immunospot assay

    PubMed Central

    Calarota, Sandra A; Chiesa, Antonella; Zelini, Paola; Comolli, Giuditta; Minoli, Lorenzo; Baldanti, Fausto

    2013-01-01

    Approaches to evaluate T-cell responses to Epstein–Barr virus (EBV) include enzyme-linked immunospot (ELISPOT), which quantifies cells capable of immediate interferon-? secretion upon antigen stimulation. However, evaluation of expandable EBV-specific memory T cells in an ELISPOT format has not been described previously. We quantified EBV-specific T-cell precursors with high proliferative capacity by using a peptide-based cultured interferon-? ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15-mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV-specific T-cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared with standard ELISPOT responses. T-cell responses quantified by cultured ELISPOT were mainly mediated by CD4+ T cells and a marked pattern of immunodominance to latent-phase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV-specific T-cell responses were altered in solid organ transplant recipients; in particular, cultured ELISPOT responses were almost undetectable in a lung-transplanted patient with EBV-associated diseases. Analysis of T-cell responses to EBV by ELISPOT assays might provide new insights into the pathogenesis of EBV-related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients. PMID:23560877

  10. Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes

    Microsoft Academic Search

    Shao-Zeng Zhang; Michael M. Lipsky; Benjamin F. Trump; Ih-Chang Hsu

    1990-01-01

    Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus,

  11. An improved infectivity assay combining cell culture with real-time PCR for rapid quantification of human adenoviruses 41 and semi-quantification of human adenovirus in sewage.

    PubMed

    Rodríguez, Roberto A; Polston, Patsy M; Wu, Ming Jing; Wu, Jianyong; Sobsey, Mark D

    2013-06-01

    A protocol for the rapid detection and semi-quantification of human enteric adenovirus based on the quantification of viral mRNA during cell culture infectivity assay was developed. Infectivity assays for adenovirus incorporated cell culture and reverse transcription real-time PCR, where viral mRNA detection was used to monitor the progress of adenovirus infection (CC/mRNA qPCR). The cell line used was G293. This specific infectivity assay was calibrated against different initial concentrations of human adenovirus 41. In addition, the expression of the host's housekeeping (HK) gene, GAPDH, served as internal control for the mRNA assays for quality assurance of the mRNA extraction and reverse transcription steps. The concentrations of infectious human adenoviruses in different sewage samples were estimated semi-quantitatively using the CC/mRNA qPCR assay and calibration obtained for adenovirus 41. A linear relationship between concentrations of viral mRNA (hexon gene) and infectious units was observed between 10(7) to 10(1) infectious units per assay (R(2) = 0.97) in samples analyzed 3-5 days post infection. The expressions of host cell GAPDH gene were not significantly affected by infections with different concentrations of human adenovirus 41, and between virus positive and negative cell cultures (p > 0.1). The estimated concentrations of human adenoviruses in sewage samples ranged between 10(2) to 10(3) mRNA-IU/L. Most of the viruses detected in sewage samples were from human adenovirus species F. The CC/mRNA qPCR assay can be used for quantifying infectious human adenovirus 41, estimating the levels of human adenoviruses in sewage samples, and applied to other sample settings. The CC/mRNA qPCR protocol described here represents an improvement in the detection of human enteric adenoviruses by reducing incubation time (5 days); whereas the conventional cell culture method requires longer incubation periods (10-20 days). More importantly, this protocol can be used to more rapidly and semi-quantitatively estimate the levels of infectious human adenoviruses in environmental samples. PMID:23579085

  12. 21 CFR 866.2350 - Microbiological assay culture medium.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... false Microbiological assay culture medium. 866.2350 Section 866.2350 ...2350 Microbiological assay culture medium. (a) Identification. A microbiological assay culture medium is a device that consists...

  13. Development of a combined in vitro cell culture--quantitative PCR assay for evaluating the disinfection performance of pulsed light for treating the waterborne enteroparasite Giardia lamblia.

    PubMed

    Garvey, Mary; Stocca, Alessia; Rowan, Neil

    2014-09-01

    Giardia lamblia is a flagellated protozoan parasite that is recognised as a frequent cause of water-borne disease in humans and animals. We report for the first time on the use of a combined in vitro HCT-8 cell culture-quantitative PCR assay for evaluating the efficacy of using pulsed UV light for treating G. lamblia parasites. Findings showed that current methods that are limited to using vital stains before and after cyst excystation are not appropriate for monitoring or evaluating cyst destruction post PUV-treatments. Use of the human ileocecal HCT-8 cell line was superior to that of the human colon Caco-2 cell line for in vitro culture and determining PUV sensitivity of treated cysts. G. lamblia cysts were also shown to be more resistant to PUV irradiation compared to treating similar numbers of Cryptosporidium parvum oocysts. These observations also show that the use of this HCT-8 cell culture assay may replace use of animal models for determining disinfection performances of PUV for treating both C. parvum and G. lamblia. PMID:24929148

  14. Human monocyte-derived dendritic cells from leukoreduction system chambers after plateletpheresis are functional in an in vitro co-culture assay with intestinal epithelial cells.

    PubMed

    Tiscornia, Inés; Sánchez-Martins, Viviana; Hernández, Ana; Bollati-Fogolín, Mariela

    2012-10-31

    The dendritic cells (DC) found in the intestine are involved both in the maintenance of tolerance towards commensal microbiota, and in the generation of protective immune responses against pathogens, thus contributing to gut immune homeostasis. There is an increasing interest in the use of lactic acid bacteria (LAB) as probiotics; among their beneficial effects we highlight the modulation of the immune system which is one of their fundamental properties. As these effects are strain-dependent, it is important to have in vitro systems that include DC and intestinal epithelial cells (IEC), which are crucial for intestinal homeostasis, to identify candidates by means of bacterial screening. Obtaining enough human cells, necessary to simultaneously test several bacteria, is a major challenge for researchers. In this study we analyzed the usefulness of the cellular fraction retained in leukoreduction system chambers following plateletpheresis (PP) as a source of DC. We compared the capacity of peripheral blood mononuclear cells (PBMC) from buffy coats (BC) or PP to generate DC using a short differentiation protocol. The functionality of the DC obtained was analyzed in co-cultures together with intestinal epithelial HT-29 cells, stimulating with LPS alone or with two LAB commonly used in the food industry, Streptococcus thermophilus and Lactobacillus delbrueckii. DC surface markers CD86, HLA-DR and cytokine production were measured. The behavior of DC derived from PP was similar to the behavior observed for DC derived from BC. When we tested the response of DC to bacteria, we found significant differences in cytokine secretion, especially for IL-10, suggesting that the system has the ability to discriminate LAB with different immunomodulatory properties. We also found that DC derived from both sources displayed a similar ability to phagocyte bacteria. In conclusion, we hereby propose a modification of the two-day protocol for obtaining human DC previously described, using PP as an alternative source of PBMC, to be used in co-culture systems with IEC. The novelty of this protocol is the combination of the blood monocyte source with a simple and fast differentiation method to obtain DC, and their use in a combined culture with IEC and LAB to model microbial-host interaction. Since the initial PP volume is ten times lower than that of BC, the use of PP minimizes biological residue generation and reagent consumption. In addition, monocyte-derived DC from PP were suitable for use in co-culture assays as a first screening step to study the immunomodulatory properties of LAB. PMID:22841576

  15. High density micromass cultures of a human chondrocyte cell line: A reliable assay system to reveal the modulatory functions of pharmacological agents

    PubMed Central

    Greco, K.V.; Iqbal, A.J.; Rattazzi, L.; Nalesso, G.; Moradi-Bidhendi, N.; Moore, A.R.; Goldring, M.B.; Dell’Accio, F.; Perretti, M.

    2014-01-01

    Osteoarthritis is a highly prevalent and disabling disease for which we do not have a cure. The identification of suitable molecular targets is hindered by the lack of standardized, reproducible and convenient screening assays. Following extensive comparisons of a number of chondrocytic cell lines, culture conditions, and readouts, we have optimized an assay utilizing C-28/I2, a chondrocytic cell line cultured in high-density micromasses. Utilizing molecules with known effects on cartilage (e.g. IL-1?, TGF?1, BMP-2), we have exploited this improved protocol to (i) evoke responses characteristic of primary chondrocytes; (ii) assess the pharmacodynamics of gene over-expression using non-viral expression vectors; (iii) establish the response profiles of known pharmacological treatments; and (iv) investigate their mechanisms of action. These data indicate that we have established a medium-throughput methodology for studying chondrocyte-specific cellular and molecular responses (from gene expression to rapid quantitative measurement of sulfated glycosaminoglycans by Alcian blue staining) that may enable the discovery of novel therapeutics for pharmacological modulation of chondrocyte activation in osteoarthritis. PMID:21946086

  16. Identification of Candidate Agents Active against N. ceranae Infection in Honey Bees: Establishment of a Medium Throughput Screening Assay Based on N. ceranae Infected Cultured Cells

    PubMed Central

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

    Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae. PMID:25658121

  17. Identification of Candidate Agents Active against N. ceranae Infection in Honey Bees: Establishment of a Medium Throughput Screening Assay Based on N. ceranae Infected Cultured Cells.

    PubMed

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

    Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae. PMID:25658121

  18. The importance of the microenvironment in breast cancer progression: recapitulation of mammary tumorigenesis using a unique human mammary epithelial cell model and a three-dimensional culture assay

    PubMed Central

    Weaver, V.M.; Fischer, A.H.; Peterson, O.W.; Bissell, M.J.

    2010-01-01

    The extracellular matrix (ECM) is a dominant regulator of tissue development and homeostasis. “Designer microenvironments” in culture and in vivo model systems have shown that the ECM regulates growth, differentiation, and apoptosis in murine and human mammary epithelial cells (MEC) through a hierarchy of transcriptional events involving the intricate interplay between soluble and physical signaling pathways. Furthermore, these studies have shown that these pathways direct and in turn are influenced by the tissue structure. Tissue structure is directed by the cooperative interactions of the cell–cell and cell–ECM pathways and can be modified by stromal factors. Not surprisingly then, loss of tissue structure and alterations in ECM components are associated with the appearance and dissemination of breast tumors, and malignancy is associated with perturbations in cell adhesion, changes in adhesion molecules, and a stromal reaction. Several lines of evidence now support the contention that the pathogenesis of breast cancer is determined (at least in part) by the dynamic interplay between the ductal epithelial cells, the microenvironment, and the tissue structure (acini). Thus, to understand the mechanisms involved in carcinogenesis, the role of the microenvironment (ECM as well as the stromal cells) with respect to tissue structure should be considered and studied. Towards this goal, we have established a unique human MEC model of tumorigenesis, which in concert with a three-dimensional assay, recapitulates many of the genetic and morphological changes observed in breast cancer in vivo. We are currently using this system to understand the role of the microenvironment and tissue structure in breast cancer progression. PMID:9164652

  19. Mammalian Cell Culture

    Microsoft Academic Search

    Simon P. Langdon

    Mammalian cell culture is used widely in academic, medical and industrial settings. It has provided a means to study the physiology and biochemistry of the cell and developments in the fields of cell and molecular biology have required the use of reproducible model systems that only cultured cell lines can provide. For medical use, cell culture provides test systems to

  20. New Artificial Electron Donors for in Vitro Assay of Nitrate Reductase Isolated from Cultured Tobacco Cells and Other Organisms

    PubMed Central

    Hoarau, Jackson; Hirel, Bertrand; Nato, Aimé

    1986-01-01

    The capacity of bromphenol blue and its analogs to act as electron donors for measurement of in vitro nitrate reductase activity from tobacco cells (Nicotiana tabacum var Techné SP 25 strain) was determined. Competitive inhibition was demonstrated to occur between NADH, the natural electron donor, and bromphenol blue, the artificial electron donor, suggesting that both donors bind to a similar active site on the enzyme. NADH-dependent or bromphenol blue-dependent nitrate reductase activity was carried out by a similar molecular weight protein exhibiting similar antigenic sites. Following ammonium sulfate precipitation, sucrose density gradient and two chromatographic steps, nitrate reductase activity from tobacco cells was purified near homogeneity using bromphenol blue as an electron donor in the absence of measurable NADH-dependent activity. The enzyme is composed of two identical subunits of 83 kilodaltons < M?? < 94 kilodaltons. Images Fig. 3 PMID:16664746

  1. Mutation assays involving blood cells that metabolize toxic substances

    DOEpatents

    Crespi, Charles L. (Downers Grove, IL); Thilly, William G. (Winchester, MA)

    1985-01-01

    A line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity) is disclosed. Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. Mutation assays using these cells, and other cells with similar characteristics, are also disclosed.

  2. Circulating Tumor Cell Assay Frequently Asked Questions

    Cancer.gov

    Version: July 2010 Circulating Tumor Cell Assay Frequently Asked Questions LHTP003.8.1 ?H2AX IMMUNOFLUORESCENCE ASSAY FOR CIRCULATING TUMOR CELLS USING THE CELLSEARCH SYSTEM 1. Do I need any prior experience before applying for the training

  3. Stem cell culture engineering

    Microsoft Academic Search

    Gargi Seth; Catherine M. Verfaillie

    2005-01-01

    Stem cells have the capacity for self renewal and undergo multilineage differentiation. Stem cells isolated from both blastocysts and adult tissues represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self?renewal and differentiation capacity. In generating particular cell types for specific applications,

  4. Optimizing stem cell culture

    PubMed Central

    Van Der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-01-01

    Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh’s plane. PMID:20803548

  5. Fifty Percent Tissue Culture Infective Dose Assay for Determining the Titer of Infectious Human Herpesvirus 8

    PubMed Central

    Nadgir, Sagar V.; Hensler, Heather R.; Knowlton, Emilee R.; Rinaldo, Charles R.; Rappocciolo, Giovanna

    2013-01-01

    We have developed a human herpesvirus 8 (HHV-8) 50% tissue culture infective dose (TCID50) assay using the T1H6-DC-SIGN cell line. Infection of T1H6-DC-SIGN cells with HHV-8 induces expression of ?-galactosidase, which was used to determine TCID50 levels. Validation of TCID50 values was performed by immunofluorescence assay of HHV-8 infection of immature dendritic cells at various TCID50 doses. PMID:23554189

  6. In vitro assay of cytotoxicity with cultured liver: accomplishments and possibilities.

    PubMed Central

    Grisham, J W; Charlton, R K; Kaufman, D G

    1978-01-01

    Tissue cultures offer potential advantages for assaying the toxicity of chemicals and for evaluating tissue susceptibility to toxic agents. Several properties of cultured cells hinder the immediate, widespread use of tissue cultures to assay toxicity routinely. These points are illustrated by briefly reviewing attempts to utilize different types of hepatic cultures to evaluate the actions of carcinogenic chemicals in vitro. Hepatocytes in vivo apparently can metabolize all known procarcinogenic chemicals, but the process of tissue isolation and the environmental conditions in vitro may modify drastically the responses of hepatocytes and other cultured hepatic cells to toxic chemicals. Before cell cultures can be used routinely as the basis of screening systems to detect chemical toxins, specificity and sensitivity of response to chemicals representing all chemical classes must be validated by laboratory studies. PMID:363407

  7. Culture amplification in human colon adenocarcinoma cell line (CaCo-2) combined with an ELISA as a supplementary assay for accurate diagnosis of rotavirus

    Microsoft Academic Search

    Andrea C. Cumino; Miguel O. Giordano; Laura C. Mart??nez; Silvia I. Medeot; Jorge V. Pavan; Silvia Yudowsky; Mar??a Beatriz Isa; Ariel R. Depetris; Silvia V. Nates

    1998-01-01

    Culture amplification in colon adenocarcinoma cell line (CaCo-2) combined with enzyme immunoassay (Pathfinder ELISA) was developed as a supplementary tool for rotavirus diagnosis. One hundred and thirty stools in which results by polyacrylamide gel electrophoresis (PAGE) were in agreement with those obtained by ELISA were amplified in the CaCo-2 cell line. After the first passage 100% specimens were revealed as

  8. Shortening the culture time in cytogenetic dosimetry using PCC-R assay.

    PubMed

    Romero, Ivonne; Lamadrid, Ana Ilsa; González, Jorge Ernesto; García, Omar; Voisin, Philippe; Roy, Laurence

    2015-03-01

    The fast assessment of the dose received by exposed persons is crucial in radiological accidents, so the 48 h of cell culture in conventional cytogenetic dosimetry in addition to some limitations after high doses becomes a disadvantage. The premature chromosome condensation (PCC) assay permits to analyse enough cells after high radiation exposure, and the score of PCC-R may reduce the culture time up to 40-42 h. Peripheral whole-blood samples were exposed to 1-10 Gy of gamma radiation and cultured during 40 and 42 h. No statistical difference between frequencies was obtained between 40, 42 and 48 h of culture time, and PCC index decreased with the increase of the dose and increased with the culture time. The protocol proposed allows reduce the culture time down to 40 or 42 h when using the PCC-R assay with adequate precision in dose estimation. PMID:25114320

  9. Mutation assays involving blood cells that metabolize toxic substances

    DOEpatents

    Crespi, C.L.; Thilly, W.G.

    1999-08-10

    The present invention pertains to a line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity). Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. The invention also includes mutation assays using these cells, and other cells with similar characteristics. 3 figs.

  10. Mutation assays involving blood cells that metabolize toxic substances

    DOEpatents

    Crespi, Charles L. (Marblehead, MA); Thilly, William G. (Winchester, MA)

    1999-01-01

    The present invention pertains to a line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity). Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. The invention also includes mutation assays using these cells, and other cells with similar characteristics.

  11. Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels.

    PubMed

    Dietrich, Franziska; Lelkes, Peter I

    2006-01-01

    A prerequisite for successful tissue engineering is the existence of a functional microvascular network. We hypothesized that such networks can be created and quantified in an in vitro setting by co-culturing endothelial cells (ECs) with tissue-specific 'bystander cells' in 3-D gel matrices. To test this hypothesis we adapted a previously described in vitro microcarrier-based angiogenesis assay (V. Nehls and D. Drenckhahn, 1995, Microvasc Res 50: 311-322). On optimizing this assay, we noted that the initial EC-microcarrier coverage depended on EC type and seeding technique employed to coat the microcarrier beads with the ECs. A confluent EC monolayer on the microcarrier surfaces formed only when bovine aortic endothelial cells (BAECs) were admixed to the beads under gentle agitation on an orbital shaker. After embedding BAEC-covered microcarrier beads into a sandwich-like arrangement of collagen or fibrin gels, we assessed cellular outgrowth at different serum concentrations in terms of migration distance and sprout formation. Quantifiable sprout formation was highest at 1% fetal bovine serum (FBS) in collagen matrices and at 0.1% FBS in fibrin matrices. At higher serum concentration, excess cell migration and formation of clusters prevented quantitative analysis of sprouting. Following the fine-tuning of this angiogenesis assay, we co-cultured BAECs with adipose tissue-derived fibroblasts (FBs) and vascular smooth muscle cells (SMCs). While FBs were able to increase the average migration distance of BAECs in both matrices, SMCs enhanced BAEC migration in fibrin, but not in collagen gels. By contrast, the number of newly formed sprouts in fibrin gels was increased by both cell types. We conclude that in this model bystander cells enhance EC network formation in a matrix-dependent manner. Additionally, these results stress the importance of carefully selecting the experimental parameters of a given in vitro angiogenesis model. PMID:17051343

  12. Comparison of a New Gold Immunochromatographic Assay for the Rapid Diagnosis of the Novel Influenza A (H7N9) Virus with Cell Culture and a Real-Time Reverse-Transcription PCR Assay

    PubMed Central

    Wu, Nanping; Peng, Xiaorong; Yao, Hangping; Lu, Xiangyun; Chen, Yu; Wu, Haibo; Xie, Tiansheng; Cheng, Linfang; Liu, Fumin; Kang, Keren; Tang, Shixing; Li, Lanjuan

    2014-01-01

    We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings. PMID:24822207

  13. Automatic cell tracking applied to analysis of cell migration in wound healing assay.

    PubMed

    Bise, Ryoma; Kanade, Takeo; Yin, Zhaozheng; Huh, Seung-il

    2011-01-01

    The wound healing assay in vitro is widely used for research and discovery in biology and medicine. This assay allows for observing the healing process in vitro in which the cells on the edges of the artificial wound migrate toward the wound area. The influence of different culture conditions can be measured by observing the change in the size of the wound area. For further investigation, more detailed measurements of the cell behaviors are required. In this paper, we present an application of automatic cell tracking in phase-contrast microscopy images to wound healing assay. The cell behaviors under three different culture conditions have been analyzed. Our cell tracking system can track individual cells during the healing process and provide detailed spatio-temporal measurements of cell behaviors. The application demonstrates the effectiveness of automatic cell tracking for quantitative and detailed analysis of the cell behaviors in wound healing assay in vitro. PMID:22255749

  14. Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. The RV-43 Study Group, the AIDS Clinical Trials Group Virology Committee Resistance Working Group.

    PubMed Central

    Japour, A J; Mayers, D L; Johnson, V A; Kuritzkes, D R; Beckett, L A; Arduino, J M; Lane, J; Black, R J; Reichelderfer, P S; D'Aquila, R T

    1993-01-01

    A standardized antiviral drug susceptibility assay for clinical human immunodeficiency virus type 1 (HIV-1) isolates has been developed for use in clinical trials. The protocol is a two-step procedure that first involves cocultivation of patient infected peripheral blood mononuclear cells (PBMC) with seronegative phytohemagglutinin-stimulated donor PBMC to obtain an HIV-1 stock. The virus stock is titrated for viral infectivity (50% tissue culture infective dose) by use of serial fourfold virus dilutions in donor PBMC. A standardized inoculum of 1,000 50% tissue culture infective doses per 10(6) cells is used in the second step of the procedure to acutely infect seronegative donor PBMC in a 7-day microtiter plate assay with triplicate wells containing zidovudine (ZDV) concentrations ranging from 0 to 5.0 microM. The ZDV 50% inhibitory concentrations (IC50) for reference ZDV-susceptible and ZDV-resistant HIV-1 isolates ranged from 0.002 to 0.113 microM and from 0.15 to > 5.0 microM, respectively. Use of this consensus protocol reduced interlaboratory variability for ZDV IC50 determinations with reference HIV-1 isolates. Among eight laboratories, the coefficient of variation ranged from 0.85 to 1.25 with different PBMC protocols and was reduced to 0.39 to 0.98 with the standardized assay. Among the clinical HIV-1 isolates assayed by the standardized drug susceptibility assay, the median ZDV IC50 increased gradually with more ZDV therapy. This protocol provides an efficient and reproducible means to assess the in vitro susceptibility to antiretroviral agents of virtually all clinical HIV-1 isolates. PMID:8517697

  15. Mammalian Cell Culture

    NSDL National Science Digital Library

    This "Course-in-a-Box" from Bio-Link is a good starting point for instructors to develop a course on how to maintain mammalian cells in culture. Students will learn "basic techniques of routine cell culture using common cell lines before progressing to differentiation of mouse embryonic stem cells." Laboratories include Basic Aseptic Technique, Media Preparation, and Plating Cells from Frozen Stock. Materials include an Instructor Laboratory Manual, Student Laboratory Manual, Problem Sets, and Quizzes. A free login is required to access the materials.

  16. Mammalian Cell Culture Simplified.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  17. Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

    PubMed Central

    Azari, Hassan; Louis, Sharon A.; Sharififar, Sharareh; Vedam-Mai, Vinata; Reynolds, Brent A.

    2011-01-01

    The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies ?2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies < 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are < 2mm and the ones that are ?2mm in reference to the number of cells that were initially plated. PMID:21403640

  18. Quantification of an in vitro cell-cell adhesion assay using interactive laser scanning cytometry.

    PubMed

    Newton, S C; Millette, C F

    1992-01-01

    We are interested in identifying cell-cell adhesion molecules on the surface of Sertoli cells that mediate Sertoli cell-spermatogenic cell adhesion. Numerous cell-cell adhesion assays employ microscopic observation, photomicroscopy or radioactive isotopes for quantification. Previously, we developed an in vitro assay for testicular cell interactions. This assay was, however, time consuming using photography for analysis. We have now modified this system using laser cytometry to quantify adherent cells. Rat testicular epithelial cells are cultured for approximately 6 days before labelling with fluorescein diactetate (FDA) to assess confluency by image scanning so that spermatogenic cell binding can be normalized to available epithelial cell surface area. Rat spermatogenic cells are labeled with FDA before addition to epithelial cell monolayers. In some studies, purified spermatogenic cell populations were isolated to determine average cell size. We found that spermatocyte area varied between 225-500 microns2, spermatids were 100-225 microns2 and residual bodies were less than 100 microns2. Using these parameters, scanning cytometry allows the differential analysis of adhesion by individual germ cell sub-classes from mixed cell suspensions, saving time, animals, and major expense. The scanning laser assisted assay is faster, more reproducible and less subjective than earlier cell-cell adhesion assays using light microscopy or isotopes. This experimental approach should facilitate any cell-cell adhesion assay in which one cell type is adherent to a substrate. PMID:1576888

  19. Quantum dot-based cell motility assay

    Microsoft Academic Search

    Teresa Pellegrino; Wolfgang J. Parak; Rosanne Boudreau; Mark A. Le gros; Daniele Gerion; A. Paul Alivisatos; Carolyn A. Larabell

    2003-01-01

    Motility and migration are measurable characteristics of cells that are classically associated with the invasive potential of cancer cells, but in vitro assays of invasiveness have been less than perfect. We previously developed an assay to monitor cell motility and migration using water-soluble CdSe\\/ZnS nanocrystals; cells engulf the fluorescent nanocrystals as they crawl across them and leave behind a fluorescent-free

  20. Optimization of NRU assay in primary cultures of Eisenia fetida for metal toxicity assessment.

    PubMed

    Irizar, Amaia; Duarte, Daniel; Guilhermino, Lucia; Marigómez, Ionan; Soto, Manu

    2014-09-01

    Coelomocytes, immunocompetent cells of lumbricids, have received special attention for ecotoxicological studies due to their sensibility to pollutants. Their in vitro responses are commonly quantified after in vivo exposure to real or spiked soils. Alternatively, quantifications of in vitro responses after in vitro exposure are being studied. Within this framework, the present study aimed at optimizing the neutral red uptake (NRU) assay in primary culture of Eisenia fetida coelomocytes for its application in soil toxicity testing. Optimized assay conditions were: earthworm depuration for 24 h before retrieving coelomocytes by electric extrusion; 2 × 10(5) seeded cells/well (200 µl) for the NRU assay and incubation for 1 h with neutral red dye. Supplementation of the culture medium with serum was not compatible with the NRU assay, but coelomocytes could be maintained with high viability for 3 days in a serum-free medium without replenishment. Thus, primary cultures were used for 24 h in vitro toxicity testing after exposure to different concentrations of Cd, Cu, Ni and Pb (ranging from 0.1 to 100 ?g/ml). Primary cultures were sensitive to metals, the viability declining in a dose-dependent manner. The toxicity rank was, from high to low, Pb > Ni > Cd > Cu. Therefore, it can be concluded that the NRU assay in coelomocytes in primary cultures provides a sensitive and prompt response after in vitro exposure to metals. PMID:25011921

  1. Microassay of decarboxylation reactions in cultured cells.

    PubMed

    Bartos, D; Vlessis, A A; Muller, P; Mela-Riker, L; Trunkey, D D

    1993-09-01

    The currently described methods for determination of decarboxylation reaction rates in cultured cells require large quantities of cells and often involve cell manipulation prior to assay. We describe a simple microassay for the rapid measurement of various decarboxylation reaction rates in intact cultured cells. The assay is based on the traditional measurement of 14CO2 generated from 14C-labeled substrates. Key to the method is a novel modification of the standard petri dish. Pyruvate dehydrogenase, branched chain alpha-ketoacid dehydrogenase, alpha-ketoglutarate dehydrogenase, and ornithine decarboxylase activities were determined in adult cardiomyocyte cultures containing only 0.1-0.5 mg of protein per culture dish. Efficiency of 14CO2 collection ranged between 94 and 100%. Pharmacological enhancement or inhibition of pyruvate dehydrogenase activity was easily detected in the culture system. This new method simplifies the measurement of various decarboxylation reaction rates in cultured cells and allows rapid, reproducible measurements to be made on small numbers of cells without perturbation of the culture conditions or the cells themselves. PMID:8238897

  2. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  3. Gaussia luciferase reporter assay for monitoring of biological processes in culture and in vivo

    PubMed Central

    Tannous, Bakhos A.

    2009-01-01

    Secreted reporters are a useful tool in the monitoring of different biological processes in the conditioned medium of cultured cells as well in the blood and urine of experimental animals. Described here is a protocol for detecting the recently established naturally secreted Gaussia luciferase (Gluc) in cultured cells as well as in blood and urine in vivo. Further, the assay for detecting the secreted alkaline phosphatase (SEAP), the most commonly used secreted reporter in serum is also presented. The Gluc reporter system has several advantages over the SEAP assay including: a much reduced assay time (1-10 min vs. 1.5 - 2 h); 20,000-fold (in vitro) or 1000-fold (in vivo) increased sensitivity and a linear range covering over 5 orders of magnitude of cell number. Additionally, the Gluc signal can be detected in urine and the signal can be localized in animals using in vivo bioluminescence imaging. PMID:19373229

  4. A Biochemical Assay for Acetylcholinesterase Activity in PC12 Cells

    NSDL National Science Digital Library

    Paul J. Schwartz (University of Medicine and Dentistry of New Jersey; Department of Neurological Surgery REV)

    2007-07-10

    This lab describes two biochemical assays: One for measuring acetylcholinesterase activity and one for measuring protein concentration. Students learn how to manipulate small-volume samples, use a standard spectrophotometer or a microplate reader spectrophotometer, construct a standard curve, and normalize data. The lab is intended to be used in conjunction with a cell culture lab in which PC12 cells are exposed to various agents that influence their phenotypic state.

  5. Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays

    Microsoft Academic Search

    Traver Hart; Alice Zhao; Ankit Garg; Swetha Bolusani; Edward M. Marcotte; Joanna Mary Bridger

    2009-01-01

    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other

  6. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  7. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc. has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc. is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  8. Oscillating Cell Culture Bioreactor

    NASA Technical Reports Server (NTRS)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid dynamic shear (i.e., as required for viability of shear-sensitive cells) to the developing engineered tissue construct. This bioreactor was recently utilized to show independent and interactive effects of a growth factor (IGF-I) and slow bidirectional perfusion on the survival, differentiation, and contractile performance of 3D tissue engineering cardiac constructs. The main application of this system is within the tissue engineering industry. The ideal final application is within the automated mass production of tissue- engineered constructs. Target industries could be both life sciences companies as well as bioreactor device producing companies.

  9. Notch-ligand binding assays in Drosophila cells.

    PubMed

    Xu, Aiguo; Irvine, Kenneth D

    2014-01-01

    Activation of the Drosophila transmembrane receptor protein Notch is induced by association with its transmembrane ligands, Delta and Serrate. The ability to assay binding between Notch and its ligands has been essential for characterizing the influence of posttranslational modifications, such as glycosylation, as well as for characterizing structural motifs involved in receptor-ligand interactions. We describe here a simple, widely used method for assaying receptor-ligand binding. This method involves expression of soluble forms of either Notch or its ligands, comprising the extracellular domains fused to an easily assayed tag, the enzyme alkaline phosphatase. These soluble proteins are then incubated with their binding partners, either as transmembrane proteins expressed on the surface of cultured cells or as extracellular protein domains attached to agarose beads. After washing, the amount of bound protein can be readily assayed by measuring alkaline phosphatase activity. PMID:25053497

  10. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  11. Quantum Dot-Based Cell Motility Assay

    SciTech Connect

    Gu, Weiwei; Pellegrino, Teresa; Parak Wolfgang J; Boudreau,Rosanne; Le Gros, Mark A.; Gerion, Daniele; Alivisatos, A. Paul; Larabell, Carolyn A.

    2005-06-06

    Because of their favorable physical and photochemical properties, colloidal CdSe/ZnS-semiconductor nanocrystals (commonly known as quantum dots) have enormous potential for use in biological imaging. In this report, we present an assay that uses quantum dots as markers to quantify cell motility. Cells that are seeded onto a homogeneous layer of quantum dots engulf and absorb the nanocrystals and, as a consequence, leave behind a fluorescence-free trail. By subsequently determining the ratio of cell area to fluorescence-free track area, we show that it is possible to differentiate between invasive and noninvasive cancer cells. Because this assay uses simple fluorescence detection, requires no significant data processing, and can be used in live-cell studies, it has the potential to be a powerful new tool for discriminating between invasive and noninvasive cancer cell lines or for studying cell signaling events involved in migration.

  12. Microfluidic Cell Culture Device

    NASA Technical Reports Server (NTRS)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  13. Enzyme-Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures, Extracts, and Plant Tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercially available enzyme linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross-reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L-2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of...

  14. Application of long-term cultured interferon-gamma enzyme-linked immunospot assay for assessing effector and memory T cell responses in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Effector and memory T cells are generated through developmental programing of naïve cells following antigen recognition. If the infection is controlled, up to 95% of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a l...

  15. Enzyme?Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures, Extracts, and Plant Tissues

    Microsoft Academic Search

    Robert E. Hoagland; Mark A. Weaver; C. Douglas Boyette

    2008-01-01

    A rapid technique for trichothecene detection was needed in screening tests of the potential bioherbicide Myrothecium verrucaria (MV), in order to select strains, mutants, or formulations that were void of or that possessed low amounts of these undesirable mycotoxin compounds. Commercially available enzyme?linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross?reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and

  16. Cell culture's spider silk road.

    PubMed

    Perkel, Jeffrey

    2014-06-01

    A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk. PMID:24924388

  17. Human Primary Lung Endothelial Cells in Culture

    PubMed Central

    Xu, Weiling; Mavrakis, Lori; Aldred, Micheala A.; Asosingh, Kewal; Erzurum, Serpil C.

    2012-01-01

    Pulmonary endothelial functions are critical to maintain the low pressure of the pulmonary circulation and effective diffusion capacity of the lung. To investigate pulmonary endothelial cell biology in healthy or diseased lungs, we developed methods to harvest and culture pure populations of primary pulmonary arterial endothelial cells and microvascular endothelial cells from human lung explanted at time of transplantation or from donor lungs not used in transplantation. The purity and characteristics of cultured endothelial cells is ascertained by morphologic criteria using phase contrast and electron microscopy; phenotypic expression profile for endothelial specific proteins such as endothelial nitric oxide synthase, platelet/endothelial cell adhesion molecule, and von Willbrand factor; and endothelial function assays such as Dil-acetylated low-density lipoprotein uptake and tube formation. This detailed method provides researchers with the ability to establish cells for molecular, genetic, and biochemical investigation of human pulmonary vascular diseases. PMID:22427538

  18. Cell culture purity issues and DFAT cells

    SciTech Connect

    Wei, Shengjuan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China) [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States); Bergen, Werner G. [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States)] [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States); Hausman, Gary J. [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States)] [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States); Zan, Linsen, E-mail: zanls@yahoo.com.cn [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China)] [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Dodson, Michael V., E-mail: dodson@wsu.edu [Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States)

    2013-04-12

    Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  19. DETECTION AND TITRATION OF BLUETONGUE VIRUS IN CULICOIDES INSECT CELL CULTURE BY AN ANTIGEN-CAPTURE ENZYME-LINKED IMMUNOSORBENT ASSAY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. Virus is typically isolated and characterized by infection of susceptible vertebrate cells that undergo detectable and measurable cytopathic effects. Cell lines derived from C. ...

  20. Haute Culture: Tailoring stem cells

    E-print Network

    Chou, James

    research projects and its faculty have founded five stem cell-related startup companies and serveHaute Culture: Tailoring stem cells to make us well Tuesday, April 24, 2012 6:00-7:30 p;Haute Culture: Tailoring stem cells to make us well Moderator Brock Reeve, MPhil, MBA Executive Director

  1. 3D culture assays of murine mammary branching morphogenesis and epithelial invasion.

    PubMed

    Nguyen-Ngoc, Kim-Vy; Shamir, Eliah R; Huebner, Robert J; Beck, Jennifer N; Cheung, Kevin J; Ewald, Andrew J

    2015-01-01

    Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called "organoids," to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems. PMID:25245692

  2. KERATINOCYTE CELL-MEDIATED MUTAGENESIS ASSAY: CORRELATION WITH IN VIVO TUMOR STUDIES

    EPA Science Inventory

    A murine keratinocyte cell-mediated mutagenesis assay was characterized and examined as an in vitro model system for studying the biotransformation of promutagens/procarcinogens by mouse skin. The assay used living cultured newborn SENCAR keratinocytes for the metabolic activatio...

  3. Development of a high-throughput antiviral assay for screening inhibitors of chikungunya virus and generation of drug-resistant mutations in cultured cells.

    PubMed

    Gong, Edwin Yunhao; Bonfanti, Jean-François; Ivens, Tania; Van der Auwera, Marijke; Van Kerckhove, Barbara; Kraus, Guenter

    2013-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that has already infected millions of people in recent large-scale epidemics in Africa, the islands of the Indian Ocean, South and Southeast Asia, and northern Italy. The infection is still ongoing in many countries, such as India. Although the fatal rate is approximately 0.1% in the La Réunion outbreak, it causes painful arthritis-like symptoms that can last for months or even years. Currently, neither vaccine nor approved antiviral therapy exists to protect humans from chikungunya infection. Therefore, there is an urgent unmet medical need for the development of antiviral drugs for pre-exposure prophylaxis and/or treatment of chikungunya infections. In this chapter, we describe a fully validated ATP/luminescence assay that is effective for high-throughput screening of CHIKV inhibitors. Protocols for growing CHIKV stocks and generating drug-resistant viral variants for modes of action studies of compounds are also described. PMID:23821286

  4. CONTINUOUS-FLOW ENZYME ASSAY ON A MICROFLUIDIC CHIP FOR MONITORING GLYCEROL SECRETION FROM CULTURED ADIPOCYTES

    PubMed Central

    Clark, Anna M.; Sousa, Kyle M.; Jennings, Colin; MacDougald, Ormond A.; Kennedy, Robert T.

    2009-01-01

    A dual-chip microfluidic platform that coupled perfusion of cultured adipocytes with on-line fluorescence-based enzyme assay was developed to monitor glycerol secretions in real-time from cultured adipocytes. The perfusion cell chip, which could be reversibly sealed to allow reloading of cells and reuse of the chip, was shown by modeling to generate low shear stress on the cells under study. Effluent from the perfusion chip was pumped into an enzyme assay chip for monitoring of secretion from the cells. The on-line enzyme assay had a LOD of 4 ?M glycerol. The temporal resolution of the combined system for detecting changes in glycerol concentration was 90 s. The microfluidic device was used to continuously monitor glycerol secretion from murine 3T3-L1 adipocytes, grown and differentiated on glass cover slips, for at least 2 h. An average basal glycerol concentration of 28 ?M was detected in the effluent. Pharmacological treatment with a ?-adrenergic agonist to stimulate lipolysis evoked a 3-fold increase in glycerol secretion followed by sustained release that was 40% higher than basal concentration. The ability to monitor changes in cellular secretion over time may provide insight into adipocyte metabolism and the dysregulation that occurs with obesity-related disorders. PMID:19231843

  5. Fermentation with immobilized cell cultures.

    PubMed

    Werner, R G; Merk, W; Walz, F

    1988-02-01

    For the production of monoclonal antibodies and complex recombinant human proteins or glycoproteins a number of immobilized cell culture systems have been developed. The advantages of such cell culture systems are that cells can be kept in small volumes of cell culture fluid and media can be changed continuously if necessary for induction of product synthesis or removal and harvest of metabolic products. Whereas the hollow fiber and the opticell culture systems can be limited in scaling up the microcarrier system, the fluidized bed bioreactor and the solid bed bioreactor are suitable for scaling up. In contrast to the other systems, the solid bed bioreactor requires no special manipulation for anchoring the cells to the wire springs. In situ cleaning is possible and the beads are reusable. With this cell culture fermentation system, production processes for interferon beta, monoclonal antibodies for interferon alfa and recombinant human tissue plasminogen activator were developed. PMID:3285839

  6. A new gaseous imaging detector for the assay of lymphocyte cultures

    NASA Astrophysics Data System (ADS)

    Bateman, J. E.; Joyce, A.; Knight, S. C.; Bedford, P.

    1991-12-01

    Tritium-labelled cell cultures used in studies of lymphocyte proliferation at the Clinical Research Centre are blotted in arrays of 10 × 6 spots spaced at 6 mm. An imaging detector based on the differential induction signals produced at a central amplifying electrode has been developed for the imaging and assay of these blots. A spatial resolution ˜2.5 mm FWHM attained over the aperture of 60 mm × 36 mm enables the individual spots to be reliably counted. Data is captured in a PC/AT at rates which permit an assay to be completed in typically 30-60 min. The simplicity of both the detector and the readout electronics leads to a low cost system. Images and assay results are presented.

  7. Detection of soluble T cell receptor-releasing cells by ELISPOT assay.

    PubMed

    Ishizaka, S; Kimoto, M; Nishiyama, T; Araki, T

    1995-02-01

    A specific and sensitive enzyme-linked immunospot (ELISPOT) assay has been developed for the detection and enumeration of soluble T cell receptor (TCR)-releasing cells. Using this method, we readily detected at the single cell level the release of soluble TCR by living T lymphoma cells (MT-2 and HSB-2) but not by human B lymphoma cells (DAKIKI), mouse hepatoma cells (MH134) and dead MT-2. Furthermore, distinct spots in MT-2 cell culture were not visualized using several monoclonal antibodies against antigens unrelated to TCRs as a primary antibody. The specific and quantitative detection of soluble TCR-releasing cells using ELISPOT assay will certainly provide a valuable tool to better characterize soluble TCRs and their relationship to immune regulation and a number of diseases. PMID:7775664

  8. Huanglongbing and psyllid cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We successfully established cell cultures of the Asian citrus psyllid, Diaphorina citri (Psyllidae: Hemiptera), DcHH-1. The cell culture also supported growth of Candidatus Liberibacter asiaticus. This bacterial pathogen is associated with Huanglongbing, known as citrus greening disease. Research on...

  9. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

  10. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

  11. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

  12. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section...Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used...

  13. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

  14. MATERIALS AND METHODS Cell culture

    E-print Network

    cell line hCMEC/D3 which retains the main characteristics of primary brain endothelial cells, has beenMATERIALS AND METHODS Reagents Cell culture The immortalized human brain microvessel endothelial previously described (S1). hCMEC/D3 were grown at a density of 25 000 cells per cm2 in flasks coated with 5

  15. New Assay Procedure for Separation of Mycoplasmas from Virus Pools and Tissue Culture Systems

    PubMed Central

    Zgorniak-Nowosielska, Izabella; Sedwick, W. David; Hummeler, Klaus; Koprowski, Hilary

    1967-01-01

    Presence of mycoplasma organisms in tissue culture systems and virus pools was detected by titration of the contaminated material on agarose-suspended BHK21/13S cells. The use of this method permitted isolation of mycoplasmas which could not be detected by standard assay methods. Mycoplasma colonies at concentrations ranging from 104 to 106 colony-forming units/ml in agarose-BHK21/13S media could be distinguished from virus plaques, and the two populations of microorganisms could be easily disassociated either by electron microscopy or by biological methods. All isolated mycoplasmas were identified in growth inhibition tests as belonging to the GDL group. The growth inhibition test on agarose-BHK21/13S cell suspension plates could also be applied directly to those strains which could not be isolated by standard assay procedures. Images PMID:4912246

  16. High density cell culture system

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (inventor)

    1994-01-01

    An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

  17. Orosphere Assay: A method for propagation of head and neck cancer stem cells

    PubMed Central

    Krishnamurthy, Sudha; Nör, Jacques E.

    2014-01-01

    Background Recent evidence suggests that head and neck squamous cell carcinomas (HNSCC) harbor a small sub-population of highly tumorigenic cells, named cancer stem cells. A limiting factor in cancer stem cell research is the intrinsic difficulty of expanding cells in an undifferentiated state in vitro. Methods Here, we describe the development of the orosphere assay, a method for the study of putative head and neck cancer stem cells. An orosphere is defined as a non-adherent colony of cells sorted from primary HNSCC or from HNSCC cell lines and cultured in 3-D soft agar or ultra-low attachment plates. Aldehyde dehydrogenase (ALDH) activity and CD44 expression were used here as stem cell markers. Results This assay allowed for the propagation of head and neck cancer cells that retained stemness and self-renewal. Conclusion The orosphere assay is well suited for studies designed to understand the pathobiology of head and neck cancer stem cells. PMID:22791367

  18. Ovine Carotid Artery-Derived Cells as an Optimized Supportive Cell Layer in 2-D Capillary Network Assays

    PubMed Central

    Dreier, Agnieszka; Unger, Ronald E.; Flanagan, Thomas C.; Kirkpatrick, C. James; Zenke, Martin; Klee, Doris; Jockenhoevel, Stefan

    2014-01-01

    Background Endothelial cell co-culture assays are differentiation assays which simulate the formation of capillary-like tubules with the aid of a supportive cell layer. Different cell types have been employed as a supportive cell layer, including human pulmonary artery smooth muscle cells (PASMCs) and human mammary fibroblasts. However, these sources of human tissue-derived cells are limited, and more readily accessible human or animal tissue-derived cell sources would simplify the endothelial cell co-culture assay. In the present study, we investigated the potential use of alternative, accessible supportive cells for endothelial cell co-culture assay, including human umbilical cord and ovine carotid artery. Methods and Results: Human umbilical artery SMCs (HUASMCs) and ovine carotid artery-derived cells were seeded into 96-well plates, followed by addition of human umbilical vein endothelial cells (HUVECs). Nine days after co-culture, cells were fixed, immunostained and analysed using an in vitro angiogenesis quantification tool. Capillary-like structures were detected on ovine carotid artery-derived supportive cell layers. The initial cell number, as well as pro- and anti-angiogenic factors (VEGF, PDGF-BB and Bevacizumab), had a positive or negative influence on the number of capillary-like structures. Furthermore, HUVECs from different donors showed distinct levels of VEGF receptor-2, which correlated with the amount of capillary-like structures. In the case of HUASMC supportive cell layers, HUVECs detached almost completely from the surface. Conclusions Cells of different origin have a varying applicability regarding the endothelial cell co-culture assay: under the conditions described here, ovine carotid artery-derived cells seem to be more suitable than HUASMCs for an endothelial co-culture assay. Furthermore, the ovine carotid artery-derived cells are easier to obtain and are in more abundant supply than the currently used dermal or breast tissue cells. The use of ovine carotid artery-derived cells simplifies the endothelial co-culture assay with respect to testing large amounts of pro- and anti-angiogenic factors. PMID:24621607

  19. Shellfish tissues evaluated for Perkinsus spp. using the Ray's fluid thioglycollate medium culture assay can be used for downstream molecular assays.

    PubMed

    Audemard, C; Carnegie, R B; Burreson, E M

    2008-08-01

    Ray's fluid thioglycollate medium (RFTM) culture assay is the standard, recommended method for surveillance of Perkinsus spp. infections in marine molluscs. In this assay, shellfish tissues are incubated in RFTM, stained with Lugol's iodine solution to render Perkinsus spp. cells blue-black, and evaluated microscopically to rate infection intensities. A limitation of this assay, however, is the lack of pathogen species specificity. Generally, identification of Perkinsus spp. requires DNA sequence analysis of parallel or additional samples since the exposure to iodine is believed to hamper DNA amplification from samples processed by the RFTM assay. However, we show that P. marinus DNA can be successfully amplified by PCR from Crassostrea virginica tissues cultured in RFTM and stained with Lugol's iodine. The beneficial consequence is that, where necessary, DNA sequence data may be obtained from RFTM-cultured tissues, allowing the identification of the Perkinsus sp. responsible for an observed infection. This would obviate further sampling, representing gain of time and reduction in cost, where a Perkinsus sp. is unexpectedly observed in new host(s) or location(s) but where parallel samples are not available for molecular diagnostics. Laboratories without molecular diagnostic tools for Perkinsus spp. may fix presumptive Perkinsus sp.-positive culture material in 95% ethanol for transport to, and subsequent analysis by, a laboratory that does have this capacity. PMID:18814549

  20. Viral inhibition assay: a CD8 T cell neutralization assay for use in clinical trials of HIV-1 vaccine candidates.

    PubMed

    Spentzou, Aggeliki; Bergin, Philip; Gill, Dilbinder; Cheeseman, Hannah; Ashraf, Ambreen; Kaltsidis, Harry; Cashin-Cox, Michelle; Anjarwalla, Insiyah; Steel, Alan; Higgs, Christopher; Pozniak, Anton; Piechocka-Trocha, Alicja; Wong, Johnson; Anzala, Omu; Karita, Etienne; Dally, Len; Gotch, Frances; Walker, Bruce; Gilmour, Jill; Hayes, Peter

    2010-03-01

    We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials. PMID:20132004

  1. The Molecular Bacterial Load Assay Replaces Solid Culture for Measuring Early Bactericidal Response to Antituberculosis Treatment

    PubMed Central

    Mtafya, Bariki; Phillips, Patrick P. J.; Hoelscher, Michael; Ntinginya, Elias N.; Kohlenberg, Anke; Rachow, Andrea; Rojas-Ponce, Gabriel; McHugh, Timothy D.; Heinrich, Norbert

    2014-01-01

    We evaluated the use of the molecular bacterial load (MBL) assay, for measuring viable Mycobacterium tuberculosis in sputum, in comparison with solid agar and liquid culture. The MBL assay provides early information on the rate of decline in bacterial load and has technical advantages over culture in either form. PMID:24871215

  2. Morphological characteristics of cultured olfactory bulb cells

    Microsoft Academic Search

    S. P. Fracek; L. Guo; R. Schafer

    1994-01-01

    Cultured olfactory bulb cells from embryonic mice had ultrastructural characteristics similar to those of many cell types in the intact adult mouse olfactory bulb. Identified cultured cells included mitral\\/tufted cells, granule cells, short-axon cells, and fibrous and protoplasmic astrocytes. Cultured neurons were found as individual cells, clusters or aggregates. Clusters consisted of a loose array of neurons that appeared to

  3. CYTOTOXICITY OF CHEMICAL CARCINOGENS TOWARDS HUMAN BRONCHIAL EPITHELIAL CELLS EVALUATED IN A CLONAL ASSAY

    EPA Science Inventory

    Survival of human bronchial epithelial cells after administration of four chemical carcinogens was measured in a clonal assay. Human bronchial epithelial cells were obtained from outgrowths of explanted tissue pieces. Serum-free medium was used for both explant culture and clonal...

  4. Cryopreservation of Dedifferentiated Cell Cultures

    Microsoft Academic Search

    Elke Heine-Dobbernack; Heiko Kiesecker; Heinz Martin Schumacher

    When Gottlieb Haberlandt made the first efforts to cultivate single isolated plant cells in salt solutions his goal was to\\u000a prove the totipotency of single cells (Haberlandt 1902). The cultivation of isolated plant cells in a chemically defined culture\\u000a medium became possible only after the discovery and application of auxins (Gautheret 1939). Today plant cells as well as tissues\\u000a can

  5. Maintaining dendritic cell viability in culture.

    PubMed

    Vremec, David; Hansen, Jacinta; Strasser, Andreas; Acha-Orbea, Hans; Zhan, Yifan; O'Keeffe, Meredith; Shortman, Ken

    2015-02-01

    When mouse dendritic cells (DCs) are isolated from tissues, purified and placed in a nutritive culture they die more rapidly than would be expected from their normal turnover in vivo. This can distort culture assays of DC function. We therefore tested several approaches to prolonging DC survival in culture. Of several cytokines tested granulocyte-macrophage colony stimulating factor was most effective at preserving the viability of conventional DCs (cDCs) but was ineffective for plasmacytoid DCs (pDCs). Surprisingly, Fms-like tyrosine kinase 3 ligand, crucial for DC development, produced only a marginal improvement in DC survival in culture, and interleukin-3, reported to prevent apoptosis of human pDCs, produced only a minor improvement in survival of mouse DCs. Genetic manipulation of cell death pathways was also tested, to avoid activation effects exerted by cytokine signalling. The isolation of DCs from mice overexpressing Bcl-2 was especially effective in maintaining pDC viability but gave a lesser improvement in cDC viability. DCs isolated from Bim(-/-)Noxa(-/-) mice also showed improved culture survival, but in this case with pDCs showing the least improvement. PMID:25081090

  6. Olfactory Ensheathing Cell Cultures

    Microsoft Academic Search

    Ronald Doucette

    \\u000a Over the past several years, neuroscientists have developed a considerable interest in a glial cell found only in the first\\u000a cranial nerve. These glial cells, which are referred to as “olfactory ensheath-ing cells,” provide ensheathment for the unmyelinated\\u000a axons of the olfactory nerve (Doucette, 1984, Doucette, 1986, 11,1; Raisman, 1985). Two major reasons why these cells have become so popular

  7. Kit-On-A-Lid-Assays for accessible self-contained cell assays.

    PubMed Central

    Berthier, Erwin; Guckenberger, David J.; Cavnar, Peter; Huttenlocher, Anna; Keller, Nancy P.

    2013-01-01

    Microfluidics have demonstrated the ability to improve the control of the biomechanical and biochemical properties of cell-based assays. However, microscale methods typically rely on macroscopic reagent handling and fluidic loading protocols that are technically challenging and do not scale favorably. Here, we demonstrate a microfluidic platform technology called “Kit-On-A-Lid-Assay” (KOALA), that enables the creation of self-contained microfluidic cell-based assays, integrating all the steps required to perform cell-based assays. The operation of the KOALA platform is user-friendly and consists of bringing together a lid containing the microchannels, and a base containing the pre-packaged reagents, thereby causing fluidic exchange in all the channels simultaneously. We demonstrate that the KOALA cell-based assays can be operated from start to finish without any external laboratory equipment. Finally, we demonstrate KOALA-bases with advanced function allowing the freezing, thawing, and storing of cell-suspensions. PMID:23229806

  8. Fathead minnow FHM cells for use in in vitro cytotoxicity assays of aquatic pollutants

    SciTech Connect

    Babich, H.; Borenfreund, E.

    1987-08-01

    The suitability of the fathead minnow (FHM) epithelial cell line for use as the target (indicator) system in in vitro cytotoxicity assays was evaluated using several endpoints. The organometal diethyltin dichloride served as the representative test agent. The concentration of diethyltin dichloride which resulted in a midpoint toxicity was 3.5 microM in a 3-day cell growth assay, 3.8 microM in the 24-hr neutral red assay, and 16.5 microM in a 4-hr cell detachment assay. The neutral red assay was used to compare the relative sensitivities of the FHM cells (exposed at 34/sup 0/C) with those of bluegill sunfish (BF-2) cells, a fibroblastic cell culture (exposed at 26 degrees C), in the presence of different classes of test agents frequently occurring as aquatic pollutants. For both fish species the sequence of potencies of the test agents was in the order of organometals greater than pesticides approximately equal to polychlorinated biphenyls greater than polynuclear aromatic hydrocarbons greater than phenolics. Overall, the FHM cells were more sensitive than were the BF-2 cells. However, there was a better correlation between the in vitro cytotoxicity data for the BF-2 cell culture and LC50 data for bluegill sunfish than between similar data for the FHM cell line and fathead minnows.

  9. Hematopoietic Stem Cells: Inferences-from In Vivo Assays

    E-print Network

    Zandstra, Peter W.

    Hematopoietic Stem Cells: Inferences-from In Vivo Assays CONNIEEAVES,CINDYMILLER,JOHANNE CASHMAN Columbia, Canada Key Words.Hematopoietic stem cells Transplantation Cord blood. Expansion Growthfactors murine hematopoietic stem cells to be quantitated. Measurements of murine CRU have shown

  10. Direct Fluorescent Assay of Urokinase and Plasminogen Activators of Normal and Malignant Cells: Kinetics and Inhibitor Profiles

    Microsoft Academic Search

    M. Zimmerman; J. P. Quigley; B. Ashe; C. Dorn; R. Goldfarb; W. Troll

    1978-01-01

    A direct rate assay for plasminogen activator has been developed using a synthetic fluorogenic peptide substrate, 7-(N-Cbz-glycylglycylargininamido)-4-methylcoumarin trifluoroacetate. The assay correlates well with the standard 125I-labeled fibrin plate assay using highly purified urokinase, culture fluids from WI-38, Chinese hamster ovary or HeLa cells, or Rous sarcoma virus-transformed chick fibroblasts as the source of plasminogen activator. The assay is sensitive, rapid,

  11. Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology.

    PubMed

    Miraglia; Swartzman; Mellentin-Michelotti; Evangelista; Smith; Gunawan; Lohman; Goldberg; Manian; Yuan

    1999-01-01

    High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-µmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software. PMID:10838439

  12. A novel coagulation assay incorporating adherent endothelial cells in thromboelastometry.

    PubMed

    Zipperle, J; Schlimp, C J; Holnthoner, W; Husa, A-M; Nürnberger, S; Redl, H; Schöchl, H

    2013-05-01

    Following vascular injury or activation, endothelial cells (ECs) participate in the modulation of haemostasis and fibrinolysis. Viscoelastic tests (VETs) are a potent bedside monitoring tool that reports haemostatic parameters in real time. However, VETs neglect the influence of the surrounding endothelium. Our aim was therefore to establish an assay that incorporates ECs in a whole blood VET and to assess the impact of ECs on coagulation parameters. Outgrowth endothelial cells (OECs) and human umbilical vein endothelial cells (HUVECs) were seeded onto microbeads to create transferable EC-microcarriers. Microbeads were then added to citrated whole blood in the measurement cup of a thromboelastometry device (ROTEM). After the addition of CaCl2 (star-TEM®) to the blood sample (NATEM assay), standard ROTEM parameters were analysed. Scanning electron microscopy (SEM) was carried out to visualise the interactions of the beads, whole blood components and the ROTEM pin after clotting. SEM showed that the added microbeads were effectively incorporated into the final blood clot. In the presence of activated ECs, the clotting time (CT) of the blood was shortened fourfold compared to that in uncoated control beads. A significant reduction in CT was also observed in the presence of unstimulated ECs. Interestingly, CT was also reduced by the addition of purified EC culture supernatant. CT shortening was prevented by incubating the supernatant with an inhibiting antibody against tissue factor (TF). Our findings demonstrate that ECs can be incorporated into a ROTEM assay via coated microbeads, and whole blood clotting initiation is accelerated by non-activated and activated ECs. PMID:23494019

  13. Cell stretching in extensional flows for assaying cell mechanics

    NASA Astrophysics Data System (ADS)

    Gossett, Daniel; Tse, Henry; Adeyiga, Oladunni; Yang, Otto; Rao, Jianyu; di Carlo, Dino

    2013-03-01

    There is growing evidence that cell deformability is a useful indicator of cell state and may be a label-free biomarker of metastatic potential, degree of differentiation, and leukocyte activation. In order for deformability measurements to be clinically valuable given the heterogeneity of biological samples, there exists a need for a high-throughput assay of this biophysical property. We developed a robust method for obtaining high-throughput (>1,000 cells/sec) single-cell mechanical measurements which employs coupled hydrodynamic lift forces and curvature-induced secondary flows to uniformly position cells in flow, extensional flow stretching, high-speed imaging, and automated image analysis to extract diameter and deformability parameters. Using this method we have assayed numerous in vitro models of cellular transformations and clinical fluids where malignant cells manifest. We found transformations associated with increased motility or invasiveness increased deformability and the presence of large and deformable cells within clinical pleural fluids correlated well with cytological diagnoses of malignancy. This agrees with the hypothesis that cancerous cells are deformable by necessity--to be able to transverse tight endothelial gaps and invade tissues.

  14. Use of immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex from liquid culture

    PubMed Central

    Považan, Anika; Vukeli?, Anka; Savkovi?, Tijana; Kurucin, Tatjana

    2012-01-01

    A new, simple immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in liquid cultures has been developed. The principle of the assay is binding of the Mycobacterium tuberculosis complex specific antigen to the monoclonal antibody conjugated on the test strip. The aim of this study is evaluation of the performance of immunochromatographic assay in identification of Mycobacterium tuberculosis complex in primary positive liquid cultures of BacT/Alert automated system. A total of 159 primary positive liquid cultures were tested using the immunochromatographic assay (BD MGIT TBc ID) and the conventional subculture, followed by identification using biochemical tests. Of 159 positive liquid cultures, using the conventional method, Mycobacterium tuberculos is was identified in 119 (74.8%), nontuberculous mycobacteria were found in 4 (2.5%), 14 (8.8%) cultures were contaminated and 22 (13.8%) cultures were found to be negative. Using the immunochromatographic assay, Mycobacterium tuberculosis complex was detected in 118 (74.2%) liquid cultures, and 41 (25.8%) tests were negative. Sensitivity, specificity, positive and negative predictive values of the test were 98.3%; 97.5%; 99.15%; 95.12%, respectively. The value of kappa test was 0.950, and McNemar test was 1.00. The immunochromatographic assay is a simple and rapid test which represents a suitable alternative to the conventional subculture method for the primary identification of Mycobacterium tuberculosis complex in liquid cultures of BacT/Alert automated system. PMID:22364301

  15. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  16. Hawthorn (Crataegus monogyna Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay.

    PubMed

    Salehi, Satin; Long, Shannon R; Proteau, Philip J; Filtz, Theresa M

    2009-01-01

    Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases. Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. To test the hypothesis that hawthorn is acting via muscarinic receptors, the effect of hawthorn extract on atrial versus ventricular cardiomyocytes in culture was evaluated. As would be expected for activation of muscarinic receptors, hawthorn extract had a greater effect in atrial cells. Atrial and/or ventricular cardiomyocytes were then treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, L-[benzylic-4,4'-(3)H] ([(3)H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [(3)H]-QNB binding to mouse heart membranes. Taken together, these findings suggest that decreased contraction frequency by hawthorn extracts in neonatal murine cardiomyocytes may be mediated via muscarinic receptor activation. PMID:18696181

  17. The use of macrophages stimulated by immune interferon as indicator cells in the mononuclear phagocyte assay.

    PubMed

    Wiener, E; Garner, S F

    1987-01-01

    Macrophages obtained from human monocytes by monolayer culture were stimulated with recombinant immune interferon. They were compared with unstimulated macrophages and monocytes as indicator cells in the mononuclear phagocyte assay, using both IgG anti-Rh(D)-coated complement-coated red cells. The presence of the interferon in the culture medium improved the adherence of macrophages in monolayers. The interferon markedly augmented the number of IgG-coated red cells which became attached to the macrophages and reduced the amount of antibody needed for red cell-macrophage interaction. This stimulatory effect occurred regardless of the IgG subclass composition of the anti-Rh(D) antibody. The activity of the stimulated macrophages to interact with IgG- or complement-coated red cells was considerably greater than that of monocytes. The results imply that macrophages treated with recombinant immune interferon are more sensitive than monocytes as indicator cells in the mononuclear phagocyte assay. PMID:3127106

  18. Cell culture compositions

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

    2014-03-18

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  19. Behavior of endothelial cells on Matrigel and development of a method for a rapid and reproducible in vitro angiogenesis assay.

    PubMed

    Crabtree, Benedict; Subramanian, Vasanta

    2007-02-01

    During the process of angiogenesis, the normally quiescent endothelial cells that line the vasculature are induced to proliferate, migrate and align to form new blood vessels by angiogenic stimuli. Assays for angiogenic factors mostly involve in vivo approaches. The two most commonly used in vivo assays-the chick chorioallantoic membrane (CAM) assay and the rabbit corneal assay are tedious to perform and are technically demanding. Several in vitro assays have also been developed, based on the ability of endothelial cells to form tubes in 3-D matrices. Here, we describe the modification of a microcarrier bead-based assay. This assay combines cells grown on Cytodex-3 microcarrier beads with Matrigel to provide an easy, rapid, and reliable method for evaluating and measuring angiogenic activity. We also describe the differential behavior of normal and transformed endothelial cells cultured in Matrigel. PMID:17570022

  20. Mesenchymal Progenitor Cells: Tissue Origin, Isolation And Culture

    Microsoft Academic Search

    Philippe Bourin; Mélanie Gadelorge; Julie-Anne Peyrafitte; Sandrine Fleury-Cappellesso; Marilyn Gomez; Christine Rage; Luc Sensebe

    2008-01-01

    SummarySince the pioneering work of Alexander Friedenstein on multipotent mesenchymal stromal cells (MSCs), a tremendous amount of work has been done to isolate, characterize and culture such cells. Assay of colony forming unit-fibroblasts (CFU-Fs), the hallmark of MSCs, is used to estimate their frequency in tissue. MSCs are adherent cells, so they are easy to isolate, and they show contact

  1. Production of tropane alkaloids in cultured cells of Hyoscyamus niger.

    PubMed

    Yamada, Y; Hashimoto, T

    1982-04-01

    Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS. PMID:24259019

  2. A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis

    PubMed Central

    Patra, Bishnubrata; Chen, Ying-Hua; Peng, Chien-Chung; Lin, Shiang-Chi; Lee, Chau-Hwang; Tung, Yi-Chung

    2013-01-01

    Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell–cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers. PMID:24396525

  3. Isolation and culture of protoplasts from cotton cell cultures

    E-print Network

    Finer, John James

    1981-01-01

    ISOLATION AND CULTURE OF PROTOPLASTS FROM COTTON CELL CULTURES A Thesis by JOHN JAMES FINER Submitted to the Graduate College of Texas ASM University in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE May 1981... Major Subject: Plant Physiology ISOLATION AND CULTURE OF PROTOPLASTS FROM COTTON CELL CULTURES A Thesis by John James Finer Approved as tc style and content by: (Chairman ot Committee) (Member) (Member) (Member) (Head oF Department) May 1981...

  4. A paper-based invasion assay: Assessing chemotaxis of cancer cells in gradients of oxygen.

    PubMed

    Mosadegh, Bobak; Lockett, Matthew R; Minn, Kyaw Thu; Simon, Karen A; Gilbert, Karl; Hillier, Shawn; Newsome, David; Li, Howard; Hall, Amy B; Boucher, Diane M; Eustace, Brenda K; Whitesides, George M

    2015-06-01

    This work describes a 3D, paper-based assay that can isolate sub-populations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O2). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel. Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates sub-populations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells. PMID:25818432

  5. Morphological characteristics of cultured olfactory bulb cells

    Microsoft Academic Search

    S. P. Fracek; L. Guo; R. Schafer

    1994-01-01

    Cultured olfactory bulb cells from embryonic mice had ultrastructural characteristics similar to those of many cell types\\u000a in the intact adult mouse olfactory bulb. Identified cultured cells included mitral\\/tufted cells, granule cells, short-axon\\u000a cells, and fibrous and protoplasmic astrocytes. Cultured neurons were found as individual cells, clusters or aggregates. Clusters\\u000a consisted of a loose array of neurons that appeared to

  6. Modified procedure for labelling target cells in a europium release assay of natural killer cell activity.

    PubMed

    Pacifici, R; Di Carlo, S; Bacosi, A; Altieri, I; Pichini, S; Zuccaro, P

    1993-05-01

    Lanthanide europium chelated to diethylenetriaminopentaacetate (EuDTPA) can be used to label target cells such as tumor cells and lymphocytes (Blomberg et al., 1986a,b; Granberg et al., 1988). This procedure has permitted the development of new non-radioactive methods for the detection of target cell cytolysis by natural killer (NK) cells (Blomberg et al., 1986a,b), cytotoxic T lymphocytes (CTL) (Granberg et al., 1988) or complement-mediated cytolysis (Cui et al., 1992). However, we had no success with this method because of a lack of comparability between human NK cell activity simultaneously measured by a classical 51Cr release assay (Seaman et al., 1981) and EuDTPA release assay (Blomberg et al., 1986a). Furthermore, cell division and cell viability were significantly impaired by the suggested concentrations of EuCl3. In this paper, we present a modified non-cytotoxic method for target cell labelling with EuDTPA while cells are growing in culture medium. PMID:8486925

  7. Culturing adult stem cells from mouse small intestinal crypts.

    PubMed

    Hamilton, Kathryn E; Crissey, Mary Ann S; Lynch, John P; Rustgi, Anil K

    2015-01-01

    In recent years, the study of primary cells in culture has evolved from an extraphysiological, two-dimensional platform to novel, three-dimensional platforms in which the addition of matrix components and/or supporting cells provide an ex vivo niche. Such studies have provided the basis on which to study more advanced physiological processes in detail, including multilayered, long-term cultures, epithelial-stromal interactions, and stem cell behaviors that more closely recapitulate normal morphology than two-dimensional culture. Various techniques for three-dimensional organotypic culture and crypt culture of primary cells from mouse and human small intestine and colon have been described. These methods have allowed for the study of specific stem cell characteristics, including survival, self-renewal, and long-term growth in culture, as well as the ability to propagate all the appropriate progenitor and postmitotic lineages. These assays have become a widely accepted functional measure of "stemness" and, in combination with lineage-tracing experiments in various genetically engineered mouse models, have been critical in the identification of specific markers of intestinal stem cells. In this protocol we draw upon recently described methods for the isolation and culture of mouse small intestinal enterospheres/enteroids from isolated crypts and/or single cells. Cultures of murine colon epithelium, as well as human small intestine and colon, require additional growth factors not discussed here. The description provided here represents current knowledge, and it is possible, if not likely, that modifications in the future will emerge. PMID:25834260

  8. Ocular irritation reversibility assessment for personal care products using a porcine corneal culture assay.

    PubMed

    Donahue, Douglas A; Avalos, Javier; Kaufman, Lewis E; Simion, F Anthony; Cerven, Daniel R

    2011-04-01

    Personal care product manufacturers have used a broad spectrum of alternative ocular irritation assays during the past two decades because these tests do not require the use of live animals, they provide reliable predictive data, and they are relatively inexpensive to conduct. To complement these assays, the ex vivo Porcine Corneal Opacity Reversibility Assay (PorCORA) was recently developed using a corneal culture model to predict reversibility of ocular irritants. Three commercially available consumer products (a shampoo, a hair color glaze, and a hair colorant system containing 12% hydrogen peroxide) were each tested in two PorCORA study replicates in order to assess potential ocular damage reversibility for surfactant-, propylene carbonate-, and peroxide-based formulations, respectively. Under the exaggerated, in vitro study conditions, the surfactant-based shampoo may cause irreversible porcine corneal damage (histological changes in the epithelial squamous cell and/or basal cell layers), whereas the hair color glaze and 12% hydrogen peroxide product caused fully reversible ocular irritation (microscopic changes only in the superficial squamous cell layer). The hair color glaze and peroxide product results correlate with established in vivo data for similar compounds, but the shampoo results contradicted previous BCOP results (expected to be only a mild irritant). Therefore, although the PorCORA protocol shows promise in predicting the extent and reversibility of potential ocular damage caused by accidental consumer eye exposure to personal care products, the contradictory results for the surfactant-based shampoo indicate that more extensive validation testing of the PorCORA is necessary to definitively establish the protocol's reliability as a Draize test replacement. PMID:21172418

  9. A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability.

    PubMed

    Welch, Aaron Z; Koshland, Douglas E

    2013-12-01

    Here we describe the first high-throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high-throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10(8) dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high-throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities?cell viability. PMID:24185677

  10. Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs

    PubMed Central

    Zustiak, Silviya; Nossal, Ralph; Sackett, Dan L.

    2013-01-01

    It is now well understood that the cell microenvironment, including the surrounding matrix, profoundly affects cell fate. This is especially true for solid tumors where, for example, matrix stiffness is believed to be an important factor in tumorogenesis. Our hypothesis is that since matrix stiffness affects cell fate, it may also be important in drug resistance. To test this hypothesis, we designed and built a multiwell polyacrylamide (PA) gel-based stiffness assay, in which the gels were coated with collagen in order to facilitate cell attachment and proliferation. This PA-based assay was used to examine the effect of stiffness on cultured cell responsiveness to cytotoxic drugs. In particular, we tested multiple cancer cell lines and their susceptibility to paclitaxel, a microtubule-targeting agent. By assessing cell proliferation, morphology, and the IC50 of the drug, we were able to establish that the stiffness affects responsiveness to cytotoxic drugs in a cell dependent manner. PMID:24018833

  11. Opsonophagocytic assay.

    PubMed

    Dwyer, Markryan; Gadjeva, Mihaela

    2014-01-01

    The opsonophagocytic killing (OPK) assay is used as a correlate for protection to measure the functional capacities of vaccine-candidate-raised antibodies. This in vitro assay aids selecting promising vaccines by demonstrating whether the vaccine-induced antibodies drive efficient complement deposition and subsequent opsonophagocytic killing. Here, we describe two protocols for an OPK assay using either human-derived PMNs or cultured HL-60 cells. PMID:24218277

  12. A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium

    PubMed Central

    Shen, Jie; Cai, Changzu; Yu, Zhilong; Pang, Yuhong; Zhou, Ying; Qian, Lili; Wei, Wensheng; Huang, Yanyi

    2015-01-01

    It is technically challenging to investigate the function of secreted protein in real time by supply of conditioned medium that contains secreted protein of interest. The internalization of anthrax toxin is facilitated by a secreted protein Dickkopf-1 (DKK1) and its receptor, and eventually leads to cell lethality. To monitor the dynamic interplay between these components in live cells, we use an integrated microfluidic device to perform the cell viability assays with real-time controlled culture microenvironment in parallel. Conditioned medium, which contains the secreted proteins from specific cell lines, can be continuously pumped towards the cells that exposed to toxin. The exogenous DKK1 secreted from distant cells is able to rescue the sensitivity to toxin for those DKK1-knocked-down cells. This high-throughput assay allows us to precisely quantify the dynamic interaction between key components that cause cell death, and provide independent evidence of the function of DKK1 in the complex process of anthrax toxin internalization. PMID:25731605

  13. A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium.

    PubMed

    Shen, Jie; Cai, Changzu; Yu, Zhilong; Pang, Yuhong; Zhou, Ying; Qian, Lili; Wei, Wensheng; Huang, Yanyi

    2015-01-01

    It is technically challenging to investigate the function of secreted protein in real time by supply of conditioned medium that contains secreted protein of interest. The internalization of anthrax toxin is facilitated by a secreted protein Dickkopf-1 (DKK1) and its receptor, and eventually leads to cell lethality. To monitor the dynamic interplay between these components in live cells, we use an integrated microfluidic device to perform the cell viability assays with real-time controlled culture microenvironment in parallel. Conditioned medium, which contains the secreted proteins from specific cell lines, can be continuously pumped towards the cells that exposed to toxin. The exogenous DKK1 secreted from distant cells is able to rescue the sensitivity to toxin for those DKK1-knocked-down cells. This high-throughput assay allows us to precisely quantify the dynamic interaction between key components that cause cell death, and provide independent evidence of the function of DKK1 in the complex process of anthrax toxin internalization. PMID:25731605

  14. An improved method for staining cell colonies in clonogenic assays

    Microsoft Academic Search

    Kishore Guda; Leanna Natale; Sanford D. Markowitz

    2007-01-01

    Clonogenic assay is a widely used experimental approach to test for the effects of drugs\\/genes on the growth and proliferative\\u000a characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability\\u000a to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown\\u000a on plastic and

  15. Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

    PubMed Central

    Azari, Hassan; Rahman, Maryam; Sharififar, Sharareh; Reynolds, Brent A.

    2010-01-01

    Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes. This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments. PMID:21113123

  16. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...2012-01-01 2012-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

  17. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...2014-01-01 2014-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

  18. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...2013-01-01 2013-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

  19. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...2011-01-01 2011-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

  20. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

  1. Reference cells and ploidy in the comet assay

    PubMed Central

    Brunborg, Gunnar; Collins, Andrew; Graupner, Anne; Gutzkow, Kristine B.; Olsen, Ann-Karin

    2015-01-01

    In the comet assay single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i) Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii) reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods, and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used – in combination with a reference curve – to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose, and they are inexpensive. PMID:25774164

  2. A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures, followed by modeling of ecdysteroid-EcR interactions and normal mode analysis.

    PubMed

    Zotti, Moisés J; De Geyter, Ellen; Swevers, Luc; Braz, Antônio S K; Scott, Luis P B; Rougé, Pierre; Coll, Josep; Grutzmacher, Anderson D; Lenardão, Eder J; Smagghe, Guy

    2013-11-01

    Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC50=3.3?M) and Bm5 cells (EC50=5.3?M), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC50=0.71?M and 0.00089?M, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC50 of 0.039?M; in Bm5 cells this effect only became visible at much higher concentrations (IC50=18?M). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure. PMID:24267692

  3. The effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells

    Microsoft Academic Search

    Yu-Chao Chang; Fu-Mei Huang; Kuo-Wei Tai; Ming-Yung Chou

    2001-01-01

    Objective: The objective of this study was to examine the effects of sodium hypochlorite (NaOCl) and chlorhexidine (CHx) on cultured human periodontal ligament (PDL) cells in vitro. Study Design: The effects of irrigation solutions on human PDL cells were evaluated by propidium iodide fluorescence cytotoxicity assay, protein synthesis assay, and mitochondrial activity. Results: Both NaOCl and CHx were cytotoxic to

  4. One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

    PubMed Central

    Walker, Tara L.; Kempermann, Gerd

    2014-01-01

    The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage. PMID:24637893

  5. Are in vitro estimates of cell diffusivity and cell proliferation rate sensitive to assay geometry?

    PubMed

    Treloar, Katrina K; Simpson, Matthew J; McElwain, D L Sean; Baker, Ruth E

    2014-09-01

    Cells respond to various biochemical and physical cues during wound-healing and tumour progression. in vitro assays used to study these processes are typically conducted in one particular geometry and it is unclear how the assay geometry affects the capacity of cell populations to spread, or whether the relevant mechanisms, such as cell motility and cell proliferation, are somehow sensitive to the geometry of the assay. In this work we use a circular barrier assay to characterise the spreading of cell populations in two different geometries. Assay 1 describes a tumour-like geometry where a cell population spreads outwards into an open space. Assay 2 describes a wound-like geometry where a cell population spreads inwards to close a void. We use a combination of discrete and continuum mathematical models and automated image processing methods to obtain independent estimates of the effective cell diffusivity, D, and the effective cell proliferation rate, ?. Using our parameterised mathematical model we confirm that our estimates of D and ? accurately predict the time-evolution of the location of the leading edge and the cell density profiles for both assay 1 and assay 2. Our work suggests that the effective cell diffusivity is up to 50% lower for assay 2 compared to assay 1, whereas the effective cell proliferation rate is up to 30% lower for assay 2 compared to assay 1. PMID:24787651

  6. Electrolytic valving isolation of cell co-culture microenvironment with controlled cell pairing ratios.

    PubMed

    Chen, Yu-Chih; Ingram, Patrick; Yoon, Euisik

    2014-12-21

    Cancer-stromal interaction is a critical process in tumorigenesis. Conventional dish-based co-culture assays simply mix two cell types in the same dish; thus, they are deficient in controlling cell locations and precisely tracking single cell behavior from heterogeneous cell populations. Microfluidic technology can provide a good spatial-temporal control of microenvironments, but the control has been typically realized by using external pumps, making long-term cultures cumbersome and bulky. In this work, we have presented a cell-cell interaction microfluidic platform that can accurately control the co-culture microenvironment by using a novel electrolytic cell isolation scheme without using any valves or pneumatic pumps. The proposed microfluidic platform can also precisely control the number of interacting cells and pairing ratios to emulate cancer niches. More than 80% of the chambers captured the desired number of cells. The duration of cell isolation can be adjusted by electrolytic bubble generation and removal. We have verified that the electrolytic process has a negligible effect on cell viability and proliferation in our platform. To the best of our knowledge, this work is the first attempt to incorporate electrolytic bubble generation as a cell isolation method in microfluidics. For proof of feasibility, we have performed cell-cell interaction assays between prostate cancer (PC3) cells and myoblast (C2C12) cells. The preliminary results demonstrated the potential of using electrolysis for micro-environmental control during cell culture. Also, the ratio controlled cell-cell interaction assays were successfully performed which showed that the cell pairing ratios of PC3 to C2C12 affected the proliferation rate of myoblast cells due to increased secretion of growth factors from prostate cancer cells. PMID:25118341

  7. DETECTION OF ANEUPLOIDY BY A MONOCHROMOSOMAL HYBRID CELL ASSAY

    EPA Science Inventory

    A short-term assay utilizing human/mouse monochromosomal hybrid cells to detect chemically-induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome segregation fol...

  8. Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures

    E-print Network

    Farahat, Waleed A.

    We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables ...

  9. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

  10. Metabolomic profiling of cultured cancer cells.

    PubMed

    Scoazec, Marie; Durand, Sylvere; Chery, Alexis; Galluzzi, Lorenzo; Kroemer, Guido

    2014-01-01

    Quantitative proteomics approaches have been developed-and now begin to be implemented on a high-throughput basis-to fill-in the large gap between the genomic/transcriptomic setup of (cancer) cells and their phenotypic/behavioral traits, reflecting a significant degree of posttranscriptional regulation in gene expression as well as a robust posttranslational regulation of protein function. However, proteomic profiling assays not only fail to detect labile posttranslational modifications as well as unstable protein-to-protein interactions but also are intrinsically incapable of assessing the enzymatic activity, as opposed to the mere abundance, of a given protein. Thus, determining the abundance of theoretically all the metabolites contained in a cell/tissue/organ/organism may significantly improve the informational value of proteomic approaches. Several techniques have been developed to this aim, including high-performance liquid chromatography (HPLC) coupled to quadrupole time-of-flight (Q-TOF) high-resolution mass spectrometry (HRMS). This approach is particularly advantageous for metabolomic profiling as it offers elevated accuracy and improved sensitivity. Here, we describe a simple procedure to determine the complete complement of intracellular metabolites in cultured malignant cells by HPLC coupled to Q-TOF HRMS. According to this method, (1) cells are collected and processed to minimize contaminations as well as fluctuations in their metabolic profile; (2) samples are separated by HPLC and analyzed on a Q-TOF spectrometer; and (3) data are extracted, normalized, and deconvoluted according to refined mathematical methods. This protocol constitutes a simple approach to determine the intracellular metabolomic profile of cultured cancer cells. With minimal variations (mostly related to sample collection and processing), this method is expected to provide reliable metabolomic data on a variety of cellular samples. PMID:24924132

  11. Growth Factor-Dependent Proliferation of the Pancreatic ?-cell Line ?TC-tet: An Assay for ?-cell Mitogenic Factors

    PubMed Central

    Milo-Landesman, Dalit

    2002-01-01

    The ability to expand normal pancreatic islet ? cells in culture would significantly advance the prospects of cell therapy for diabetes. A number of growth factors can stimulate limited islet cell replication, however other factors may exist which are more effective ?-cell-specific mitogens. The search for novel ?-cell growth factors has been hampered by the lack of a ?-cell-specific proliferation assay. We developed a simple and sensitive assay for ?-cell growth factors based on a conditionally-transformed mouse ?-cell line (?TC-tet). These cells express the SV40 T antigen (Tag) oncoprotein under control of the tetracycline (Tc) operon regulatory system. In the presence of Tc, Tag expression is tightly shut off and the cells undergo complete growth arrest. Here we show that the growth-arrested cells can proliferate in response to growth factors in the absence of Tag. Using this assay, a number of growth factors previously shown to be mitogenic to a mixed islet cell population were found to induce proliferation of pure ? cells. We conclude that growth-arrested ?TC-tet cells can be employed in a survey of factors from various sources for identifying novel factors with ?-cell mitogenic activity. PMID:11900281

  12. Comet assay, cloning assay, and light and electron microscopy on one preselected cell

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Oehring, Hartmut; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl-Otto

    1998-01-01

    In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular redox state, impaired cell division, formation of giant cells and cell shrinking, swelling of mitochondria and loss of cristae as well as DNA damage.

  13. Comet assay, cloning assay, and light and electron microscopy on one preselected cell

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Oehring, H.; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl O.

    1997-12-01

    In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular redox state, impaired cell division, formation of giant cells and cell shrinking, swelling of mitochondria and loss of cristae as well as DNA damage.

  14. A cell viability assay based on monitoring respiration by optical oxygen sensing.

    PubMed

    O'Riordan, T C; Buckley, D; Ogurtsov, V; O'Connor, R; Papkovsky, D B

    2000-02-15

    A cell viability assay based on monitoring of the metabolic activity of living cells via their consumption of dissolved oxygen has been developed. It uses a microwell plate format and disposable phosphorescent sensor inserts incorporated into each sample. The wells are subsequently sealed from ambient oxygen using a layer of mineral oil, and periodically scanned from underneath with a simple fiber-optic phosphorescent phase detector. Thus, dissolved oxygen levels and time profiles of cell respiration can be determined noninvasively and compared to each other. The system was tested by monitoring the viability of the fission yeast Schizosaccharomyces pombe. In comparison with the conventional cell densitometry assay, the optical oxygen sensor method could reliably monitor lower numbers of cells (10(4)-10(5) vs 10(6)-10(7) cells/ml for densitometry), and accurately determine culture viability within 1 h. The assay was then applied to determine the viability of samples treated with toxic agents such as azide and in response to expression of a physiological inducer of cell death, the Bcl-2 family member Bak. The results obtained confirm that measurement of cell respiration by this assay can serve as a predictable, reliable, and fast method for high-throughput determination of cell viability and growth. PMID:10660466

  15. Culture and differentiation of embryonic stem cells

    Microsoft Academic Search

    Austin G. Smith

    1991-01-01

    Summary Techniques are described for the culture of murine embryonic stem cells in the absence of heterologous feeder cells and for the induction of differentiation programs. The regulatory factor differentiation inhibiting activity\\/ leukaemia inhibitory factor (DIA\\/LIF) is produced at high concentration by transient expression in Cos cells and is used to suppress stem cell differentiation by addition to the culture

  16. Use of a New Tetrazolium-Based Assay to Study the Production of Superoxide Radicals by Tobacco Cell Cultures Challenged with Avirulent Zoospores of Phytophthora parasitica var nicotianae1

    PubMed Central

    Able, Amanda J.; Guest, David I.; Sutherland, Mark W.

    1998-01-01

    The relationship between the production of reactive oxygen species and the hypersensitive response (HR) of tobacco (Nicotiana tabacum L.) toward an incompatible race of the Oomycete Phytophthora parasitica var nicotianae has been investigated. A new assay for superoxide radical (O2?) production based on reduction of the tetrazolium dye sodium,3?-(1-[phenylamino-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) has enabled the quantitative estimation of perhydroxyl/superoxide radical acid-base pair (HO2·/O2?) production during the resistant response. Tobacco suspension cells were inoculated with zoospores from compatible or incompatible races of the pathogen. Subsequent HO2·/O2? production was monitored by following the formation of XTT formazan. In the incompatible interaction only, HO2·/O2? was produced in a minor burst between 0 and 2 h and then in a major burst between 8 and 10 h postinoculation. During this second burst, rates of XTT reduction equivalent to a radical flux of 9.9 × 10?15 mol min?1 cell?1 were observed. The HO2·/O2? scavengers O2? dismutase and Mn(III)desferal each inhibited dye reduction. An HR was observed in challenged, resistant cells immediately following the second burst of radical production. Both scavengers inhibited the HR when added prior to the occurrence of either radical burst, indicating that O2? production is a necessary precursor to the HR. PMID:9625702

  17. Rapid, sensitive, and validated method for detection of Salmonella in food by an enrichment broth culture – Nested PCR combination assay

    Microsoft Academic Search

    Sunil D. Saroj; R. Shashidhar; Manisha Karani; Jayant R. Bandekar

    2008-01-01

    A rapid nested PCR assay for detection of Salmonella from food was developed. The sensitivity of the assay developed was comparable to the traditional culture based methods with an advantage in reduction of assay time. The assay procedure with artificially contaminated samples was able to detect as low as 4CFU Salmonella\\/25g of food samples (sprout, carrot, cucumber and poultry meat).

  18. Dynamized Preparations in Cell Culture

    PubMed Central

    Sunila, Ellanzhiyil Surendran; Preethi, Korengath Chandran; Kuttan, Girija

    2009-01-01

    Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties. PMID:18955237

  19. Miniature Bioreactor System for Long-Term Cell Culture

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.

    2010-01-01

    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  20. Inflight Assay of Red Blood Cell Deformability

    NASA Technical Reports Server (NTRS)

    Ingram, M.; Paglia, D. E.; Eckstein, E. C.; Frazer, R. E.

    1985-01-01

    Studies on Soviet and American astronauts have demonstrated that red blood cell production is altered in response to low gravity (g) environment. This is associated with changes in individual red cells including increased mean cell volume and altered membrane deformability. During long orbital missions, there is a tendency for the red cell mass deficit to be at least partly corrected although the cell shape anomalies are not. Data currently available suggest that the observed decrease in red cell mass is the result of sudden suppression of erythropoieses and that the recovery trend observed during long missions reflects re-establishment of erythropoietic homeostasis at a "set point" for the red cell mass that is slightly below the normal level at 1 g.

  1. Evaluation of Verigene Gram-positive blood culture assay performance for bacteremic patients.

    PubMed

    Dodémont, M; De Mendonça, R; Nonhoff, C; Roisin, S; Denis, O

    2015-03-01

    The Verigene Gram-Positive Blood Culture (BC-GP) Assay (Nanosphere Inc., Northbrook, IL) is a microarray-based test designed to rapidly identify directly from positive blood cultures multiple bacterial species and their antimicrobial resistance markers. Nonduplicate blood cultures from 118 patients admitted to Erasme Hospital were prospectively enrolled. All but six organisms were members of the panel (95.6 %). For the identification of pathogens and detection of the mecA gene, the agreement with routine methods was 87.6 % and 97.7 %, respectively. The performance of the BC-GP assay was lower with polymicrobial than with monomicrobial blood cultures. Another concern of the BC-GP assay was the misidentification of Streptococcus mitis as S. pneumoniae (3/8). The BC-GP assay is a rapid and accurate tool for the simultaneous detection of multiple sepsis-causing bacteria and resistant genes from blood cultures, which could have an impact on patient management and healthcare cost. PMID:25260788

  2. Genotoxic effects of oestrogens in breast cells detected by the micronucleus assay and the Comet assay

    Microsoft Academic Search

    Edom Yared; Trevor J. McMillan; Francis L. Martin

    2002-01-01

    Cumulative exposure to oestrogen has been linked to increased risk of breast cancer. Whilst oestrogens induce cancers in rodent bioassays it is unclear whether the mechanisms involved are genotoxic and\\/or epigenetic. The cytokinesis block micronucleus (CBMN) and the alkaline single cell-gel electrophoresis 'Comet' assays were used to examine MCF-7 cells for chromosomal damage and DNA single-strand breaks (SSBs), respectively. The

  3. Comparison of saliva PCR assay versus rapid culture for detection of congenital cytomegalovirus infection.

    PubMed

    Pinninti, Swetha G; Ross, Shannon A; Shimamura, Masako; Novak, Zdenek; Palmer, April L; Ahmed, Amina; Tolan, Robert W; Bernstein, David I; Michaels, Marian G; Sánchez, Pablo J; Fowler, Karen B; Boppana, Suresh B

    2015-05-01

    As part of the CMV and Hearing Multicenter Screening (CHIMES) study, 72,239 newborns were screened for cytomegalovirus by rapid culture and real-time PCR of saliva samples. Of the 266 infants with congenital cytomegalovirus infection, discordance between rapid culture and PCR was observed in 14 children, and 13 were identified only by PCR, demonstrating the superiority of the PCR assay. PMID:25876092

  4. Cell culture techniques in honey bee research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell culture techniques are indispensable in most if not all life science disciplines to date. Wherever cell culture models are lacking scientific development is hampered. Unfortunately this has been and still is the case in honey bee research because permanent honey bee cell lines have not yet been...

  5. Cell Culture as an Alternative in Education.

    ERIC Educational Resources Information Center

    Nardone, Roland M.

    1990-01-01

    Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

  6. Characterization of cultured multipotent zebrafish neural crest cells.

    PubMed

    Kinikoglu, Beste; Kong, Yawei; Liao, Eric C

    2014-02-01

    The neural crest is a unique cell population associated with vertebrate evolution. Neural crest cells (NCCs) are characterized by their multipotent and migratory potentials. While zebrafish is a powerful genetic model organism, the isolation and culture of zebrafish NCCs would provide a useful adjunct to fully interrogate the genetic networks that regulate NCC development. Here we report for the first time the isolation, in vitro culture, and characterization of NCCs from zebrafish embryos. NCCs were isolated from transgenic sox10:egfp embryos using fluorescence activated cell sorting and cultured in complex culture medium without feeder layers. NCC multilineage differentiation was determined by immunocytochemistry and real-time qPCR, cell migration was assessed by wound healing assay, and the proliferation index was calculated by immunostaining against the mitosis marker phospho-histone H3. Cultured NCCs expressed major neural crest lineage markers such as sox10, sox9a, hnk1, p75, dlx2a, and pax3, and the pluripotency markers c-myc and klf4. We showed that the cultured NCCs can be differentiated into multiple neural crest lineages, contributing to neurons, glial cells, smooth muscle cells, melanocytes, and chondrocytes. We applied the NCC in vitro model to study the effect of retinoic acid on NCC development. We showed that retinoic acid had a profound effect on NCC morphology and differentiation, significantly inhibited proliferation and enhanced cell migration. The availability of high numbers of NCCs and reproducible functional assays offers new opportunities for mechanistic studies of neural crest development, in genetic and chemical biology applications. PMID:24326414

  7. Normal and leukemic human stem cells assayed in SCID mice

    Microsoft Academic Search

    John E. Dick

    1996-01-01

    Understanding the processes that regulate the developmental program of normal stem cells and those that initiate proliferative diseases such as leukemia remains one of the major challenges in biology. Progress to address these major questions in the human hematopoietic system have been hampered, until recently, by the lack of in-vivo assays for normal and leukemic stem cells. The recent development

  8. Single cell gel electrophoresis assay: methodology and applications

    Microsoft Academic Search

    E Rojas; M. C Lopez; M Valverde

    1999-01-01

    The single cell gel electrophoresis or Comet assay is a sensitive, reliable, and rapid method for DNA double- and single-strand breaks, alkali-labile sites and delayed repair site detection, in eukariotic individual cells. Given its overall characteristics, this method has been widely used over the past few years in several different areas. In this paper we review the studies published to

  9. Use of aggregating cell cultures for toxicological studies.

    PubMed

    Honegger, P; Werffeli, P

    1988-10-15

    Relatively simple techniques are now available which allow the preparation of large quantities of highly reproducible aggregate cultures from fetal rat brain or liver cells, and to grow them in a chemically defined medium. Since these cultures exhibit extensive histotypic cellular reorganization and maturation, they offer unique possibilities for developmental studies. Therefore, the purpose of the present study was to investigate the usefulness of these cultures in developmental toxicology. Aggregating brain cell cultures were exposed at different developmental stages to model drugs (i.e., antimitotic, neurotoxic, and teratogenic agents) and assayed for their responsiveness by measuring a set of biochemical parameters (i.e., total protein and DNA content, cell type-specific enzyme activities) which permit a monitoring of cellular growth and maturation. It was found that each test compound elicited a distinct, dose-dependent response pattern, which may ultimately serve to screen and classify toxic drugs by using mechanistic criteria. In addition, it could be shown that aggregating liver cell cultures are capable of toxic drug activation, and that they can be used in co-culture with brain cell aggregates, providing a potential model for complementary toxicological and metabolic studies. PMID:3141206

  10. Cell-based assays for Parkinson's disease using differentiated human LUHMES cells

    PubMed Central

    Zhang, Xiao-min; Yin, Ming; Zhang, Min-hua

    2014-01-01

    Aim: Lund human mesencephalic (LUHMES) cells can be differentiated to post-mitotic cells with biochemical, morphological and functional features of dopaminergic (DAergic) neurons. Given the limited scale of primary DAergic neuron culture, we developed differentiated LUHMES cell-based cytotoxicity assays for identifying neuroprotective agents for Parkinson's disease (PD). Methods: LUHMES cells were incubated in a differentiation medium containing cAMP and GDNF for 6 d, and then differentiated cells were treated with MPP+ or infected with baculovirus containing ?-synuclein. Cytotoxicity was determined by measuring intracellular ATP levels and caspase 3/7 activity in the cells. DAergic neuron-specific marker protein and mRNA levels in the cells were analyzed using Western blotting and RT-PCR, respectively. Results: LUHMES cells grew extensive neurites and became post-mitotic neuron-like cells during differentiation period, and three DAergic neuron markers TH, DAT and Nurr1 exhibited different expression profiles. MPP+ dose-dependently reduced ATP levels in the cells with an IC50 value of 65 ?mol/L. MPP+ (80 ?mol/L) significantly increased caspase 3/7 activity in the cells. Both the CDK inhibitor GW8510 and the GSK3? inhibitor SB216763 effectively rescued MPP+-induced reduction of ATP levels with EC50 values of 12 and 205 nmol/L, respectively. Overexpression of ?-synuclein also significantly decreased intracellular ATP levels and increased caspase 3/7 activity in the cells. GW8510 and SB216763 effectively rescued ?-synuclein overexpression-induced reduction of ATP levels, whereas GW8510, but not SB216763, ameliorated ?-synuclein overexpression-induced increase of caspase 3/7 activity. Conclusion: MPP+- and ?-synuclein overexpression-induced cytotoxicity of differentiated LUHMES cells may serve as good alternative systems for identifying neuroprotective compounds for PD. PMID:24989254

  11. Air pollutant production by algal cell cultures

    NASA Technical Reports Server (NTRS)

    Fong, F.; Funkhouser, E. A.

    1982-01-01

    The production of phytotoxic air pollutants by cultures of Chlorella vulgaris and Euglena gracilis is considered. Algal and plant culture systems, a fumigation system, and ethylene, ethane, cyanide, and nitrogen oxides assays are discussed. Bean, tobacco, mustard green, cantaloupe and wheat plants all showed injury when fumigated with algal gases for 4 hours. Only coleus plants showed any resistance to the gases. It is found that a closed or recycled air effluent system does not produce plant injury from algal air pollutants.

  12. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    PubMed

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan

    2015-05-15

    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 ?M DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay. PMID:25577125

  13. Culture of Cells from Amphibian Embryos.

    ERIC Educational Resources Information Center

    Stanisstreet, Martin

    1983-01-01

    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  14. A hybrid microfluidic platform for cell-based assays via diffusive and convective trans-membrane perfusion

    PubMed Central

    Vereshchagina, Elizaveta; Mc Glade, Declan; Glynn, Macdara; Ducrée, Jens

    2013-01-01

    We present a novel 3D hybrid assembly of a polymer microfluidic chip with polycarbonate track-etched membrane (PCTEM) enabling membrane-supported cell culture. Two chip designs have been developed to establish either diffusive or convective reagent delivery using the integrated PCTEM. While it is well suited to a range of cell-based assays, we specifically employ this platform for the screening of a common antitumor chemotoxic agent (mitomycin C – MMC) on the HL60 myeloid leukemia cell line. The toxic activity of MMC is based on the generation of severe DNA damage in the cells. Using either mode of operation, the HL60 cells were cultured on-chip before, during, and after exposure to MMC at concentrations ranging from 0 to 50??M. Cell viability was analysed off-chip by the trypan blue dye exclusion assay. The results of the on-chip viability assay were found to be consistent with those obtained off-chip and indicated ca. 40% cell survival at MMC concentration of 50??M. The catalogue of capabilities of the here described cell assay platform comprises of (i) the culturing of cells either under shear-free conditions or under induced through-membrane flows, (ii) the tight time control of the reagent exposure, (iii) the straightforward assembly of devices, (iv) the flexibility on the choice of the membrane, and, prospectively, (v) the amenability for large-scale parallelization. PMID:24404021

  15. Automation of 3-dimensional cell culture in arrayed microfluidic devices

    PubMed Central

    Montanez-Sauri, Sara I.; Sung, Kyung Eun; Puccinelli, John P.; Pehlke, Carolyn; Beebe, David J.

    2011-01-01

    The increasing interest in studying the interactions between cells and the extracellular matrix (ECM) has created a need for high throughput low cost three-dimensional (3D) culture systems. The recent development of tubeless microfluidics via passive pumping provides a high throughput microchannel culture platform compatible with existing high throughput infrastructures (e.g. automated liquid handlers). Here we build on a previously reported high throughput two-dimensional (2D) system to create a robust automated system for 3D culture. Operational controls including temperature and sample handling have been characterized and automated. Human mammary fibroblasts (HMFs) suspended in type-I collagen are loaded and cultured in microchannel arrays, and used to optimize the system operational parameters. A Peltier cooler maintains the collagen as a liquid at 4°C during cell seeding, followed by polymerization at 37°C. Optimization of this platform is discussed (e.g. controlling collagen contraction, increasing cell viability, preventing the removal of microchannel contents), and 3D distribution of HMFs is examined by fluorescent microscopy. Finally, we validate the platform by automating a previously developed 3D breast carcinoma co-culture assay. The platform allows more efficient 3D culture experiments and lays the foundation for high throughput studies of cell-ECM interactions. PMID:21609700

  16. Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures

    PubMed Central

    Cui, Xiaohong; Lee, Ronzo; Adler, Stuart P.; McVoy, Michael A.

    2013-01-01

    Anti-cytomegalovirus (CMV) antibodies reduce the incidence of CMV transmission and ameliorate the severity of CMV-associated disease. Neutralizing activity, measured as the ability of antibodies to prevent entry of cell-free virus, is an important component of natural immunity. However, in vivo CMV amplification may occur mainly via spread between adjacent cells within tissues. Thus, inhibition of cell-to-cell spread may be important when evaluating therapeutic antibodies or humoral responses to infection or immunization. In vitro CMV cell-to-cell spread is largely resistant to antibodies in fibroblast cultures but sensitive in endothelial cell cultures. In the present study antibodies in CMV hyperimmuneglobulin or seropositive human sera inhibited CMV cell-to-cell spread in epithelial cell cultures. Spread inhibition activity was quantitated with a GFP reporter assay employing GFP-tagged epithelialtropic variants of CMV strains Towne or AD169. Measurement of spread inhibition provides an additional parameter for the evaluation of candidate vaccines or immunotherapeutics and to further characterize the role of antibodies in controlling CMV transmission and disease. PMID:23669101

  17. Label-free cell-based dynamic mass redistribution assays.

    PubMed

    Gitschier, Hannah J; Bergeron, Audrey B; Randle, David H

    2014-01-01

    Label-free cell-based assays offer a powerful approach to drug discovery and compound profiling for endogenously expressed receptors in a variety of cell types, including primary and stem cells. Dynamic mass redistribution (DMR) responses in whole cells following receptor stimulation provide phenotypic activity profiles that are readily amenable to evaluation of compound pharmacology. Protocols are provided in this unit to obtain DMR response profiles in adherent and suspension cells, and then to use known tool compounds to delineate the biology of the underlying signaling pathways from the information-rich kinetic traces that are recorded. PMID:24652622

  18. Embryonic Stem Cells: Isolation, Characterization and Culture

    NASA Astrophysics Data System (ADS)

    Amit, Michal; Itskovitz-Eldor, Joseph

    Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

  19. Progress in Cell Based Assays for Botulinum Neurotoxin Detection

    PubMed Central

    2013-01-01

    Botulinum neurotoxins (BoNTs) are the most potent human toxins known and the causative agent of botulism, and are widely used as valuable pharmaceuticals. The BoNTs are modular proteins consisting of a heavy chain and a light chain linked by a disulfide bond. Intoxication of neuronal cells by BoNTs is a multi-step process including specific cell binding, endocytosis, conformational change in the endosome, translocation of the enzymatic light chain into the cells cytosol, and SNARE target cleavage. The quantitative and reliable potency determination of fully functional BoNTs produced as active pharmaceutical ingredient (API) requires an assay that considers all steps in the intoxication pathway. The in vivo mouse bioassay has for years been the ‘gold standard’ assay used for this purpose, but it requires the use of large numbers of mice and thus causes associated costs and ethical concerns. Cell-based assays are currently the only in vitro alternative that detect fully functional BoNTs in a single assay and have been utilized for years for research purposes. Within the last 5 years, several cell-based BoNT detection assays have been developed that are able to quantitatively determine BoNT potency with similar or greater sensitivity than the mouse bioassay. These assays now offer an alternative method for BoNT potency determination. Such quantitative and reliable BoNT potency determination is a crucial step in basic research, in the development of pharmaceutical BoNTs, and in the quantitative detection of neutralizing antibodies. PMID:23239357

  20. J Cell Biochem . Author manuscript Optimizing stem cell culture

    E-print Network

    Paris-Sud XI, Université de

    J Cell Biochem . Author manuscript Page /1 7 Optimizing stem cell culture Boudewijn Van Der Sanden * Correspondence should be adressed to: Didier Wion Abstract Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical

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

    EPA Science Inventory

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

  2. Standardization of the comet assay technique on FRTL5 cells.

    PubMed

    Francesconi, A; Del Terra, E; Meli, A; Ambesi-Impiombato, F S

    2001-01-01

    The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells. The principle of break detection, using either the alkaline or neutral version of the assay, makes it a good technique for studying both double and single strand DNA breaks. Furthermore, the possibility of following DNA damage at different time moments also makes it possible to investigate the cell repair mechanisms. This explains why in the last few years there has been a tremendous increase in the number of laboratories which started to use this technique. The technique was first created for lymphocyte cells and later on has been used on many other cell types, growing both in suspension and adherent. To date, no one has applied this technique on normal differentiated endocrine cells, such as FRTL5 cells (Fisher Rat Thyroid Cells). The aim of this study has been to standardize the alkaline version of the Comet Assay technique on FRTL5 cells by studying the kinetics of DNA-damage and DNA-repair after different doses of UV-C (254 nm). FRTL-5 cells not only resulted very sensitive to UV-C (p<0.05 at 5 J/m2), but were also able to repair most of their DNA damage very rapidly (within one hour) as shown by a significant exponential regression in comet length. Finally, the successful measurement of biomarkers of UV-C on thyroid cells established the comet assay as a valuable tool in measurement of DNA damage and repair. Any radiation, or other damaging agents, interacting with living organisms could cause DNA damages which, depending upon dosages and kinetics of exposure, may or may not be completely repaired. PMID:11776984

  3. Single-cell growth analysis in a mixed cell culture

    NASA Astrophysics Data System (ADS)

    Ando, Jun; Bato, Mary Grace P.; Daria, Vincent Ricardo

    2008-06-01

    We perform single cell analysis of cell growth in a mixed cell culture. Two species of yeast cells: Saccharomyces cerevisiae and Candida albicans, are optically trapped using focused continuous-wave near infrared laser. Cell growth for both cells is inhibited only when the two species of cells are in contact with each other. This indicates cell-cell interaction mediated cell growth inhibition mechanism. Single cell level analysis of cell growth studied here contributes to the further understanding of yeast growth arrest in a mixed yeast culture.

  4. A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment Culture

    Microsoft Academic Search

    Andrew D. Sails; Andrew J. Fox; Frederick J. Bolton; David R. A. Wareing; David L. A. Greenway

    2003-01-01

    A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately

  5. AMMONIA REMOVAL FROM MAMMALIAN CELL CULTURE MEDIUM

    EPA Science Inventory

    Metabolites such as ammonia and lactic formed during mammalian cell culture can frequently be toxic to the cells themselves beyond a threshold concentration of the metabolites. ell culture conducted in the presence of such accumulated metabolites is therefore limited in productiv...

  6. Gametogony of Sarcocystis sp. in Cell Culture

    Microsoft Academic Search

    Ronald Fayer

    1972-01-01

    Sexual stages and cystlike bodies of Sarcocystis sp., a protozoan parasite found in muscles of reptiles, birds, and mammals, including man, developed in cell culture. Motile organisms, obtained from leg muscles of wild grackles, were inoculated into cell line cultures of embryonic bovine kidney. Mature micro- and macrogametes and the cystlike forms were found 30 and 42 hours after inoculation,

  7. Assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling

    SciTech Connect

    Prins, A.P.; Kiljan, E.; v.d. Stadt, R.J.; v.d. Korst, J.K.

    1986-02-01

    A method is described for determination of inorganic pyrophosphate (PPi) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate (/sup 32/Pi). Intra- and extracellular /sup 32/PPi formed was measured using high-performance liquid chromatographic (HPLC) separation of the PPi from orthophosphate (Pi) and other phosphate-containing compounds. The chromatographic separation on a weak anion-exchange column is based on the extent to which various phosphate compounds form complexes with Mg2+ at low pH and the rate at which such formation occurs. These complexes are eluted more readily than the uncomplexed compounds. Best results were obtained using a simultaneous gradient of Mg2+ ions and ionic strength. In this case separation of small amounts of PPi from a large excess of Pi was possible without prior removal of Pi or extraction of the PPi fraction. The assay is also useful for measurement of inorganic pyrophosphatase activity. The sensitivity of the assay depends on the specific activity of the added /sup 32/Pi and on the culture conditions, but is comparable with the most sensitive of the enzymatic assays. Sample preparation, particularly deproteinization, proved to be of importance. The losses of PPi which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated.

  8. Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

    PubMed Central

    Ribbens, Jameson; Zheng, Wei; Southall, Noel; Hu, Xin; Marugan, Juan J.; Ferrer, Marc; Maegawa, Gustavo H. B.

    2011-01-01

    Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets. PMID:22216298

  9. EVALUATION OF MIXED CELL TYPES AND 5-IODO-2'-DEOXYURIDINE TREATMENT UPON PLAQUE ASSAY TITERS OF HUMAN ENTERIC VIRUSES (JOURNAL VERSION)

    EPA Science Inventory

    Four continuous cell lines BGM, L-132, HEL-299, and RD were compared both when cultured separately and as mixtures for use in plaque assay titrations of human Adenovirus 1 and six human enterovirus serotypes. The effect of incubating these cell cultures in media containing IDU (5...

  10. MAMMALIAN CELL GENE MUTATION ASSAYS WORKING GROUP REPORT

    EPA Science Inventory

    Mammalian cell gene mutation assays have been used for many years and the diversity of the available systems attests to the varied methods found to grow mammalian dells and detect mutations. s part of the International Workshop on Standardization of Genotoxicity Test Procedures, ...

  11. Cancer Phylogenetics from Single-Cell Assays Gregory Pennington

    E-print Network

    Cancer Phylogenetics from Single-Cell Assays Gregory Pennington Stanley Shackney Russell Schwartz the Carnegie Mellon University Berkman Faculty Development Fund. #12;Keywords: computational biology, cancer, FISH, phylogeny #12;Abstract In the field of cancer biology, there is currently great interest

  12. Mammalian cell cultures for biologics manufacturing.

    PubMed

    Kantardjieff, Anne; Zhou, Weichang

    2014-01-01

    Biopharmaceuticals represent a growing sector of the pharmaceutical industry, and are used for a wide range of indications, including oncology and rheumatology. Cultured mammalian cells have become the predominant expression system for their production, partly due to their ability to complete the posttranslational modifications required for drug safety and efficacy. Over the past decade, the productivity of mammalian cell culture production processes has growth dramatically through improvements in both volumetric and specific productivities. This article presents an overview of the biologics market, including analysis of sales and approvals; as well as a review of industrial production cell lines and cell culture operations. PMID:24258145

  13. HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE

    PubMed Central

    Gimbrone, Michael A.; Cotran, Ramzi S.; Folkman, Judah

    1974-01-01

    Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [3H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [3H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [3H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration. PMID:4363161

  14. Longterm cultures of sheep thyroid cells.

    PubMed

    O'Connor, M K; Malone, J F; Cullen, M J

    1980-01-01

    A thyroid tissue culture system has been established in which follicular morphology can be preserved for at least 30 days. The system is highly dependent on whether or not the medium supporting the cells is changed during the culture period. The high levels of TSH (40 mU/ml) normally used in thyroid culture systems enhance follicular morphology but are not a prerequisite for differentiation. In the absence of medium changes follicular morphology improves for up to 20 days after initiation of the culture. Thereafter, the cells die unless the medium is changed. If differentiation is to be preserved after 20 days the medium into which the cells are transferred must be "conditioned" by preincubation with thryoid cultures. Regular medium changes into fresh medium causes the cultures to lose their differentiated characteristics and revert to a conventional monolayer. The capacity of these cultures to trap iodide has been quantified using a new method. The method is based on comparison of the 125I--iodide retained by thyroid cells with that retained in a undifferentiated established cell line (CHO--K1). The results demonstrated that trapping can be preserved for at least 20 days in cells cultured in the presence of TSH, provided the medium is not changed; and that under appropriate conditions the cells can trap iodide even in the absence of TSH stimulation. The extent to which the above cultures proliferate is also investigated. At the relatively high innoculation of cells used in primary cultures little proliferation takes place even when the cells are stimulated by TSH. However, regular medium changed induce some growth. In the absence of medium changes the cells die after 15-20 days. Those grown in the presence of TSH live slightly longer than those grown in its absence. Subculturing thyroid cells and innoculating them at low densities in Petri dishes leads to substantial cell proliferation whether or not TSH is present. The doubling time of cells in these cultures is the order of 1 to 2 days. The population resulting from this growth exhibit both epithelial and fibroblast like morphology although the former predominates. When cells from primary thryoid cultures are reseeded at very low concentrations (approximately 10(3) cells/Petri dish) about 3-10 per cent of the population give rise to viable macroscopic clones. PMID:6996408

  15. Emulsions Containing Perfluorocarbon Support Cell Cultures

    NASA Technical Reports Server (NTRS)

    Ju, Lu-Kwang; Lee, Jaw Fang; Armiger, William B.

    1990-01-01

    Addition of emulsion containing perfluorocarbon liquid to aqueous cell-culture medium increases capacity of medium to support mammalian cells. FC-40 Fluorinert (or equivalent) - increases average density of medium so approximately equal to that of cells. Cells stay suspended in medium without mechanical stirring, which damages them. Increases density enough to prevent cells from setting, and increases viscosity of medium so oxygen bubbled through it and nutrients stirred in with less damage to delicate cells.

  16. Constructing a High Density Cell Culture System

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor)

    1996-01-01

    An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

  17. Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues

    Microsoft Academic Search

    Valeria M. Rodas; Fabiano H. Marques; Marcelo T. Honda; Daniela M. Soares; Soraia A. C. Jorge; Marta M. Antoniazzi; Claudia Medugno; Maria E. B. Castro; Bergmann M. Ribeiro; Marlinda L. Souza; Aldo Tonso; Carlos A. Pereira

    2005-01-01

    We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors.\\u000a We have assayed the kLa of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients\\u000a consumption, and

  18. Integrating live cells with semiconductor devices: A biocompatibility assay.

    PubMed

    Cioffi, M; Giordano, C; Gusmeroli, R; Raimondi, M T; Spinelli, A S; Baranauskas, G

    2005-01-01

    There is increasing interest in the development of small hybrid cell-semiconductor systems for the non-invasive evaluation of the physiological state of a cell population. These miniature devices can be used in many areas of biomedical applications, ranging from basic research to drug screening during cancer chemosensitivity testing in clinics. A prerequisite for the biological and medical application of these devices is that cells retain their functional and growth properties when in contact with the semiconductor sensor material. The sensor surface is usually coated with dielectric silicon dioxide (SiO2 ) or a silicon nitride layer (Si3 N4 ); therefore, cellular adhesion to these materials and cellular viability on these surfaces are of crucial im-portance. This is especially true for bone cells that are sensitive to the surface microstructure. Therefore, we investigated the short-term (1-7 days) behavior of model bone cells (MG63 human osteosarcoma cells) grown on silicon samples coated with SiO2 . Cell adhesion and morphology were evaluated by scanning electron microscopy (SEM) 1 day after seeding and cell pro-liferation was evaluated by Alamar Blue assay at 2, 3 and 7 days after seeding. No adverse cellular reactions could be detected with these assays suggesting that the tested substrate is suitable for the hybrid cell-semiconductor systems that test bone tumor chemosensitivity. PMID:20799231

  19. Evaluation of four cell lines for assay of infectious adenoviruses in water samples.

    PubMed

    Jiang, Sunny C; Han, Jijun; He, Jian-Wen; Chu, Weiping

    2009-12-01

    Human viral contamination in drinking and recreational waters poses health risks. The application of PCR-based molecular technology has advanced our knowledge of the occurrence and prevalence of human viruses in water; however, it has provided no information on viral viability and infectivity. Four human cell lines were compared for their sensitivity to different serotypes of human adenoviruses using the TCID50 test. The sensitivity of each cell line varied with different serotypes of adenovirus. Human embryonic kidney cell line 293A and human lung carcinoma cell line A549 were the most sensitive, especially to enteric adenovirus 40 and 41. Plaque assay of primary sewage samples showed 293A can detect viral plaques in 7 of 13 primary sewage samples tested. Adenoviruses were also isolated using 293A from environmental water concentrates. Cloning and sequencing of environmental adenoviral isolates indentified them to be aligned with adenoviruses serotype 40 and serotype 5. The result of this study suggests that plaque assay with 293A cell line is suitable for detection of adenovirus in the aquatic environment. Combining this cell culture with molecular methods for viral assay in the aquatic environment will provide critical information for risk assessment. PMID:19590132

  20. Culture of Rodent Spermatogonial Stem Cells, Male Germline Stem Cells of the Postnatal Animal

    PubMed Central

    Kubota, Hiroshi; Brinster, Ralph L.

    2014-01-01

    Spermatogonial stem cells (SSCs), postnatal male germline stem cells, are the foundation of spermatogenesis, during which an enormous number of spermatozoa is produced daily by the testis throughout life of the male. SSCs are unique among stem cells in the adult body because they are the only cells that undergo self-renewal and transmit genes to subsequent generations. In addition, SSCs provide an excellent and powerful model to study stem cell biology because of the availability of a functional assay that unequivocally identifies the stem cell. Development of an in vitro culture system that allows an unlimited supply of SSCs is a crucial technique to manipulate genes of the SSC to generate valuable transgenic animals, to study the self-renewal mechanism, and to develop new therapeutic strategies for infertility. In this chapter, we describe a detailed protocol for the culture of mouse and rat SSCs. A key factor for successful development of the SSC culture system was identification of in vitro growth factor requirements for the stem cell using a defined serum-free medium. Because transplantation assays using immunodeficient mice demonstrated that extrinsic factors for self-renewal of SSCs appear to be conserved among many mammalian species, culture techniques for SSCs of other species, including farm animals and humans, are likely to be developed in the coming 5–10 years. PMID:18442644

  1. Hawthorn ( Crataegus monogyna Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay

    Microsoft Academic Search

    Satin Salehi; Shannon R. Long; Philip J. Proteau; Theresa M. Filtz

    2009-01-01

    Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases.\\u000a Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine\\u000a cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the\\u000a effect of hawthorn extract to

  2. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

  3. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

  4. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

  5. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

  6. 21 CFR 864.2280 - Cultured animal and human cells.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

  7. Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB/c 3T3 cell transformation assay.

    PubMed

    Muramatsu, Dai; Sasaki, Kiyoshi; Kuroda, Sachiko; Hayashi, Kumiko; Tanaka, Noriho; Sakai, Ayako

    2009-04-30

    A short-term cell transformation assay has recently been developed, using Bhas 42 cells which were established from BALB/c 3T3 cells transfected by v-Ha-ras gene and postulated to be initiated in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay has been reported to be capable of detecting initiating and promoting activities of chemical carcinogens, according to the different protocols, initiation assay and promotion assay, respectively. The assay is superior to classical transformation assays in cost and labor performance. The present study was carried out to compare its sensitivity with that of a classical BALB/c 3T3 cell system. We performed the Bhas 42 cell transformation assay with inorganic arsenic compounds which are potent environmental carcinogens in human but not mutagens in bacteria or weak mutagens in mammalian cells in vitro. Sodium arsenite, disodium arsenate, and their metabolites, monomethylarsonic acid and dimethylarsinic acid (DMAA) were included in the study. Sodium arsenite was positive in the initiation assay and all compounds except for DMAA were positive in the promotion assay. These results were compared with reported data in a two-stage BALB/c 3T3 cell transformation assay. The sensitivity of Bhas 42 cell transformation assay was found to be similar to that of the conventional BALB/c 3T3 cell transformation assay for the detection of initiating activities of arsenic compounds. For the detection of promoting activities, its sensitivity was equivalent to that of the two-stage BALB/c 3T3 cell transformation assay where the target cells were initiated with sub-threshold dose of 3-methylcholanthrene, confirming that Bhas 42 cells behave as initiated cells in the transformation assay. PMID:19386250

  8. Comparison of the illumigene Mycoplasma DNA Amplification Assay and Culture for Detection of Mycoplasma pneumoniae

    PubMed Central

    Ratliff, Amy E.; Duffy, Lynn B.

    2014-01-01

    A loop-mediated isothermal amplification (LAMP) system, the illumigene Mycoplasma DNA amplification assay (Meridian Bioscience, Inc., Cincinnati, OH) was evaluated to determine its analytical sensitivity, specificity, and clinical application in comparison to historic culture in a collection of archived respiratory specimens. The illumigene limit of detection was ?88 CFU/reaction for 10 Mycoplasma pneumoniae reference strains. This assay correctly identified 36 M. pneumoniae reference strains and clinical isolates from various geographic origins, including both of the main subtypes. No cross-reactions were detected with other mycoplasmas, ureaplasmas, other bacterial species, viruses, yeasts, or human DNA. Among 214 respiratory specimens previously cultured for M. pneumoniae, when real-time PCR with bidirectional sequencing of the PCR products was used to resolve discrepancies, the sensitivity was 22 of 22 (100%) and the specificity was 190 of 192 (99%). This commercial LAMP assay is a useful rapid method for detecting M. pneumoniae in clinical specimens. Additional prospective clinical trials with direct comparison to culture and PCR are warranted. PMID:24430454

  9. Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

    PubMed

    Webb, Sarah J; Zychowski, Gregory V; Bauman, Sandy W; Higgins, Benjamin M; Raudsepp, Terje; Gollahon, Lauren S; Wooten, Kimberly J; Cole, Jennifer M; Godard-Codding, Céline

    2014-12-16

    Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 ?M following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 ?M benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells. PMID:25384208

  10. Bioprocessing technology for plant cell suspension cultures

    Microsoft Academic Search

    Wei wen Su

    1995-01-01

    Considering various forms of in vitro plant tissue cultures, cell suspension culture is most amenable to large-scale production\\u000a of natural compounds, owing primarily to its superior culture homogeneity. This fact has already been demonstrated in several\\u000a largescale applications, including the commercial shikonin process. The scope of this work is to review the state of the art\\u000a in bioprocessing technologies pertinent

  11. An ELISA assay for cytochrome P4501A in fish liver cells

    SciTech Connect

    Brueschweiler, B.J.; Fent, K. [Swiss Federal Inst. for Environmental Science and Technology, Duebendorf (Switzerland)]|[Swiss Federal Inst. of Technology Zuerich, Duebendorf (Switzerland); Wuergler, F.E. [Swiss Federal Inst. of Tech., Schwerzenbach (Switzerland). Inst. of Toxicology]|[Univ. of Zuerich, Schwerzenbach (Switzerland)

    1996-04-01

    An enzyme-linked immunosorbent assay (ELISA) for measuring cytochrome P4501A (CYP1A) expression in vitro in fish hepatoma cells is described. Cells were cultured as monolayers in 96-microwell cell culture plates and exposed to polychlorinated biphenyl (PCB) congeners 77, 105, 153, and 169; 3-methylcholanthrene (3-MC), and {beta}-naphthoflavone (BNF) for 3 d. Relative CYP1A protein content, CYP1A enzymatic activity, and total protein content were determined directly within the wells. At low concentrations of PCB 77, PCB 169, and 3-MC, the ethoxyresorufin-O-deethylase (EROD) activity was induced, but it was inhibited at high concentrations of these compounds. However, CYP1A protein content measured in an ELISA performed with intact cells increased monotonically in response to the concentration. No CYP1A induction was observed for PCB 105 and PCB 153. Because comparison between EROD activity and CYP1A amount gives information about the catalytic efficiency of CYP1A in the cells, this noncompetitive, solid-phase ELISA is recommended as a complementary method to the EROD assay. This novel ELISA method may be an accurate in vitro technique for a rapid and sensitive screening of CYP1A-inducible compounds.

  12. Rotating cell culture systems for human cell culture: human trophoblast cells as a model.

    PubMed

    Zwezdaryk, Kevin J; Warner, Jessica A; Machado, Heather L; Morris, Cindy A; Höner zu Bentrup, Kerstin

    2012-01-01

    The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines support our understanding of invasion of the uterine wall and remodeling of uterine spiral arteries by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS. The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies. The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu. We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS. PMID:22297395

  13. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    SciTech Connect

    Walter, M.N.M. [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom) [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom); School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom); Wright, K.T.; Fuller, H.R. [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom)] [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom); MacNeil, S. [Kroto Research Institute and Centre for Nanoscience and Technology, Sheffield University, Sheffield, S1 2UE (United Kingdom)] [Kroto Research Institute and Centre for Nanoscience and Technology, Sheffield University, Sheffield, S1 2UE (United Kingdom); Johnson, W.E.B., E-mail: w.e.johnson@aston.ac.uk [School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom)] [School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom)

    2010-04-15

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-{beta}1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  14. Stamp wound assay for studying coupled cell migration and cell debris clearance.

    PubMed

    Lee, Jiyeon; Wang, Yu-Lin; Ren, Fan; Lele, Tanmay P

    2010-11-16

    A new method for studying wound healing under realistic conditions in vitro was developed. The method involves creating defined patterns of damaged cell debris with poly(dimethyl)siloxane (PDMS) stamping. This novel assay permitted the quantification of wound healing rates in the presence of cell debris. Experimental results with this assay suggest that cell migration in the presence of cell debris is a two step process requiring (1) non-muscle myosin II-dependent cell clearance followed by (2) cell migration into newly cleared wound areas. The novel stamp wound assay allows the study of coupled cell migration and debris clearance and is a more realistic wound healing assay in vitro. PMID:20961056

  15. Differentiation of mouse iPS cells into ameloblast-like cells in cultures using medium conditioned by epithelial cell rests of Malassez and gelatin-coated dishes.

    PubMed

    Yoshida, Koki; Sato, Jun; Takai, Rie; Uehara, Osamu; Kurashige, Yoshihito; Nishimura, Michiko; Chiba, Itsuo; Saitoh, Masato; Abiko, Yoshihiro

    2014-10-16

    Induced pluripotent stem (iPS) cells are generated from adult cells and are potentially of great value in regenerative medicine. Recently, it was shown that iPS cells can differentiate into ameloblast-like cells in cultures using feeder cells. In the present study, we sought to induce differentiation of ameloblast-like cells from iPS cells under feeder-free conditions using medium conditioned by cultured epithelial cell rests of Malassez (ERM) cells and gelatin-coated dishes. Two culture conditions were compared: co-cultures of iPS cells and ERM cells; and, culture of iPS cells in ERM cell-conditioned medium. Differentiation of ameloblast-like cells in the cultures was assessed using real-time RT-PCR assays of expression of the marker genes keratin 14, amelogenin, and ameloblastin and by immunocytochemical staining for amelogenin. We found greater evidence of ameloblast-like cell differentiation in the cultures using the conditioned medium. In the latter, the level of amelogenin expression increased daily and was significantly higher than controls on the 7th, 10th, and 14th days. Expression of ameloblastin also increased daily and was significantly higher than controls on the 14th day. The present study demonstrates that mouse iPS cells can be induced to differentiate into ameloblast-like cells in feeder-free cell cultures using ERM cell-conditioned medium and gelatin-coated dishes. PMID:25319805

  16. Effects of several salt marsh plants on mouse spleen and thymus cell proliferation using mtt assay

    NASA Astrophysics Data System (ADS)

    Seo, Youngwan; Lee, Hee-Jung; Kim, You Ah; Youn, Hyun Joo; Lee, Burm-Jong

    2005-12-01

    In the present study, we have tested the effects of 21 salt marsh plants on cell proliferation of mouse immune cells (spleen and thymus) using MTT assay in culture. The methanolic extracts of six salt marsh plants ( Rosa rugosa, Ixeris tamagawaensis, Artemisia capillaris, Tetragonia tetragonoides, Erigeron annus, and Glehnia littoralis) showed very powerful suppressive effects of mouse immune cell death and significant activities of cell proliferation in vitro. Especially, the methanolic extract of Rosa rugosa was found to have fifteen times compared to the control treatment, demonstrating that Rosa rugosa may have a potent stimulation effect on immune cell proliferation. These results suggest that several salt marsh plants including Rosa rugosa could be useful for further study as an immunomodulating agent.

  17. Culture and Manipulation of Embryonic Cells

    PubMed Central

    Edgar, Lois G.; Goldstein, Bob

    2012-01-01

    The direct manipulation of embryonic cells is an important tool for addressing key questions in cell and developmental biology. C. elegans is relatively unique among genetic model systems in being amenable to manipulation of embryonic cells. Embryonic cell manipulation has allowed the identification of cell interactions by direct means, and it has been an important technique for dissecting mechanisms by which cell fates are specified, cell divisions are oriented, and morphogenesis is accomplished. Here, we present detailed methods for isolating, manipulating and culturing embryonic cells of C. elegans. PMID:22226523

  18. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    PubMed

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases. PMID:25502925

  19. SNP panel identification assay (SPIA): a genetic-based assay for the identification of cell lines

    PubMed Central

    Demichelis, Francesca; Greulich, Heidi; Macoska, Jill A.; Beroukhim, Rameen; Sellers, William R.; Garraway, Levi; Rubin, Mark A.

    2008-01-01

    Translational research hinges on the ability to make observations in model systems and to implement those findings into clinical applications, such as the development of diagnostic tools or targeted therapeutics. Tumor cell lines are commonly used to model carcinogenesis. The same tumor cell line can be simultaneously studied in multiple research laboratories throughout the world, theoretically generating results that are directly comparable. One important assumption in this paradigm is that researchers are working with the same cells. However, recent work using high throughput genomic analyses questions the accuracy of this assumption. Observations by our group and others suggest that experiments reported in the scientific literature may contain pre-analytic errors due to inaccurate identities of the cell lines employed. To address this problem, we developed a simple approach that enables an accurate determination of cell line identity by genotyping 34 single nucleotide polymorphisms (SNPs). Here, we describe the empirical development of a SNP panel identification assay (SPIA) compatible with routine use in the laboratory setting to ensure the identity of tumor cell lines and human tumor samples throughout the course of long term research use. PMID:18304946

  20. Cell Culture on MEMS Platforms: A Review

    E-print Network

    Ni, Ming

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods ...

  1. Effect of plasma needle on cultured cells

    Microsoft Academic Search

    I. E. Kieft; N. A. Dvinskikh; Jos L. V. Broers; Dick W. Slaaf; Eva Stoffels

    2004-01-01

    To investigate a possible application of plasma in fine surgery, we studied the effects of a small atmospheric glow discharge on living cultured cells. The plasma source used for this purpose was the \\

  2. Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting.

    PubMed

    Bausinger, Julia; Speit, Günter

    2014-11-01

    The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells. PMID:25771724

  3. Cell-free Assays for HIV-1 Uncoating

    PubMed Central

    Aiken, Christopher

    2013-01-01

    Summary/Abstract Uncoating is an essential step in the retrovirus life cycle about which little is known. Uncoating is defined as the specific dissociation of the capsid shell from the viral core in the host cell cytoplasm. In this chapter, biochemical assays for studying HIV-1 uncoating in vitro are described. These techniques have proven useful for characterizing HIV-1 mutants that exhibit defects in the uncoating step of infection. PMID:19020817

  4. An improved method for staining cell colonies in clonogenic assays

    PubMed Central

    Natale, Leanna; Markowitz, Sanford D.

    2007-01-01

    Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale clonogenic experiments. PMID:19003022

  5. Cell assay using a two-photon-excited europium chelate

    PubMed Central

    Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

    2011-01-01

    We report application of two-photon excitation of europium chelates to immunolabeling of epidermal growth factor receptor (EGFR) cell surface proteins on A431 cancer cells. The europium chelates are excited with two photons of infrared light and emit in the visible. Europium chelates are conjugated to antibodies for EGFR. A431 (human epidermoid carcinoma) cells are labeled with this conjugate and imaged using a multiphoton microscope. To minimize signal loss due to the relatively long-lived Eu3+ emission, the multiphoton microscope is used with scanning laser two-photon excitation and non-scanning detection with a CCD. The chelate labels show very little photobleaching (less than 1% during continuous illumination in the microscope for 20 minutes) and low levels of autofluorescence (less than 1% of the signal from labeled cells). The detection limit of the europium label in the cell assay is better than 100 zeptomoles. PMID:21833362

  6. Impact of static magnetic fields on human myoblast cell cultures.

    PubMed

    Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Kassner, Stefan S; Faber, Anne; Sauter, Alexander; Schulz, Johannes D; Hörmann, Karl; Kinscherf, Ralf; Goessler, Ulrich Reinhart

    2011-12-01

    Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle ?1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal muscle tissue engineering approaches. PMID:21837362

  7. White Blood Cell-Based Detection of Asymptomatic Scrapie Infection by Ex Vivo Assays

    PubMed Central

    Halliez, Sophie; Jaumain, Emilie; Huor, Alvina; Douet, Jean-Yves; Lugan, Séverine; Cassard, Hervé; Lacroux, Caroline; Béringue, Vincent; Andréoletti, Olivier; Vilette, Didier

    2014-01-01

    Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods - in vitro, ex vivo and in vivo assays - to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA) and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages). However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease. PMID:25122456

  8. Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells

    PubMed Central

    2013-01-01

    Background Huntington’s disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD. Results An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course. Conclusions The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials. PMID:24274906

  9. Immunological and virological studies of cultured labial biopsy cells from patients with Sjögren's syndrome

    PubMed Central

    Cremer, Natalie E.; Daniels, T. E.; Oshiro, L. S.; Marcus, F.; Claypool, R.; Sylvester, R. A.; Talal, N.

    1974-01-01

    Labial salivary gland tissues from twenty-five patients were cultured in vitro for virus studies and for use as target cells in cellular and antibody-mediated cytotoxicity assays. Fourteen patients had definite Sjögren's Syndrome (SS), four had possible SS and seven did not have SS. No evidence for the presence of a virus in the cultured cells or after chemical treatment of the cultured cells was obtained. Tubuloreticular structures were present in three of the original biopsies but were not seen in the corresponding cultured cells, although in two of these cell lines rare bundles of intranuclear microfibrils occurred. The significance of these structures is unknown. Autologous serum and autologous lymphocytes were not cytotoxic for the cultured cells. ImagesFig. 1Fig. 2 PMID:4468195

  10. A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

    PubMed Central

    2010-01-01

    Background Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase) is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine)-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU) and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here. Results Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice. Conclusions In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems. PMID:20181034

  11. Quasi-spherical microwells on superhydrophobic substrates for long term culture of multicellular spheroids and high throughput assays.

    PubMed

    Liu, Tianqing; Winter, Marnie; Thierry, Benjamin

    2014-07-01

    Multicellular tumour spheroids closely recapitulate the physiological environment of tumour tissues. However, their implementation in drug screening assays remains limited due to the technological challenges of forming large numbers of high quality spheroids in platforms compatible with high throughput screening. A simple bench-top microfabrication strategy is demonstrated here based on the principle of ice lithography carried out on superhydrophobic substrates to fabricate quasi-spherical microwells (spheriwells). The microwells shapes and dimensions are directly controlled by the hydrophobicity of the substrate and the volume of the water droplets. The prepared concave microwells enable the formation of dense and homogeneous multicellular tumour spheroids. Spheroids formed within spheriwells are trapped within the microwells, which eliminate loss during media manipulation and facilitate long-term on-chip culture. Morphological and phenotypical changes associated with the growth of MCF-7 adenocarcinoma cells in spheriwells were characterised using imaging flow cytometry and revealed the appearance of heterogeneous populations with loss of E-Cadherin expression. The compatibility of the spheriwells with an on-chip MTT assay is demonstrated. The very unusual shape of the spheriwells, prepared using materials and methods routinely used in most research laboratories, provides a straightforward and scalable platform to prepare high quality multicellular tumour spheroids compatible with high throughput biological screening assays. PMID:24797879

  12. Detection of DNA damage in individual cells from marine organisms using the single cell gel assay

    Microsoft Academic Search

    Diane E. Nacci; Stephanie Cayula; Eugene Jackim

    1996-01-01

    The single cell gel (SCG) or comet assay is a simple method by which DNA damage is expressed as relative nuclear ‘tail’ length of gel-embedded cells following alkaline electrophoresis. While potentially applicable to any cell type, laboratory experiments were conducted to examine the utility of the SCG method for the detection of genotoxicity in cells of marine fish and invertebrates.

  13. Homogeneous Cell and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

    Microsoft Academic Search

    Sheri Miraglia; Elana E. Swartzman; Julia Mellentin-Michelotti; Lolita Evangelista; Christopher Smith; Iwan Gunawan; Kenton Lohman; Edward M. Goldberg; Bala Manian; Pau-Miau Yuan

    1999-01-01

    High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay

  14. Genotoxicity of complex mixtures: CHO cell mutagenicity assay

    SciTech Connect

    Frazier, M.E.; Samuel, J.E.

    1985-02-01

    A Chinese hamster ovary (CHO) mammalian cell assay was used to evaluate the genotoxicity of complex mixtures (synthetic fuels). The genotoxicity (mutagenic potency) of the mixtures increased as the temperature of their boiling range increased. Most of the genotoxicity in the 750/sup 0/F+ boiling-range materials was associated with the neutral polycyclic aromatic hydrocarbon (PAH) fractions. Chemical analysis data indicate that the PAH fractions of high-boiling coal liquids contain a number of known chemical carcinogens, including five- and six-ring polyaromatics (e.g., benzo(a)pyrene) as well as four- and five-ring alkyl-substituted PAH (e.g., methylchrysene and dimethylbenzanthracenes); concentrations are a function of boiling point (bp). In vitro genotoxicity was also detected in fractions of nitrogen-containing polyaromatic compounds, as well as in those with aliphatics of hydroxy-containing PAH. Mutagenic activity of some fractions was detectable in the CHO assay in the absence of an exogenous metabolic activation system; in some instances, addition of exogenous enzymes and cofactors inhibited expression of the direct-acting mutagenic potential of the fraction. These data indicate that the organic matrix of the chemical fraction determines whether, and to what degree, various mutagens are expressed in the CHO assay. Therefore, the results of biological assays of these mixtures must be correlated with chemical analyses for proper interpretation of these data. 29 references, 16 figures, 4 tables.

  15. Establishment and Characterization of a Madin-Darby Canine Kidney Reporter Cell Line for Influenza A Virus Assays?

    PubMed Central

    Hossain, M. Jaber; Perez, Sandra; Guo, Zhu; Chen, Li-Mei; Donis, Ruben O.

    2010-01-01

    Influenza virus diagnosis has traditionally relied on virus isolation in chicken embryo or cell cultures. Many laboratories have adopted rapid molecular methods for detection of influenza viruses and discontinued routine utilization of the relatively slow viral culture methods. We describe an influenza A virus reporter cell line that contributes to more efficient viral detection in cell culture. Madin-Darby canine kidney (MDCK) cells were engineered to constitutively produce an influenza virus genome-like luciferase reporter RNA driven by the canine RNA polymerase I promoter. Induction of a high level of luciferase activity was detected in the Luc9.1 cells upon infection with various strains of influenza A virus, including 2009 H1N1 pandemic and highly pathogenic H5N1 virus. In contrast, infection with influenza B virus or human adenovirus type 5 did not induce significant levels of reporter expression. The reporter Luc9.1 cells were evaluated in neutralizing antibody assays with convalescent H3N2 ferret serum, yielding a neutralization titer comparable to that obtained by the conventional microneutralization assay, suggesting that the use of the reporter cell line might simplify neutralization assays by facilitating the establishment of infectious virus endpoints. Luc9.1 cells were also used to determine the susceptibility of influenza A viruses to a model antiviral drug. The equivalence to conventional antiviral assay results indicated that the Luc9.1 cells could provide an alternative cell-based platform for high-throughput drug discovery screens. In summary, the MDCK-derived Luc9.1 reporter cell line is highly permissive for influenza A virus replication and provides a very specific and sensitive approach for simultaneous detection and isolation of influenza A viruses as well as functional evaluation of antibodies and antiviral molecules. PMID:20504984

  16. Illuminations: A Cell Notes Publication | January 2014 32 Illuminations: A Cell Notes Publication | January 2014 How to Choose a Cell Health Assay

    E-print Network

    Cai, Long

    a signal. This approach, exemplified by the CellTiter-FluorTM Cell Viability Assay, is homogeneous, more | January 2014 How to Choose a Cell Health Assay Choosing the Right Cell Health Assay Depends on What You Want to Measure by Terry Riss Introduction Promega has a large portfolio of cell health assays, which

  17. Infrared Spectroscopy of Plant Cell Cultures 1

    PubMed Central

    Sowa, Sharon; Towill, Leigh E.

    1991-01-01

    Infrared spectroscopy was used to examine suspension-cultured pear (Pyrus communis L.) and Spartina pectinata cells. Noninvasive measurements were made using internal reflectance sampling. Spectra of actively growing cells exhibited a pronounced absorbance at 2343 reciprocal centimeters. The absorbance peak was identified and verified as CO2 dissolved in water. This peak was absent in nonviable cells. Peak height was directly proportional to percent viability in artificial mixtures of viable and nonviable cells, indicating that the level of intracellular CO2 production could be used as a viability determinant for plant cells. Suspension-cultured cells were slowly cooled to subzero temperatures and analyzed for viability using infrared spectroscopy and tetrazolium staining. Both methods showed similar trends in viability assessment. Infrared spectroscopy could provide a more detailed understanding of cell viability and allow measurement on a noninvasive basis. PMID:16668026

  18. Use of a Panfungal PCR Assay for Detection of Fungal Pathogens in a Commercial Blood Culture System

    PubMed Central

    Iwen, Peter C.; Freifeld, Alison G.; Bruening, Tricia A.; Hinrichs, Steven H.

    2004-01-01

    A panfungal PCR assay was used to evaluate the ability of the ESP blood culture system to detect fungemia. The results showed that the ESP system is reliable for the detection of fungi and showed the applicability of using a molecular-based assay as a potential rapid and reliable method for the identification of fungi. PMID:15131216

  19. Use of a panfungal PCR assay for detection of fungal pathogens in a commercial blood culture system.

    PubMed

    Iwen, Peter C; Freifeld, Alison G; Bruening, Tricia A; Hinrichs, Steven H

    2004-05-01

    A panfungal PCR assay was used to evaluate the ability of the ESP blood culture system to detect fungemia. The results showed that the ESP system is reliable for the detection of fungi and showed the applicability of using a molecular-based assay as a potential rapid and reliable method for the identification of fungi. PMID:15131216

  20. Cell transformation assays: are we barking up the wrong tree?

    PubMed

    Combes, Robert D

    2012-05-01

    There has been a current resurgence of interest in the use of cell transformation for predicting carcinogenicity, which is based mainly on rodent carcinogenicity data. In view of this renewed interest, this paper critically reviews the published literature concerning the ability of the available assays to detect IARC Group 1 agents (known human carcinogens) and Group 2A agents (probable human carcinogens). The predictivity of the available assays for human and rodent non-genotoxic carcinogens (NGCs), in comparison with standard and supplementary in vitro and in vivo genotoxicity tests, is also discussed. The principal finding is that a surprising number of human carcinogens have not been tested for cell transformation across the three main assays (SHE, Balb/c 3T3 and C3H10T1/2), confounding comparative assessment of these methods for detecting human carcinogens. This issue is not being addressed in the ongoing validation studies for the first two of these assays, despite the lack of any serious logistical issues associated with the use of most of these chemicals. In addition, there seem to be no plans for using exogenous bio-transformation systems for the metabolic activation of pro-carcinogens, as recommended in an ECVAM workshop held in 1999. To address these important issues, it is strongly recommended that consideration be given to the inclusion of more human carcinogens and an exogenous source of xenobiotic metabolism, such as an S9 fraction, in ongoing and future validation studies. While cell transformation systems detect a high level of NGCs, it is considered premature to rely only on this endpoint for screening for such chemicals, as recently suggested. This is particularly important, in view of the fact that there is still doubt as to the relevance of morphological transformation to tumorigenesis in vivo, and the wide diversity of potential mechanisms by which NGCs are known to act. Recent progress with regard to increasing the objectivity of scoring the transformed phenotype, and prospects for developing human cell-based transformation assays, are reviewed. PMID:22762196

  1. Human cell culture in a space bioreactor

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.

    1988-01-01

    Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

  2. Cell culture experiments planned for the space bioreactor

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.; Cross, John H.

    1987-01-01

    Culturing of cells in a pilot-scale bioreactor remains to be done in microgravity. An approach is presented based on several studies of cell culture systems. Previous and current cell culture research in microgravity which is specifically directed towards development of a space bioprocess is described. Cell culture experiments planned for a microgravity sciences mission are described in abstract form.

  3. Pitfalls in cell culture work with xanthohumol.

    PubMed

    Motyl, M; Kraus, B; Heilmann, J

    2012-01-01

    Xanthohumol, the most abundant prenylated chalcone in hop (Humulus lupulus L.) cones, is well known to exert several promising pharmacological activities in vitro and in vivo. Among these, the chemopreventive, anti-inflammatory and anti-cancer effects are probably the most interesting. As xanthohumol is hardly soluble in water and able to undergo conversion to isoxanthohumol we determined several handling characteristics for cell culture work with this compound. Recovery experiments revealed that working with xanthohumol under cell culture conditions requires a minimal amount of 10% FCS to increase its solubility to reasonable concentrations (-50-75 micromol/l) for pharmacological in vitro tests. Additionally, more than 50% of xanthohumol can be absorbed to various plastic materials routinely used in the cell culture using FCS concentrations below 10%. In contrast, experiments using fluorescence microscopy in living cells revealed that detection of cellular intake of xanthohumol is hampered by concentrations above 1% FCS. PMID:22393838

  4. High density cell culture by membrane-based cell recycle.

    PubMed

    Chang, H N; Yoo, I K; Kim, B S

    1994-01-01

    Enhancement of productivity of a bioprocess necessitates continuous operation of bioreactors with high biomass concentrations than are possible in conventional batch, fedbatch or continuous modes of culture. Membrane-based cell recycle has been effectively used to maintain high cell concentrations in bioreactors. This review compares membranebased cell recycle operation with other such high density cell culture systems as immobilized cell reactors and reactors with cell recycle by centrifugation or gravity sedimentation. A theoretical of production of primary and secondary metabolites in membrane-based recycle systems is presented. Operation of this type of system is discussed with examples from aerobic and anaerobic fermentations. PMID:14548467

  5. Additional survey on genotoxicity of natural anthraquinones in the hepatocyte primary culture/DNA repair assay.

    PubMed

    Mori, H; Yoshimi, N; Iwata, H; Tanaka, T; Kawai, K; Sankawa, U

    1988-08-01

    Genotoxicity of fungal anthraquinones of islandicin, iridoskyrin and (-) rubroskyrin, and a colorant of insect origin, cochineal and its component, carminic acid, an anthraquinone, was examined in the hepatocyte primary culture/DNA repair test. The results were compared with that of versicolorin A, an anthraquinone with bisfuran ring, which had been proved to be genotoxic on this assay. All of these anthraquinones, differently from versicolorin A did not show clear response of DNA repair. The results suggest that these agents are not genotoxic carcinogens. PMID:3193483

  6. Enhanced growth medium and method for culturing human mammary epithelial cells

    DOEpatents

    Stampfer, Martha R. (7290 Sayre Dr., Oakland, CA 94611); Smith, Helene S. (5693 Cabot Dr., Oakland, CA 94611); Hackett, Adeline J. (82 Evergreen Dr., Orinda, CA 94563)

    1983-01-01

    Methods are disclosed for isolating and culturing human mammary epithelial cells of both normal and malignant origin. Tissue samples are digested with a mixture including the enzymes collagenase and hyaluronidase to produce clumps of cells substantially free from stroma and other undesired cellular material. Growing the clumps of cells in mass culture in an enriched medium containing particular growth factors allows for active cell proliferation and subculture. Clonal culture having plating efficiencies of up to 40% or greater may be obtained using individual cells derived from the mass culture by plating the cells on appropriate substrates in the enriched media. The clonal growth of cells so obtained is suitable for a quantitative assessment of the cytotoxicity of particular treatment. An exemplary assay for assessing the cytotoxicity of the drug adriamycin is presented.

  7. Agarose mold embedding of cultured cells for tissue microarrays.

    PubMed

    Moskaluk, Christopher A; Stoler, Mark H

    2002-12-01

    There are several indications for the placement of samples of cultured cells in tissue microarrays (TMAs). To optimize this technique, three embedding procedures were compared: embedding of fixed cells pelleted by centrifugation, embedding of cells dispersed in an agarose matrix, and embedding of pelleted cells packed into the center of hollow agarose molds. TMAs were made from these preparations. The number of cells per tissue spot and the number of histologic sections that could be obtained from the preparations were determined. The agarose matrix and agarose mold techniques resulted in the longest core samples, while the cell pellet and agarose mold methods resulted in the greatest cell density. Thus, the use of cylindrical agarose molds optimizes both the number of cells present on a histologic section of a TMA, and the number of histologic sections that can be obtained from a TMA. This technique results in a paraffin-embedded cell preparation that yields a cell density of approximately 1000 cells per 0.6-mm diameter circular histologic section, and that produces uniform core samples the full thickness of the donor block. Histologic sections of TMAs prepared in this manner were validated in immunohistochemical and in situ hybridization assays. PMID:12459640

  8. Electrophoretic mobilities of cultured human embryonic kidney cells in various buffers

    NASA Technical Reports Server (NTRS)

    1985-01-01

    Data on the electrophoretic mobility distributions of cells in the new D-1 buffer and the interlaboratory standardization of urokinase assay methods are presented. A table of cell strains and recent data on cell dispersal methods are also included. It was decided that glycerol in A-1 electrophoretic mobility data on cultured human embryonic kidney cells subjected to electrophoresis in this buffer. The buffer composition is presented.

  9. The consensus mechanics of cultured mammalian cells

    PubMed Central

    Hoffman, Brenton D.; Massiera, Gladys; Van Citters, Kathleen M.; Crocker, John C.

    2006-01-01

    Although understanding cells' responses to mechanical stimuli is seen as increasingly important for understanding cell biology, how to best measure, interpret, and model cells' mechanical properties remains unclear. We determine the frequency-dependent shear modulus of cultured mammalian cells by using four different methods, both unique and well established. This approach clarifies the effects of cytoskeletal heterogeneity, ATP-dependent processes, and cell regional variations on the interpretation of such measurements. Our results clearly indicate two qualitatively similar, but distinct, mechanical responses, corresponding to the cortical and intracellular networks, each having an unusual, weak power-law form at low frequency. The two frequency-dependent responses we observe are remarkably similar to those reported for a variety of cultured mammalian cells measured with different techniques, suggesting it is a useful consensus description. Finally, we discuss possible physical explanations for the observed mechanical response. PMID:16793927

  10. Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme-Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

    PubMed Central

    Els, C.; Sprong, L.; van Beek, R.; van der Vries, E.; Osterhaus, A. D. M. E.; Rimmelzwaan, G. F.

    2014-01-01

    To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate. PMID:24622097

  11. Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays.

    PubMed

    Jose, Seena; Mohandas, A; Philip, Rosamma; Bright Singh, I S

    2010-11-01

    Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 gl?¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 ?g ml?¹ chloramphenicol, 100 ?g ml?¹ streptomycin and 100 IU ml?¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2'-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24h. Susceptibility of the cells to WSSV was confirmed by immunofluorescence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT(50)/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC(50). The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals. PMID:20807537

  12. Sensitivity of edge detection methods for quantifying cell migration assays.

    PubMed

    Treloar, Katrina K; Simpson, Matthew J

    2013-01-01

    Quantitative imaging methods to analyze cell migration assays are not standardized. Here we present a suite of two-dimensional barrier assays describing the collective spreading of an initially-confined population of 3T3 fibroblast cells. To quantify the motility rate we apply two different automatic image detection methods to locate the position of the leading edge of the spreading population after 24, 48 and 72 hours. These results are compared with a manual edge detection method where we systematically vary the detection threshold. Our results indicate that the observed spreading rates are very sensitive to the choice of image analysis tools and we show that a standard measure of cell migration can vary by as much as 25% for the same experimental images depending on the details of the image analysis tools. Our results imply that it is very difficult, if not impossible, to meaningfully compare previously published measures of cell migration since previous results have been obtained using different image analysis techniques and the details of these techniques are not always reported. Using a mathematical model, we provide a physical interpretation of our edge detection results. The physical interpretation is important since edge detection algorithms alone do not specify any physical measure, or physical definition, of the leading edge of the spreading population. Our modeling indicates that variations in the image threshold parameter correspond to a consistent variation in the local cell density. This means that varying the threshold parameter is equivalent to varying the location of the leading edge in the range of approximately 1-5% of the maximum cell density. PMID:23826283

  13. Buffer Combinations for Mammalian Cell Culture

    Microsoft Academic Search

    Harry Eagle

    1971-01-01

    The growth and metabolism of cultured mammalian cells are markedly affected by the pH variation in ordinary bicarbonate-buffered media(pH 8.0 to 6.9). Those pH swings can be reduced and the pH of the culture can be stabilized as desired in the range pH 6.4 to 8.3 by appropriate combinations of two or three organic buffers, each at 10 to 15

  14. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

    PubMed Central

    Galluzzi, L; Aaronson, SA; Abrams, J; Alnemri, ES; Andrews, DW; Baehrecke, EH; Bazan, NG; Blagosklonny, MV; Blomgren, K; Borner, C; Bredesen, DE; Brenner, C; Castedo, M; Cidlowski, JA; Ciechanover, A; Cohen, GM; De Laurenzi, V; De Maria, R; Deshmukh, M; Dynlacht, BD; El-Deiry, WS; Flavell, RA; Fulda, S; Garrido, C; Golstein, P; Gougeon, M-L; Green, DR; Gronemeyer, H; Hajn?czky, G; Hardwick, JM; Hengartner, MO; Ichijo, H; Jäättelä, M; Kepp, O; Kimchi, A; Klionsky, DJ; Knight, RA; Kornbluth, S; Kumar, S; Levine, B; Lipton, SA; Lugli, E; Madeo, F; Malorni, W; Marine, J-CW; Martin, SJ; Medema, JP; Mehlen, P; Melino, G; Moll, UM; Morselli, E; Nagata, S; Nicholson, DW; Nicotera, P; Nuñez, G; Oren, M; Penninger, J; Pervaiz, S; Peter, ME; Piacentini, M; Prehn, JHM; Puthalakath, H; Rabinovich, GA; Rizzuto, R; Rodrigues, CMP; Rubinsztein, DC; Rudel, T; Scorrano, L; Simon, H-U; Steller, H; Tschopp, J; Tsujimoto, Y; Vandenabeele, P; Vitale, I; Vousden, KH; Youle, RJ; Yuan, J; Zhivotovsky, B; Kroemer, G

    2009-01-01

    Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells. PMID:19373242

  15. Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

    PubMed Central

    Azari, Hassan; Millette, Sebastien; Ansari, Saeed; Rahman, Maryam; Deleyrolle, Loic P.; Reynolds, Brent A.

    2011-01-01

    Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 ?m in diameter can be observed and are ready for further passaging and expansion. PMID:22064695

  16. Isolation of Nuclei from Skeletal Muscle Satellite Cells and Myofibers for Use in Chromatin lmmunoprecipitation Assays

    PubMed Central

    Ohkawa, Yasuyuki; Mallappa, Chandrashekara; Dacwag Vallaster, Caroline S.; lmbalzano, Anthony N.

    2014-01-01

    Studies investigating mechanisms controlling gene regulation frequently examine specific DNA sequences using chromatin immunoprecipitation (ChIP) assays to determine whether specific regulatory factors or modified histones are present. While use of primary cells or cell line models for differentiating or differentiated tissue is widespread, the ability to assess factor binding and histone modification in tissue defines the events that occur in vivo and provides corroboration for studies in cultured cells. Many tissues can be analyzed with minimal modification to existing ChIP protocols that are designed for cultured cells; however, some tissues, such as skeletal muscle, are problematic in that accessibility of the cross-linking agent is limited. We describe a method to isolate skeletal muscle tissue nuclei suitable for use in ChIP protocols. Furthermore, we utilize a simple fractionation of digested skeletal muscle tissue that can separate mature myofibers from satellite cells, which are responsible for postnatal skeletal muscle regeneration, thereby allowing simultaneous preparation of nuclei from both cell types. PMID:22130858

  17. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

    USGS Publications Warehouse

    Elliott, D.G.; Applegate, L.J.; Murray, A.L.; Purcell, M.K.; McKibben, C.L.

    2013-01-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

  18. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture.

    PubMed

    Elliott, D G; Applegate, L J; Murray, A L; Purcell, M K; McKibben, C L

    2013-09-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test. PMID:23346868

  19. Estrogenic activity of phthalate esters by in vitro VTG assay using primary-cultured Xenopus hepatocytes.

    PubMed

    Nomura, Yuji; Mitsui, Naoko; Bhawal, Ujjal Kumar; Sawajiri, Masahiko; Tooi, Osamu; Takahashi, Toru; Okazaki, Masayuki

    2006-09-01

    Estrogenic activity of phthalate esters in dental soft resins was evaluated with an amphibian system consisting of a vitellogenin (VTG)-detecting Enzyme-Linked Immunosorbent Assay and a primary-cultured hepatocyte assay using adult male Xenopus laevis. In particular, phthalate esters--Di-n-butyl phthalate (DBP), Butyl phthalyl butyl glycolate (BPBG), Benzyl butyl phthalate (BBP), and Benzyl benzoate (BB)--were investigated. Bisphenol A (BPA) was prepared for comparison with these chemicals, and 17beta-estradiol (E2) was used as a positive control. The chemicals were diluted in dimethyl sulfoxide (DMSO) to obtain final concentrations ranging from 10(-11) to 10(-4) mol/l. BPA induced estrogenic activity at a concentration of 1.1x10(-6) mol/l, while E2 showed at 4.1x10(-11) mol/l. DBP, BBP, BB, and BPBG showed no estrogenic activity at concentrations between 4x10(-7) mol/l and 1x10(-4) mol/l. The latter result indicated that these phthalate esters might be metabolically transformed into non-estrogenic substances in Xenopus hepatocytes. Furthermore, this study demonstrated that through in vitro metabolism assessment, the estrogenic activity of chemical substances could be directly detected in terms of VTG secretion in primary-cultured Xenopus hepatocytes. PMID:17076324

  20. Performance evaluation of 3D polystyrene 96-well plates with human neural stem cells in a calcium assay.

    PubMed

    Lai, Yinzhi; Kisaalita, William S

    2012-08-01

    In this study, we have generated a high-throughput screening (HTS)-compatible 3D cell culture platform by chemically "welding" polystyrene scaffolds into standard 2D polystyrene 96-well plates. The variability of scaffolds was minimized by introducing automation into the fabrication process. The fabricated 3D cell culture plates were compared with several commercially available 3D cell culture platforms with light and scanning electron microscopy. Voltage-gated calcium channel functionality was used to access the Z' factors of all plates, including a 2D standard plate control. It was found that with the No-Wash Fluo-4 calcium assay and neural progenitor cells, all plates display acceptable Z' factors for use in HTS. The plates with "welded" polystyrene scaffolds have several advantages, such as being versatile and economical, and are ready to use off the shelf. These characteristics are especially desired in HTS preclinical drug discovery applications. PMID:22496208

  1. Cell Culture on MEMS Platforms: A Review

    PubMed Central

    Ni, Ming; Tong, Wen Hao; Choudhury, Deepak; Rahim, Nur Aida Abdul; Iliescu, Ciprian; Yu, Hanry

    2009-01-01

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented. PMID:20054478

  2. Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

    PubMed

    Feizi, Sepehr; Soheili, Zahra-Soheila; Bagheri, Abouzar; Balagholi, Sahar; Mohammadian, Azam; Rezaei-Kanavi, Mozhgan; Ahmadieh, Hamid; Samiei, Shahram; Negahban, Kambiz

    2014-05-01

    The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. PMID:24726921

  3. Micro3D cell culture devices for single cell analysis

    Microsoft Academic Search

    M. R. Dusseillerl; D. Schlaepfer; A. Ferrari; R. Kroschewski; M. Textorl

    2005-01-01

    In this paper we present two novel devices for the culturing of single cells or small clusters of cells in an array format. The focus of these devices is to create local 3-dimensional microenvironments for the cells which mimic more closely the adhesion state in an in vivo situation. The devices were manufactured using fast processes and cost-effective materials like

  4. Immunological evidence for thyroliberin (TRH) neurons in primary cultures of fetal mouse brain cells. Ontogenic aspects.

    PubMed

    Faivre-Bauman, A; Nemeskeri, A; Tougard, C; Tixier-Vidal, A

    1980-03-10

    Primary cultures of dissociated cells were initiated from fetal mouse hypothalami and brain hemispheres, on the 13th and the 16th day of gestation (respectively 12-day and 15-day-old fetuses). After 10 days in vitro, the cultured cells were collected, pooled in an appropriate medium and thyroliberin (TRH) was assayed in the cell extracts using a specific radioimmunoassay. TRH was found in every type of culture. For hypothalami, higher levels of TRH were found when when starting from older embryos, while in brain hemisphere cultures the TRH content increased in culture of 12-day-old fetal cells only, whereas it decreased in cultures of 16-day-old fetal cells. Immunocytochemical staining allows visualization of TRH positive cells in all cultures except in hypothalamic cultures from 12-day-old embryos. This is consistent with the radioimmunoassay data. TRH was localized exclusively in some of the overlying cells, whereas the basal cells were always negative. Specificity of the staining was assessed by immunochemistry and radioimmunoassay. At the electron microscope level, the positive cells display neuronal features. The immunoprecipitate was found in both perikaryon and axons as well as in axonal dilatations. PMID:6766779

  5. Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells

    PubMed Central

    Rath, Subha N.; Brandl, Andreas; Hiller, Daniel; Hoppe, Alexander; Gbureck, Uwe; Horch, Raymund E.; Boccaccini, Aldo R.; Kneser, Ulrich

    2014-01-01

    Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches. PMID:25470000

  6. Bioactive copper-doped glass scaffolds can stimulate endothelial cells in co-culture in combination with mesenchymal stem cells.

    PubMed

    Rath, Subha N; Brandl, Andreas; Hiller, Daniel; Hoppe, Alexander; Gbureck, Uwe; Horch, Raymund E; Boccaccini, Aldo R; Kneser, Ulrich

    2014-01-01

    Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches. PMID:25470000

  7. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    PubMed Central

    Nikfarjam, Laleh; Farzaneh, Parvaneh

    2012-01-01

    One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importance of mycoplasma tests in cell culture; 4. different methods to identify mycoplasma contamination; 5. the consequences of mycoplasma contamination in cell culture and 6. available methods to eliminate mycoplasma contamination. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture. PMID:23508237

  8. Culture media from hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury.

    PubMed

    Hummitzsch, Lars; Zitta, Karina; Bein, Berthold; Steinfath, Markus; Albrecht, Martin

    2014-03-10

    Remote ischemic preconditioning (RIPC) is a phenomenon, whereby short episodes of non-lethal ischemia to an organ or tissue exert protection against ischemia/reperfusion injury in a distant organ. However, there is still an apparent lack of knowledge concerning the RIPC-mediated mechanisms within the target organ and the released factors. Here we established a human cell culture model to investigate cellular and molecular effects of RIPC and to identify factors responsible for RIPC-mediated intestinal protection. Human umbilical vein cells (HUVEC) were exposed to repeated episodes of hypoxia (3 × 15 min) and conditioned culture media (CM) were collected after 24h. Human intestinal cells (CaCo-2) were cultured with or without CM and subjected to 90 min of hypoxia/reoxygenation injury. Reverse transcription-polymerase chain reaction, Western blotting, gelatin zymography, hydrogen peroxide measurements and lactate dehydrogenase (LDH) assays were performed. In HUVEC cultures hypoxic conditioning did not influence the profile of secreted proteins but led to an increased gelatinase activity (P<0.05) in CM. In CaCo-2 cultures 90 min of hypoxia/reoxygenation resulted in morphological signs of cell damage, increased LDH levels (P<0.001) and elevated levels of hydrogen peroxide (P<0.01). Incubation of CaCo-2 cells with CM reduced the hypoxia-induced signs of cell damage and LDH release (P<0.01) and abrogated the hypoxia-induced increase of hydrogen peroxide. These events were associated with an enhanced phosphorylation status of the prosurvival kinase Erk1/2 (P<0.05) but not Akt and STAT-5. Taken together, CM of hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury. The established culture model may help to unravel RIPC-mediated cellular events and to identify molecules released by RIPC. PMID:24394542

  9. Computerized microfluidic cell culture using elastomeric channels and Braille displays

    PubMed Central

    Gu, Wei; Zhu, Xiaoyue; Futai, Nobuyuki; Cho, Brenda S.; Takayama, Shuichi

    2004-01-01

    Computer-controlled microfluidics would advance many types of cellular assays and microscale tissue engineering studies wherever spatiotemporal changes in fluidics need to be defined. However, this goal has been elusive because of the limited availability of integrated, programmable pumps and valves. This paper demonstrates how a refreshable Braille display, with its grid of 320 vertically moving pins, can power integrated pumps and valves through localized deformations of channel networks within elastic silicone rubber. The resulting computerized fluidic control is able to switch among: (i) rapid and efficient mixing between streams, (ii) multiple laminar flows with minimal mixing between streams, and (iii) segmented plug-flow of immiscible fluids within the same channel architecture. The same control method is used to precisely seed cells, compartmentalize them into distinct subpopulations through channel reconfiguration, and culture each cell subpopulation for up to 3 weeks under perfusion. These reliable microscale cell cultures showed gradients of cellular behavior from C2C12 myoblasts along channel lengths, as well as differences in cell density of undifferentiated myoblasts and differentiation patterns, both programmable through different flow rates of serum-containing media. This technology will allow future microscale tissue or cell studies to be more accessible, especially for high-throughput, complex, and long-term experiments. The microfluidic actuation method described is versatile and computer programmable, yet simple, well packaged, and portable enough for personal use. PMID:15514025

  10. Calcitonin receptors on neoplastic mononuclear cells cultured from a human giant-cell tumor of the sacrum

    Microsoft Academic Search

    Akio Maeda; Hisao Matsui; Masahiko Kanamori; Kazuo Yudoh; Haruo Tsuji

    1994-01-01

    Saturable, specific, high-affinity calcitonin receptors were demonstrated in cultured neoplastic mononuclear spindle cells from a giant-cell tumor of the sacrum of a 38-year-old woman. The receptor was analyzed by autoradiography and125I-calcitonin binding assay. Binding reversibility of125I-calcitonin to the cells was not complete and the structural specificity was indicated by the inability of unrelated hormones to compete with calcitonin. The 24

  11. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    NASA Technical Reports Server (NTRS)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  12. Date Palm Cell and Protoplast Culture

    Microsoft Academic Search

    A. Assani; D. Chabane; H. Shittu; N. Bouguedoura

    \\u000a This chapter describes the current status of cell and protoplast cultures in date palm (Phoenix dactylifera L.). Critically important steps toward plant regeneration from recalcitrant date palm protoplasts have been achieved in the\\u000a recent past. Callus regeneration was achieved in commercial cvs. Deglet Noor, Takerboucht, Barhee and Zaghloul. The use of\\u000a feeder layer was the main factor for inducing cell

  13. Cytotoxicity effects of amiodarone on cultured cells

    Microsoft Academic Search

    Emna El Golli-Bennour; Amel Bouslimi; Olfa Zouaoui; Safa Nouira; Abdellatif Achour; Hassen Bacha

    Amiodarone is a potent anti-arrhythmic drug used for the treatment of cardiac arrhythmias. Although, the effects of amiodarone are well characterized on post-ischemic heart and cardiomyocytes, its toxicity on extra-cardiac tissues is still poorly understood. To this aim, we have monitored the cytotoxicity effects of this drug on three cultured cell lines including hepatocytes (HepG2), epithelial cells (EAhy 926) and

  14. The Effect of Spaceflight on Bone Cell Cultures

    NASA Technical Reports Server (NTRS)

    Landis, William J.

    1999-01-01

    Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural methods (conventional and high voltage electron microscopy, inununocytochemistry, stereomicroscopy, and 3D image reconstruction). The studies have provided new knowledge of aspects of bone cell development and structural regulation, extracellular matrix assembly, and mineralization during spaceflight and under normal gravity. The information has contributed to insights into the means in general by which cells respond and adapt to different conditions of gravity (loading). The data may as well have suggested an underlying basis for the observed loss of bone by vertebrates, including man, in microgravity; and these scientific results may have implications for understanding bone loss following fracture healing and extended periods of inactivity such as during long-term bedrest.

  15. Determining cell number during cell culture using the Scepter cell counter.

    PubMed

    Ongena, Kathleen; Das, Chandreyee; Smith, Janet L; Gil, Sónia; Johnston, Grace

    2010-01-01

    Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed(1) who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate(2). To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry(1). For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments(1). The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection(3) in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram. PMID:22158024

  16. ANTHOCYANIN (ACN) STABILITY IN CELL CULTURE MEDIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anthocyanins (ACNs) are potential oxygen radical scavengers that have coronary vasoactive and vasoprotective properties. Cell or tissue culture systems have been used to examine the bioactivity and mechanisms of action of ACNs on the vascular system. However, due to their unique chemical structure, ...

  17. Plant cell suspension cultures: some engineering considerations.

    PubMed

    Kieran, P M; MacLoughlin, P F; Malone, D M

    1997-12-17

    Higher plants are the source of a vast array of biochemicals which are used as drugs, pesticides, flavourings and fragrances. For some of these compounds, plant cell culture can provide a potential production alternative to traditional cultivation methods or chemical synthesis routes. Many systems have been patented and the last 20 years have seen considerable industrial and academic interest in the development of large scale cultures to produce pharmaceutically active, high value substances. However, the industrial application of plant cell suspension cultures has, to date, been limited. Commercialisation has essentially been impeded by economic feasibility, arising from both biological and engineering considerations. This paper reviews the commercial development of the technology to date and focuses on the impact of specific engineering-related factors, in particular, the shear sensitivity of plant cell suspension cultures. Evidence of sensitivity to hydrodynamic shear in bioreactors has generally been attributed to the physical characteristics of the suspended cells. Recent studies indicate that shear sensitivity may not be as important, in some cases, as initially anticipated. PMID:9487717

  18. Human endothelial cell-based assay for endotoxin as sensitive as the conventional Limulus Amebocyte Lysate assay.

    PubMed

    Unger, Ronald E; Peters, Kirsten; Sartoris, Anne; Freese, Christian; Kirkpatrick, C James

    2014-03-01

    Endotoxin, also known as lipopolysaccharide (LPS) produced by bacteria can be present in any liquid or on any biomaterial even if the material is sterile. Endotoxin in mammals can cause fever, inflammation, cell and tissue damage and irreversible septic shock and death. In the body, endothelial cells making up the blood vasculature and endothelial cells in vitro rapidly react to minute amounts of endotoxin resulting in a rapid induction of the cell adhesion molecule E-selectin. In this study we have used immunofluorescent staining to evaluate the expression of E-selectin on human microvascular endothelial cells from the skin (HDMEC) and human umbilical vein endothelial cells (HUVEC) exposed to various concentrations of LPS. In addition, the sensitivity of detection was compared with the most widely used assay for the presence of endotoxin, the Limulus Amebocyte Lysate assay (LAL). The detection of E-selectin on endothelial cells in the presence of LPS for 4 h was found to be at least as sensitive in detecting the same concentration using the LAL assay. A cell adhesion molecule-enzyme immunosorbent assay was also developed and used to quantify LPS using the endothelial cell model. A comparison of LAL and the immunofluorescent staining method was carried out with solutions, nanoparticles, biomaterial extracts and endothelial cells grown directly on biomaterials. Under all conditions, the endothelial/E-selectin model system was positive for the test samples that were positive by LAL. Thus, we propose the use of this highly sensitive, rapid, reproducible assay for the routine testing of endotoxin in all steps in the manufacturing process of materials destined for use in humans. This can give a rapid feedback and localization of bacterial contamination sources with the LAL being reserved for the testing of the final product. PMID:24456607

  19. Proliferation assay of mouse embryonic stem (ES) cells exposed to atmospheric-pressure plasmas at room temperature

    NASA Astrophysics Data System (ADS)

    Miura, Taichi; Ando, Ayumi; Hirano, Kazumi; Ogura, Chika; Kanazawa, Tatsuya; Ikeguchi, Masamichi; Seki, Atsushi; Nishihara, Shoko; Hamaguchi, Satoshi

    2014-11-01

    Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H2O2), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells.

  20. UVA-induced oxidative stress in single cells probed by autofluorescence modifications, cloning assay, and comet assay

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Krasieva, Tatjana; Bauer, Eckhard; Fiedler, Ulrich; Berns, Michael W.; Tromberg, Bruce J.; Greulich, Karl O.

    1996-01-01

    Cell damage by low-power 365 nm radiation of a 50 W high-pressure mercury microscopy lamp was studied. UVA exposure to CHO cells resulted for radiant exposures greater than 10 kJ/m2 in significant modifications of NADH-attributed autofluorescence and in inhibition of cell division. Single cell gel electrophoresis (comet assay) revealed UVA-induced single strand DNA breaks. According to these results, UVA excitation radiation in fluorescence microscopy may damage cells. This has to be considered in vital cell microscopy, e.g. in calcium measurements.

  1. Cell damage by UVA radiation of a mercury microscopy lamp probed by autofluorescence modifications, cloning assay, and comet assay

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Krasieva, Tatiana B.; Bauer, Eckhard; Fiedler, Ursula; Berns, Michael W.; Tromberg, Bruce J.; Greulich, Karl O.

    1996-04-01

    Cell damage by low-power 365-nm radiation of a 50-W high-pressure mercury microscopy lamp was studied. Exposure of Chinese hamster ovary cells to ultraviolet-A (UVA) radiation > 10 kJ/m2 resulted in significant modifications of nicotinamide adenine dinucleotide attributed autofluorescence and inhibition of cell division. Single-cell gel electrophoresis (comet assay) revealed UVA-induced single-strand DNA breaks. According to these results, UVA excitation radiation in fluorescence microscopy may damage cells. This has to be considered in vital cell microscopy, e.g., in calcium measurements.

  2. Comparison of Loop-Mediated Isothermal Amplification Assay and Conventional Culture Methods for Detection of Campylobacter jejuni and Campylobacter coli in Naturally Contaminated Chicken Meat Samples

    Microsoft Academic Search

    Wataru Yamazaki; Masumi Taguchi; Takao Kawai; Kentaro Kawatsu; Junko Sakata; Kiyoshi Inoue; Naoaki Misawa

    2009-01-01

    We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for detection of chicken meat samples naturally contaminated with Campylobacter jejuni and Campylobacter coli. A total of 144 Preston enrichment broth cultures from chicken meat samples were assessed by using the LAMP assay and conventional culture methods, which consist of a combination of Preston enrichment culturing and plating onto

  3. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

    NASA Astrophysics Data System (ADS)

    Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

    2013-10-01

    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

  4. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

    PubMed Central

    Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

    2013-01-01

    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures. PMID:24141454

  5. Biotransformation of bavachinin by three fungal cell cultures.

    PubMed

    Luo, Jianmei; Liang, Qikun; Shen, Yanbing; Chen, Xi; Yin, Zhinan; Wang, Min

    2014-02-01

    Biotransformation of bavachinin (1) was investigated using three fungal cell cultures of Aspergillus flavus ATCC 30899, Cunninghamella elegans CICC 40250 and Penicillium raistrickii ATCC 10490, respectively. Two major converted products were identified by LC/MS, (1)H NMR and (13)C NMR and X-ray diffraction. Two biocatalyst systems, A. flavus ATCC 30899 and C. elegans CICC 40250 cell cultures, showed a great capacity of hydroxylation and two hydroxyl groups were attached at C-2? and C-3? positions in the side chain of the bavachinin A-ring, resulting in the formation of the same compound with a name, (S)-6-((R)-2,3-dihydroxy-3-methylbutyl)-2-(4-hydroxyphenyl)-7-methoxychromen-4-one (2). On the other hand, P. raistrickii ATCC 10490 cell cultures possessed the ability to reduction at C-4 of the substrate C-ring, resulting in the production of (2S,4R)-2-(4-hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chromen-4-ol (3). Furthermore, the in vitro anti-tumor activities of the above compounds were evaluated by MTT assay. Compared with the substrate (1), product 3 possessed stronger inhibition activity on the human breast cancer cell line (MCF-7) and slightly lower inhibition activities against Hep G2, HeLa, Hep-2 and A549 cells lines; while the hydroxyl product 2 possessed much lower inhibition activity on tumor cells lines, which might be related to the insertion of two hydroxyl groups. Compounds 2 and 3 were considered to be novel. It was also the first time to biotransform bavachinin (1) by these three fungi, which suggested the potential role of microbial enzymes to synthesize novel compounds from plant secondary metabolites. PMID:24012108

  6. Identification of Hedgehog Pathway Components by RNAi in Drosophila Cultured Cells

    Microsoft Academic Search

    Lawrence Lum; Shenqin Yao; Brian Mozer; Alessandra Rovescalli; Doris Von Kessler; Marshall Nirenberg; Philip A. Beachy

    2003-01-01

    Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of

  7. Catalytic Inhibition of Human DNA Topoisomerase II by Interactions of Grape Cell Culture Polyphenols

    Microsoft Academic Search

    Jeong-Youn Jo; Elvira Gonzalez de Mejia; Mary Ann Lila

    2006-01-01

    Previously, we isolated mixed polyphenolic fractions on a toyopearl matrix (TP-2 to TP-6) from grape cell cultures that were highly potent catalytic inhibitors in a human DNA topoisomerase II assay for cancer chemoprevention. The objectives of this study were to evaluate the potency of, and potential interactions between, individual fractions and some of the purified bioactive polyphenols that comprise these

  8. UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR

    Microsoft Academic Search

    Gwangpyo Ko; Theresa L. Cromeans; Mark D. Sobsey

    2005-01-01

    Adenoviruses are among the most resistant waterborne pathogens to UV disinfection, yet of the 51 serologically distinct human adenoviruses, only a few have been evaluated for their sensitivities to UV irradiation. Human enteric adenoviruses (Ad40 and Ad41) are difficult to cultivate and reliably assay for infectivity, requiring weeks to obtain cytopathogenic effects (CPE). Inoculated cell cultures often deteriorate before the

  9. Probing nanoparticle interactions in cell culture media.

    PubMed

    Sabuncu, Ahmet C; Grubbs, Janna; Qian, Shizhi; Abdel-Fattah, Tarek M; Stacey, Michael W; Beskok, Ali

    2012-06-15

    Nanoparticle research is often performed in vitro with little emphasis on the potential role of cell culture medium. In this study, gold nanoparticle interactions with cell culture medium and two cancer cell lines (human T-cell leukemia Jurkat and human pancreatic carcinoma PANC1) were investigated. Gold nanoparticles of 10, 25, 50, and 100 nm in diameter at fixed mass concentration were tested. Size distributions and zeta potentials of gold nanoparticles suspended in deionized (DI) water and Dulbecco's Modified Eagle's Media (DMEM) supplemented with fetal calf serum (FCS) were measured using dynamic light scattering (DLS) technique. In DI water, particle size distributions exhibited peaks around their nominal diameters. However, the gold nanoparticles suspended in DMEM supplemented with FCS formed complexes around 100 nm, regardless of their nominal sizes. The DLS and UV-vis spectroscopy results indicate gold nanoparticle agglomeration in DMEM that is not supplemented by FCS. The zeta potential results indicate that protein rich FCS increases the dispersion quality of gold nanoparticle suspensions through steric effects. Cellular uptake of 25 and 50 nm gold nanoparticles by Jurkat and PANC1 cell lines were investigated using inductively coupled plasma-mass spectroscopy. The intracellular gold level of PANC1 cells was higher than that of Jurkat cells, where 50 nm particles enter cells at faster rates than the 25 nm particles. PMID:22421416

  10. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    SciTech Connect

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.

    2005-10-14

    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows surface-linked ligands to diffuse freely in two dimensions. Ligands can become reorganized beneath cells, by reaction-diffusion processes, and may also adopt spatial configurations reflecting those of their cognate receptors on the cell surface (Figure 1B). This provides a significant benefit over conventional cell signaling and culturing systems that present inflexible distributions of signaling molecules. In this study, we observe marked differences in the response of cells to membrane surface displayed soluble ligands as a function of membrane fluidity. Tethering of soluble signaling molecules to fluid supported membranes opens up opportunities to use already developed membrane fabrication technologies to present soluble components within a surface array format.

  11. Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

    NASA Technical Reports Server (NTRS)

    Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

    1998-01-01

    The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

  12. Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines

    PubMed Central

    Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W.; Stringer, Brett W.; Boyd, Andrew W.; Johns, Terrance G.; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A.

    2015-01-01

    Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. PMID:25806119

  13. Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines.

    PubMed

    Rahman, Maryam; Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W; Stringer, Brett W; Boyd, Andrew W; Johns, Terrance G; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A

    2015-03-01

    Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. PMID:25806119

  14. High-Content Assays for Hepatotoxicity Using Induced Pluripotent Stem Cell–Derived Cells

    PubMed Central

    Sirenko, Oksana; Hesley, Jayne; Rusyn, Ivan

    2014-01-01

    Abstract Development of predictive in vitro assays for early toxicity evaluation is extremely important for improving the drug development process and reducing drug attrition rates during clinical development. High-content imaging-based in vitro toxicity assays are emerging as efficient tools for safety and efficacy testing to improve drug development efficiency. In this report we have used an induced pluripotent stem cell (iPSC)–derived hepatocyte cell model having a primary tissue-like phenotype, unlimited availability, and the potential to compare cells from different individuals. We examined a number of assays and phenotypic markers and developed automated screening methods for assessing multiparameter readouts of general and mechanism-specific hepatotoxicity. Endpoints assessed were cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis. We assayed compounds with known mechanisms of toxicity and also evaluated a diverse hepatotoxicity library of 240 compounds. We conclude that high-content automated screening assays using iPSC-derived hepatocytes are feasible, provide information about mechanisms of toxicity, and can facilitate the safety assessment of drugs and chemicals. PMID:24229356

  15. An Approach for Assessing the Signature Quality of Various Chemical Assays when Predicting the Culture Media Used to Grow Microorganisms

    SciTech Connect

    Holmes, Aimee E.; Sego, Landon H.; Webb-Robertson, Bobbie-Jo M.; Kreuzer, Helen W.; Anderson, Richard M.; Unwin, Stephen D.; Weimar, Mark R.; Tardiff, Mark F.; Corley, Courtney D.

    2013-02-01

    We demonstrate an approach for assessing the quality of a signature system designed to predict the culture medium used to grow a microorganism. The system was comprised of four chemical assays designed to identify various ingredients that could be used to produce the culture medium. The analytical measurements resulting from any combination of these four assays can be used in a Bayesian network to predict the probabilities that the microorganism was grown using one of eleven culture media. We evaluated combinations of the signature system by removing one or more of the assays from the Bayes network. We measured and compared the quality of the various Bayes nets in terms of fidelity, cost, risk, and utility, a method we refer to as Signature Quality Metrics

  16. Antibiotic utilization improvement with the Nanosphere Verigene Gram-Positive Blood Culture assay.

    PubMed

    Beal, Stacy G; Thomas, Cody; Dhiman, Neelam; Nguyen, Daniel; Qin, Huanying; Hawkins, Jennifer M; Dekmezian, Mhair; Benavides, Raul

    2015-04-01

    New technologies offer rapid identification of organisms and antimicrobial resistance markers in blood cultures several hours faster than conventional methods. We sought to determine whether implementation of the Verigene® Gram-Positive Blood Culture (BC-GP) assay paired with a well-defined results reporting algorithm would lead to earlier deescalation of empiric therapy for inpatients with methicillin-sensitive Staphylococcus aureus (MSSA) and vancomycin-resistant Enterococcus (VRE) bacteremia. The algorithm design focused on lessening the demand for pharmacist time by using electronic communications where possible. Our study compared inpatients with MSSA and VRE bacteremia from the time period before (pre-BC-GP) and after (post-BC-GP) implementation of the assay on June 25, 2013. The time from blood draw to identification and susceptibility results was decreased by 36.4 hours (P < 0.001) in the post-BC-GP group. The mean time from collection to the first dose of optimal antibiotics was reduced in the post-BC-GP group by 18.9 hours (P = 0.004) overall, with a 20.6-hour reduction (P = 0.009) for patients with MSSA and a 20.7-hour reduction (P = 0.077) for patients with VRE. Additionally, the percent of patients on empiric therapy who were placed on optimal antibiotics at any time after the Gram stain result was available increased from 64% (45/70) pre-BC-GP to 80% (43/54) post-BC-GP. The BC-GP led to an increased rate of deescalation of empiric antibiotics and a reduction in the time to optimal antibiotics for patients with MSSA and VRE bacteremia. PMID:25829639

  17. Antibiotic utilization improvement with the Nanosphere Verigene Gram-Positive Blood Culture assay

    PubMed Central

    Beal, Stacy G.; Thomas, Cody; Dhiman, Neelam; Nguyen, Daniel; Qin, Huanying; Hawkins, Jennifer M.; Dekmezian, Mhair

    2015-01-01

    New technologies offer rapid identification of organisms and antimicrobial resistance markers in blood cultures several hours faster than conventional methods. We sought to determine whether implementation of the Verigene® Gram-Positive Blood Culture (BC-GP) assay paired with a well-defined results reporting algorithm would lead to earlier deescalation of empiric therapy for inpatients with methicillin-sensitive Staphylococcus aureus (MSSA) and vancomycin-resistant Enterococcus (VRE) bacteremia. The algorithm design focused on lessening the demand for pharmacist time by using electronic communications where possible. Our study compared inpatients with MSSA and VRE bacteremia from the time period before (pre-BC-GP) and after (post-BC-GP) implementation of the assay on June 25, 2013. The time from blood draw to identification and susceptibility results was decreased by 36.4 hours (P < 0.001) in the post-BC-GP group. The mean time from collection to the first dose of optimal antibiotics was reduced in the post-BC-GP group by 18.9 hours (P = 0.004) overall, with a 20.6-hour reduction (P = 0.009) for patients with MSSA and a 20.7-hour reduction (P = 0.077) for patients with VRE. Additionally, the percent of patients on empiric therapy who were placed on optimal antibiotics at any time after the Gram stain result was available increased from 64% (45/70) pre-BC-GP to 80% (43/54) post-BC-GP. The BC-GP led to an increased rate of deescalation of empiric antibiotics and a reduction in the time to optimal antibiotics for patients with MSSA and VRE bacteremia. PMID:25829639

  18. Human T cell priming assay: depletion of peripheral blood lymphocytes in CD25(+) cells improves the in vitro detection of weak allergen-specific T cells.

    PubMed

    Vocanson, Marc; Achachi, Amine; Mutez, Virginie; Cluzel-Tailhardat, Magalie; Varlet, Béatrice Le; Rozières, Aurore; Fournier, Philippe; Nicolas, Jean-François

    2014-01-01

    To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-? secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was possible to induce T cell priming to most of the moderate/weak allergens, including lipophilic molecules highly insoluble in culture media. Therefore, the present optimized in vitro human T cell priming assay is a valuable method to detect the sensitizing properties of chemical allergens. PMID:24214620

  19. Rapid Detection of Methicillin-Resistant Staphylococci from Blood Culture Bottles by Using a Multiplex PCR Assay

    Microsoft Academic Search

    L. Louie; J. Goodfellow; P. Mathieu; A. Glatt; M. Louie; A. E. Simor

    2002-01-01

    Rapid detection and accurate identification of methicillin-resistant staphylococci are critical for the effective management of infections caused by these organisms. We describe a multiplex PCR-based assay for the direct detection of methicillin-resistant staphylococci from blood culture bottles (BacT\\/Alert; Organon-Teknika, Durham, N.C.). A simple lysis method followed by a multiplex PCR assay designed to detect the nuc, mecA, and bacterial 16S

  20. Establishing midgut cell culture from Rhynchophorus ferrugineus (Olivier) and toxicity assessment against ten different insecticides.

    PubMed

    Aljabr, Ahmed Mohammed; Rizwan-ul-Haq, Muhammad; Hussain, Abid; Al-Mubarak, Abdullah I; Al-Ayied, Hassan Y

    2014-04-01

    Midgut epithelial cell culture was successfully developed from red palm weevil (Rhynchophorus ferrugineus) during this study and named as RPW-1. Optimum conditions for four different commercial media were also worked out to successfully maintain the culture. Grace's medium was found to be the most effective for RPW-1 culturing which resulted in the highest cell density of 7.5 × 10(6) cells/ml after 72 h of cell seeding with 96% cell viability. It was followed by Schneider's medium and TNM-FH medium where cell densities reached up to 7.4 × 10(6) and 5.9 × 10(6) cells/ml, respectively, after 72 h having 91 and 89% cell viability. Comparatively, Media-199 was least effective for RPW-1 cell culturing. As a whole, temperature at 27°C and pH 6.3 were the best for RPW-1 culturing where the highest cell density and maximum cell viability were noted. Individually, Grace's medium, Schneider's medium, TNM-FH medium, and Media-199 produced better results at 27°C, 27°C, 24°C, and 21°C and pH 6.3, 6.4, 5.3, and 7.1, respectively. The toxicity assay and MTT cell proliferation assay revealed that, out of the ten insecticides used in this study, emamectin benzoate was the most toxic insecticide to RPW-1 cells resulting in 92% cell mortality and 74% cell growth inhibition. Dieldrin was the least potent, causing only 19% cell mortality and 18% cell growth inhibition. PMID:24197670

  1. Effect of methisoprinol on virus replication in cell cultures.

    PubMed

    Ma?aczewska, J; Rotkiewicz, Z

    2004-01-01

    The effect of Methisoprinol (active substance: isoprinozine) on the replication of two animal viruses, the TK900 strain of Aujeszky's disease virus and the Roakin strain of the Newcastle disease virus was investigated. When the maximal tolerable doses of the drug were added to two cell cultures (CECC and GMK), its effect on the level of infectious titres of theviruses and their adsorption were assayed. Investigations were also performed to assess the direct effect of Methisoprinol on the viral strains used. The final stage of the experiment aimed at analysing of the replication dynamics of the viruses in the presence of Methisoprinol. Methisoprinol showed no direct effect on the viruses used in the study. Nor did it affect their adsorption. The preparation applied to the culture 24 hours before infection did not influence the replication of viruses, but administered simultaneously with the infection significantly lowered the final titres of viruses. The highest inhibitory effect of the drug was observed during the analysis of the replication dynamics of both viruses in CECC and of pseudorabies virus in GMK cell culture upon the application of the maximal tolerable doses of Methisoprinol and low infectious doses of the viruses. PMID:15230539

  2. A Rapid and Sensitive Method for Measuring N-Acetylglucosaminidase Activity in Cultured Cells

    PubMed Central

    Mauri, Victor; Lotfi, Parisa; Segatori, Laura; Sardiello, Marco

    2013-01-01

    A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination therapies. PMID:23840811

  3. The Standard Scrapie Cell Assay: Development, Utility and Prospects

    PubMed Central

    van der Merwe, Jacques; Aiken, Judd; Westaway, David; McKenzie, Debbie

    2015-01-01

    Prion diseases are a family of fatal neurodegenerative diseases that involve the misfolding of a host protein, PrPC. Measuring prion infectivity is necessary for determining efficacy of a treatment or infectivity of a prion purification procedure; animal bioassays are, however, very expensive and time consuming. The Standard Scrapie Cell Assay (SSCA) provides an alternative approach. The SSCA facilitates quantitative in vitro analysis of prion strains, titres and biological properties. Given its robust nature and potential for high throughput, the SSCA has substantial utility for in vitro characterization of prions and can be deployed in a number of settings. Here we provide an overview on establishing the SSCA, its use in studies of disease dissemination and pathogenesis, potential pitfalls and a number of remaining challenges. PMID:25602372

  4. Genetic reprogramming of human amniotic cells with episomal vectors: neural rosettes as sentinels in candidate selection for validation assays.

    PubMed

    Wilson, Patricia G; Payne, Tiffany

    2014-01-01

    The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for ?III-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays. PMID:25426336

  5. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)

    EPA Science Inventory

    Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation ...

  6. Enzymatic measurement of phosphatidylserine in cultured cells

    PubMed Central

    Morita, Shin-ya; Shirakawa, Sachimi; Kobayashi, Yukiko; Nakamura, Keiko; Teraoka, Reiko; Kitagawa, Shuji; Terada, Tomohiro

    2012-01-01

    Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0–50 µM) and high (50–1000 µM) concentrations, respectively, and the detection limit was 5 µM (50 pmol in the reaction mixture). This assay quantified PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLC-phosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS. PMID:22100437

  7. An embryogenic cell suspension culture of Picea glauca (White spruce)

    Microsoft Academic Search

    I. Hakman; L. C. Fowke

    1987-01-01

    A cell suspension culture of Picea glauca (White spruce) which continuously produces somatic embryos has been established. Embryogenic callus derived from cultured zygotic embryos was used to initiate the culture. Numerous embryos at various early stages of development were recognized; they exhibited a meristematic embryonic region and suspensor consisting of elongate, vacuolated cells. The culture also contained clumps of meristematic

  8. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.

    PubMed

    Halldorsson, Skarphedinn; Lucumi, Edinson; Gómez-Sjöberg, Rafael; Fleming, Ronan M T

    2015-01-15

    Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture. PMID:25105943

  9. Enzyme-linked immunosorbent assay using monoclonal antibodies for identification of mycobacteria from early cultures.

    PubMed Central

    Verstijnen, C P; Ly, H M; Polman, K; Richter, C; Smits, S P; Maselle, S Y; Peerbooms, P; Rienthong, D; Montreewasuwat, N; Koanjanart, S

    1991-01-01

    A simple enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii has been developed (R. Schöningh, C. P. H. J. Verstijnen, S. Kuijper, and A. H. J. Kolk. J. Clin. Microbiol. 28:708-713, 1990). The test for the routine identification of cultured mycobacteria was introduced in five clinical laboratories located in Tanzania, Thailand, Vietnam, and The Netherlands. The ELISA can be conducted without an ELISA reader since the test can be read visually. The results of identification of 255 strains of the M. tuberculosis complex by microbiological means and by ELISA were compared; the specificity and the sensitivity were 100%. For the M. avium complex, the specificity was 100% and the sensitivity was 64%. All 26 M. kansasii strains tested could be identified as M. kansasii. The ELISA described here proved to be useful in both well- and modestly equipped laboratories and may replace the microbiological method of identification of M. tuberculosis and M. kansasii. PMID:1909344

  10. Quantification of antigen specific CD8 + T cells using an ELISPOT assay

    Microsoft Academic Search

    Yasushi Miyahira; Kenichiro Murata; Dolores Rodriguez; Juan R. Rodriguez; Mariano Esteban; Mauricio M. Rodrigues; Fidel Zavala

    1995-01-01

    An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-? secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by

  11. Triethyllead treatment of cultured brain cells. Effect on accumulation of radioactive precursors in galactolipids

    SciTech Connect

    Grundt, I.K.; Ammitzboll, T.; Clausen, J.

    1981-02-01

    Cultured cells from chick embryo brains were studied for their sensitivity to triethyllead. Triethyllead chloride (3.16 microM) was added to the nutrient medium and incubated for 48 hr with the cells. Morphological changes in light microscope and radioactive labeling of galactolipids were assayed. Triethyllead treatment reduced the number of neuronal cells with processes. Morphological changes were not observed in glial cells. The (/sup 35/S)sulfate labeling of sulfatides was reduced to 50%. The (/sup 3/H)serine labeling of cerebrosides with alpha-hydroxy fatty acids was not influenced, while the (/sup 3/H)serine labeling of cerebrosides with nonhydroxy fatty acids was inhibited 40% in one- and two- but not in three-week-old cultures. The results indicate that the nerve cell response to triethyllead in cultures is selective, since the neurons are more sensitive than the glia cells and the labeling of sulfatides is more sensitive than that of cerebrosides.

  12. Clonal culture of adult mouse lung epithelial stem/progenitor cells.

    PubMed

    McQualter, Jonathan L; Bertoncello, Ivan

    2015-01-01

    Clonal culture of stem cells is crucial for their identification, and the characterization of the cellular and molecular mechanisms that regulate their proliferation and differentiation. In the adult mouse lung, epithelial stem/progenitor cells are defined by the phenotype CD45(neg) CD31(neg) EpCAM(pos) CD104(pos) CD24(low). Here we describe a tissue dissociation and flow cytometry strategy for the detection and isolation of adult mouse lung epithelial stem/progenitor cells, and a three-dimensional colony-forming assay for their clonal culture in vitro. PMID:25388397

  13. T-cell costimulatory capacity of oral and skin epithelial cells in vitro: presence of suppressive activity in supernatants from skin epithelial cell cultures.

    PubMed

    Hasséus, B; Jontell, M; Bergenholtz, G; Dahlgren, U I

    2004-02-01

    Oral Langerhans cells (LC) have better T-cell costimulatory capacity than skin LC. In this study factors affecting this capacity have been assessed in a mixed epithelial cell lymphocyte reaction (MELR) assay. Flow cytometry analysis of freshly recovered cells revealed major histocompatibility complex (MHC) class II molecule expression on 7.5% of the oral epithelial cells and 9.7% of the skin epithelial cells. Monoclonal anti class II antibodies significantly reduced the T-cell proliferation in the MELR. Pretreatment of skin epithelial cells with interleukin-1beta, tumour necrosis factor-alpha or interferon (IFN)-gamma did not affect the MELR proliferation, but incubation with IFNgamma significantly suppressed the T-cell response. Transfer of supernatants from cultures of skin epithelial cells and allogeneic T cells to cultures of oral epithelial cells and T cells resulted in a reduced T-cell proliferation while supernatants from oral epithelial cells and T cells did not reduce proliferation. The higher proliferation in cultures of T cells and oral epithelial cells than in cultures containing skin epithelial cells may be due to the presence of a suppressive factor in the skin epithelial cell suspensions. PMID:14871193

  14. Acetaldehyde and hexanaldehyde from cultured white cells

    PubMed Central

    Shin, Hye-Won; Umber, Brandon J; Meinardi, Simone; Leu, Szu-Yun; Zaldivar, Frank; Blake, Donald R; Cooper, Dan M

    2009-01-01

    Background Noninvasive detection of innate immune function such as the accumulation of neutrophils remains a challenge in many areas of clinical medicine. We hypothesized that granulocytes could generate volatile organic compounds. Methods To begin to test this, we developed a bioreactor and analytical GC-MS system to accurately identify and quantify gases in trace concentrations (parts per billion) emitted solely from cell/media culture. A human promyelocytic leukemia cell line, HL60, frequently used to assess neutrophil function, was grown in serum-free medium. Results HL60 cells released acetaldehyde and hexanaldehyde in a time-dependent manner. The mean ± SD concentration of acetaldehyde in the headspace above the cultured cells following 4-, 24- and 48-h incubation was 157 ± 13 ppbv, 490 ± 99 ppbv, 698 ± 87 ppbv. For hexanaldehyde these values were 1 ± 0.3 ppbv, 8 ± 2 ppbv, and 11 ± 2 ppbv. In addition, our experimental system permitted us to identify confounding trace gas contaminants such as styrene. Conclusion This study demonstrates that human immune cells known to mimic the function of innate immune cells, like neutrophils, produce volatile gases that can be measured in vitro in trace amounts. PMID:19402909

  15. Patterning of polymeric cell culture substrates.

    PubMed

    Welle, Alexander; Weigel, Simone; Bulut, Özgül Demir

    2014-01-01

    The purpose of this chapter is to provide a summary of polymer patterning technologies for biological applications and detailed instructions for resist-free deep ultraviolet (UV) patterning of poly(styrene). Photochemical modifications of this polymer yield unstable peroxides together with stable oxidized chemical groups. The altered physicochemical properties of the polymer surface influence protein adsorption and cell adhesion. HepG2 (human hepatoma cell line), fibroblasts (L929, murine fibroblast line), and other cell lines exhibit strong adhesion on areas of UV-irradiated polymer. Masked irradiations open a simple, fast (cell patterns are obtained within a few hours), and economical route to obtain chemically patterned cell culture substrates. The described protocol is advantageous compared to silane-based patterning techniques on glass or thiol-based patterning on gold because of the elimination of any chemical treatment and the small size of achieved structures. The protocol is compatible with common clean room technologies; however, even without access to a clean room, structured substrates can be produced. The described technique can be a useful tool for a variety of cell cultures used to study biological processes like intercellular communication and organogenesis and for applications like biosensing or tissue engineering. PMID:24439278

  16. Long-Term Culture of Capillary Endothelial Cells

    Microsoft Academic Search

    Judah Folkman; Christian C. Haudenschild; Bruce R. Zetter

    1979-01-01

    Capillary endothelial cells from rats, calves, and humans, have been carried in long-term culture. Bovine capillary endothelial cells have been cloned and maintained by serial passage for longer than 8 months. This prolonged culture was accomplished by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching cells in primary culture. Cultured bovine capillary endothelial cells produce Factor VIII antigen

  17. Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system.

    PubMed

    Torgan, C E; Burge, S S; Collinsworth, A M; Truskey, G A; Kraus, W E

    2000-09-01

    The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight. PMID:11094818

  18. Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

    NASA Technical Reports Server (NTRS)

    Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

    2000-01-01

    The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

  19. Evaluation of new transport medium for detection of herpes simplex virus by culture and direct enzyme-linked immunosorbent assay.

    PubMed Central

    Ogburn, J R; Hoffpauir, J T; Cole, E; Hood, K; Michael, D; Nguyen, T; Raden, S; Raju, B; Reisinger, V; Oefinger, P E

    1994-01-01

    The transport medium Multi-Microbe Media (M4) was evaluated prospectively by culture and direct enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus from 473 specimens. In addition, 377 specimens in Bartels Viral Transport Medium were evaluated. By using culture as a "gold standard," the ELISA sensitivity was approximately 85%, while the specificities exceeded 96% for both media. PMID:7883909

  20. UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR.

    PubMed

    Ko, Gwangpyo; Cromeans, Theresa L; Sobsey, Mark D

    2005-09-01

    Adenoviruses are among the most resistant waterborne pathogens to UV disinfection, yet of the 51 serologically distinct human adenoviruses, only a few have been evaluated for their sensitivities to UV irradiation. Human enteric adenoviruses (Ad40 and Ad41) are difficult to cultivate and reliably assay for infectivity, requiring weeks to obtain cytopathogenic effects (CPE). Inoculated cell cultures often deteriorate before the appearance of distinctive CPE making it difficult to obtain reliable and reproducible data regarding UV inactivation. Adenovirus is a double-stranded DNA virus and produces messenger RNA (mRNA) during replication in host cells. The presence of viral mRNA in host cells is definitive evidence of infection. We recently developed a rapid and reliable cell culture-mRNA RT-PCR assay to detect and quantify adenovirus infectivity. Viral mRNA recovered from cell cultures 5-7 days after infection was purified on oligo-dT latex, treated with DNase, and amplified by RT-PCR using the primers specific for a conserved region of the hexon late mRNA transcript. Treatment of approximately 10(4) Ad41 with different doses of 254 nm germicidal UV radiation resulted in a dose-dependent loss of infectivity. As UV doses were increased from 75 to 200 mJ/cm2, virus survival decreased and no virus infectivity (measured by detectable mRNA) was found at a dose of 225 mJ/cm2 or higher. Our results using the cell culture mRNA RT-PCR assay indicate that Ad41 is more resistant to UV radiation than in a previous study using a conventional cell culture infectivity assay. Results were more similar to those found for Ad 40 using CPE as a measure of infectivity in another previous study. PMID:16046229

  1. Cell-based protein stabilization assays for the detection of interactions between small-molecule inhibitors and BRD4.

    PubMed

    Schulze, Jessica; Moosmayer, Dieter; Weiske, Joerg; Fernández-Montalván, Amaury; Herbst, Christopher; Jung, Marie; Haendler, Bernard; Bader, Benjamin

    2015-02-01

    Bromodomain protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) protein family, acts as a central element in transcriptional elongation and plays essential roles in cell proliferation. Inhibition of BRD4 binding to acetylated histone tails via its two bromodomains, BD1 and BD2, with small-molecule inhibitors has been shown to be a valid strategy to prevent cancer growth. We have evaluated and established two novel assays that quantify the interaction of transfected BRD4 BD1 with chemical inhibitors inside cultured cells. Both methods are based on the principle of ligand-induced protein stabilization by which the binding of a small-molecule inhibitor stabilizes intracellular BRD4 BD1 and protects it from proteolytic degradation. We demonstrate the universal character of this principle by using two orthogonal, highly sensitive detection technologies for the quantification of BRD4 BD1 levels in cellular lysates: enzyme fragment complementation and time-resolved fluorescence resonance energy transfer (TR-FRET). Upon optimization of both assays to a miniaturized high-throughput format, the methods were validated by testing a set of small-molecule BET inhibitors and comparing the results with those from a cell-free binding assay and a biophysical thermal shift assay. In addition, point mutations were introduced into BRD4 BD1, and the corresponding mutants were characterized in the TR-FRET stabilization assay. PMID:25266565

  2. Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices

    DOEpatents

    An, Yuehuei H. (Charleston, SC); Mironov, Vladimir A. (Mt. Pleasant, SC); Gutowska, Anna (Richland, WA)

    2000-01-01

    A gelling cell culture medium useful for forming a three dimensional matrix for cell culture in vitro is prepared by copolymerizing an acrylamide derivative with a hydrophilic comonomer to form a reversible (preferably thermally reversible) gelling linear random copolymer in the form of a plurality of linear chains having a plurality of molecular weights greater than or equal to a minimum gelling molecular weight cutoff, mixing the copolymer with an aqueous solvent to form a reversible gelling solution and adding a cell culture medium to the gelling solution to form the gelling cell culture medium. Cells such as chondrocytes or hepatocytes are added to the culture medium to form a seeded culture medium, and temperature of the medium is raised to gel the seeded culture medium and form a three dimensional matrix containing the cells. After propagating the cells in the matrix, the cells may be recovered by lowering the temperature to dissolve the matrix and centrifuging.

  3. Infection on a chip: a microscale platform for simple and sensitive cell-based virus assays

    E-print Network

    Beebe, David J.

    responses to minimize the spread of viral diseases. While nucleic acid- based assays have dominated newInfection on a chip: a microscale platform for simple and sensitive cell-based virus assays Ying sensitivity, uses excessive reagents, and is hard to automate. Recent modification of the assay to exploit

  4. Sequencing technologies for animal cell culture research.

    PubMed

    Kremkow, Benjamin G; Lee, Kelvin H

    2015-01-01

    Over the last 10 years, 2nd and 3rd generation sequencing technologies have made the use of genomic sequencing within the animal cell culture community increasingly commonplace. Each technology's defining characteristics are unique, including the cost, time, sequence read length, daily throughput, and occurrence of sequence errors. Given each sequencing technology's intrinsic advantages and disadvantages, the optimal technology for a given experiment depends on the particular experiment's objective. This review discusses the current characteristics of six next-generation sequencing technologies, compares the differences between them, and characterizes their relevance to the animal cell culture community. These technologies are continually improving, as evidenced by the recent achievement of the field's benchmark goal: sequencing a human genome for less than $1,000. PMID:25214225

  5. Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.

    PubMed

    Scafetta, Gaia; Tricoli, Eleonora; Siciliano, Camilla; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Cavallaro, Giuseppe; Polistena, Andrea; Frati, Giacomo; De Falco, Elena

    2013-12-01

    Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63?), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63?(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ?Np63? than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-?3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture. PMID:23832306

  6. Bacteriorhodopsin production by cell recycle culture of Halobacterium

    E-print Network

    Bacteriorhodopsin production by cell recycle culture of Halobacterium halobium Sang Yup Lee*, Ho halobium R1 was cultured with cell recycle in a bioreactor equipped with an external hollow fiber membrane- rhodopsin production. The results obtained from batch and cell recycle culture of H. halobium R1

  7. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    ERIC Educational Resources Information Center

    Mozdziak, Paul E.; Petitte, James N.; Carson, Susan D.

    2004-01-01

    Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when…

  8. Apoptosis in Batch Cultures of Chinese Hamster Ovary Cells

    E-print Network

    Sinskey, Anthony J.

    Apoptosis in Batch Cultures of Chinese Hamster Ovary Cells J. Goswami,1 A. J. Sinskey,2 H. Steller of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability. Keywords: cell culture; Chinese Hamster Ovary; apopto- sis; caspase; bcl-2 INTRODUCTION Chinese Hamster

  9. An automated cell-counting algorithm for fluorescently-stained cells in migration assays

    PubMed Central

    2011-01-01

    A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells), images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P < 0.0001 for a total of 47 images), with no difference in the measurements between methods under all conditions. We conclude that our automated method is accurate, more efficient, and void of variability and potential observer bias normally associated with manual counting. PMID:22011343

  10. A cell-based phenotypic assay to identify cardioprotective agents

    PubMed Central

    Guo, Stephanie; Olm-Shipman, Adam; Walters, Andrew; Urciuoli, William R.; Devito, Stefanie; Nadtochiy, Sergiy M.; Wojtovich, Andrew P.; Brookes, Paul S.

    2012-01-01

    Rationale Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS). Objective To develop a model of IR injury that is both physiologically relevant and amenable to HTS. Methods and Results A micro-plate based respirometry apparatus was used. Controlling gas flow in the plate head space, coupled with the instrument’s mechanical systems, yielded a 24 well model of IR injury in which H9c2 cardiomyocytes were transiently trapped in a small volume, rendering them ischemic. Following initial validation with known protective molecules, the model was used to screen a 2000 molecule library, with post IR cell death as an endpoint. pO2 and pH monitoring in each well also afforded metabolic data. Ten protective, detrimental and inert molecules from the screen were subsequently tested in a Langendorff perfused heart model of IR injury, revealing strong correlations between the screening endpoint and both recovery of cardiac function (negative r2=0.66), and infarct size (positive, r2=0.62). Relationships between the effects of added molecules on cellular bioenergetics, and protection against IR injury, were also studied. Conclusion This novel cell-based assay can predict either protective or detrimental effects on IR injury in the intact heart. Its application may help identify therapeutic or harmful molecules. PMID:22394516

  11. A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture.

    PubMed

    Sails, Andrew D; Fox, Andrew J; Bolton, Frederick J; Wareing, David R A; Greenway, David L A

    2003-03-01

    A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens. PMID:12620820

  12. Cell Culture-Derived Influenza Vaccines

    Microsoft Academic Search

    Philip R. Dormitzer

    \\u000a Conventional egg-based vaccine manufacture has provided decades of safe and effective influenza vaccines using the technologies\\u000a of the 1930–1960s. Concerns over the vulnerability of the egg supply in the case of a pandemic with a high pathogenicity avian\\u000a influenza strain have spurred the development and licensure of mammalian cell culture-based influenza vaccines, the first\\u000a major technological innovation in influenza vaccine

  13. Natural killer cells in normal pregnancy: analysis using monoclonal antibodies and single-cell cytotoxicity assays.

    PubMed Central

    Gregory, C D; Lee, H; Rees, G B; Scott, I V; Shah, L P; Golding, P R

    1985-01-01

    Peripheral blood lymphocytes (nylon wool non-adherent) from healthy pregnant women and normal non-pregnant females were tested for natural killer (NK) cell-mediated cytotoxicity against K562 target cells both by 51Cr-release assay and single-cell cytotoxicity assay in agarose. The results indicated depression of NK cytotoxicity in pregnancy due to a decrease in the proportion of target-binding lymphocytes as well as a reduction in the lytic capacity of target-bound cells. The ability of active pregnancy-associated NK lymphocytes to recycle appeared to be unimpaired. Analysis of lymphocyte populations with monoclonal antibodies recognizing NK cell-associated antigens showed that the number of Leu-11+ lymphocytes was reduced in pregnancy. Enumeration of Leu-7+ cells and correlation of NK cell subpopulation data with cytotoxicity assay data suggest that pregnancy is associated with a reduction in the number of mature NK cells and probably also an inhibition of post-binding lytic activity. PMID:3864569

  14. Heat shock induces protein tyrosine phosphorylation in cultured cells

    Microsoft Academic Search

    P. A. Maher; Elena B. Pasquale

    1989-01-01

    We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immuno- blotting and immunofluorescence microscopy experi- ments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The

  15. Cell tri-culture for cardiac vascularization.

    PubMed

    Lesman, Ayelet; Gepstein, Lior; Levenberg, Shulamit

    2014-01-01

    Poor graft survival is a critical obstacle toward production of clinically relevant engineered tissues. Here we utilize a multicellular culturing approach for induction of vascular networks embedded within cardiac tissue constructs. The construct is composed of human cardiomyocytes, endothelial cells (ECs), and embryonic fibroblast cells co-seeded onto highly porous three-dimensional (3D) scaffolds. The resulting vascularized cardiac constructs showed microstructural details characteristic of cardiomyocytes and nascent vessels and exhibited synchronous beating activity in vitro. Upon implantation, stable grafts were formed presenting intense vascularization, with evidence of anastomosis between the pre-formed endothelial capillaries and host neovessels. PMID:25070333

  16. Performance of enzymatic fuel cell in cell culture.

    PubMed

    Lamberg, P; Shleev, S; Ludwig, R; Arnebrant, T; Ruzgas, T

    2014-05-15

    Here we present the very first study of an enzymatic fuel cell (EFC) in a cell culture. An EFC with Corynascus thermophilus cellobiose dehydrogenase (CDH) based bioanode and Myrothecium verrucaria bilirubin oxidase (BOx) based biocathode was constructed at the bottom of a medusa cell culture plate. The constructed EFC had a power density of up to 25 ?W cm(-2) at 0.5 V potential in simple buffer solution and in cell culturing medium. L929 murine fibroblast cells were seeded on top of the EFC and possible effects of the EFC on the cells and vice versa were studied. It was shown that on average the power of the EFC drops by about 70% under a nearly confluent layer of cells. The EFC appeared to have a toxic effect on the L929 cell line. It was concluded that the bioanode, consisting of CDH, produced hydrogen peroxide at toxic concentrations. However, the toxic effect was circumvented by co-immobilizing catalase on the bioanode. PMID:24374299

  17. The use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus

    Microsoft Academic Search

    Jane K. A. Cook; J. H. Darbyshire; R. W. Peters

    1976-01-01

    Summary A study has been made of the use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis (AIB) virus from both naturally and experimentally infected chickens. Six strains of AIB virus were investigated, 3 of which had been isolated from natural outbreaks of disease. Two of the virus isolations from the outbreaks of AIB

  18. Effects of selenomethionine on cell growth and on S-adenosylmethionine metabolism in cultured malignant cells.

    PubMed Central

    Kajander, E O; Harvima, R J; Kauppinen, L; Akerman, K K; Martikainen, H; Pajula, R L; Kärenlampi, S O

    1990-01-01

    The effects of selenomethionine (SeMet) on the growth of 17 cultured cell lines were studied. SeMet in the culture medium of three hepatoma cell lines promoted cell growth at subcytotoxic levels (1-20 microM), but the growth of malignant lymphoid and myeloid cells was not stimulated. L-SeMet was cytotoxic to all 17 cell lines when assayed after culture for 3-10 days. A 50% growth inhibition was observed by 30-160 microM-SeMet in a culture medium containing 100 microM-methionine. SeMet cytotoxicity to normal (fibroblasts) and malignant cells was rather similar, excluding specific antineoplastic cytotoxicity. Cytotoxicity was increased by decreasing concentrations of methionine. The DL form of SeMet was less cytotoxic than the L form. L-SeMet was metabolized to a selenium analogue of S-adenosylmethionine approximately as effectively as the natural sulphur analogue methionine in malignant R1.1 lymphoblasts. Concomitantly, S-adenosylmethionine pools were decreased. This occurred early and at cytotoxic SeMet levels. Methionine adenosyltransferase activity was not altered by SeMet treatment. ATP pools were not affected early, and decreases in the synthesis of DNA and protein took place late and were apparently related to cell death. RNA synthesis was slightly stimulated at low cytotoxic SeMet levels by 24 h, but was markedly inhibited after 48 h. The SeMet analogue of S-adenosylmethionine could be effectively utilized in a specific enzymic transmethylation. Neither S-adenosylhomocysteine nor its selenium analogue accumulated in the treated cells. These findings together suggest a direct or indirect involvement of S-adenosylmethionine metabolism in SeMet cytotoxicity, but exclude a gross blockage of transmethylations. PMID:2339986

  19. Ascorbic acid transport into cultured pituitary cells

    SciTech Connect

    Cullen, E.I.; May, V.; Eipper, R.A.

    1986-05-01

    An amidating enzyme designated peptidyl-glycine ..cap alpha..-amidating monooxygenase (PAM) has been studied in a variety of tissues and is dependent on molecular oxygen and stimulated by copper and ascorbic acid. To continue investigating the relationship among cellular ascorbic acid concentrations, amidating ability, and PAM activity, the authors studied ascorbic acid transport in three cell preparations that contain PAM and produce amidated peptides: primary cultures of rat anterior and intermediate pituitary and mouse AtT-20 tumor cells. When incubated in 50 ..mu..M (/sup 14/C)ascorbic acid all three cell preparations concentrated ascorbic acid 20- to 40-fold, producing intracellular ascorbate concentrations of 1 to 2 mM, based on experimentally determined cell volumes. All three cell preparations displayed saturable ascorbic acid uptake with half-maximal initial rates occurring between 9 and 18 ..mu..M ascorbate. Replacing NaCl in the uptake buffer with choline chloride significantly diminished ascorbate uptake in all three preparations. Ascorbic acid efflux from these cells was slow, displaying half-lives of 7 hours. Unlike systems that transport dehydroascorbic acid, the transport system for ascorbic acid in these cells was not inhibited by glucose. Thus, ascorbate is transported into pituitary cells by a sodium-dependent, active transport system.

  20. Pneumocystis jirovecii Can Be Productively Cultured in Differentiated CuFi-8 Airway Cells

    PubMed Central

    Schildgen, Verena; Mai, Stephanie; Khalfaoui, Soumaya; Lüsebrink, Jessica; Pieper, Monika; Tillmann, Ramona L.; Brockmann, Michael

    2014-01-01

    ABSTRACT Although Pneumocystis jirovecii is a well-known and serious pathogen, all previous attempts to isolate, cultivate, and propagate this fungus have failed. This serious challenge in microbiology was addressed in the present study. We examined whether P. jirovecii could be cultured in a permanent three-dimensional air-liquid interface culture system formed by CuFi-8 cells, a differentiated pseudostratified airway epithelial cell line. Cultured pseudostratified cells were inoculated with bronchoalveolar fluid that had been confirmed to be positive for P. jirovecii using PCR. Five days later, the cells and basal medium were harvested and tested for P. jirovecii using quantitative PCR (qPCR), commercially available immunofluorescence detection assays, and Grocott staining of formalin-fixed, paraffin-embedded thin sections of infected-cell cultures. We successfully productively cultivated and propagated P. jirovecii from these P. jirovecii-positive bronchoalveolar lavage fluid (BALF) samples. Furthermore, we provide evidence that P. jirovecii induced cytopathic effects on lung epithelial cells and was even invasive in cell culture. To the best of our knowledge, the cell culture system developed herein represents the first methodology to enable molecular analyses of this pathogen’s life cycle and further in vitro studies of P. jirovecii, such as assessments of drug sensitivity and resistance as well as investigations of the pathogen’s stability against environmental factors and disinfectants. PMID:24825015

  1. Developing cell culture-derived pandemic vaccines.

    PubMed

    Barrett, P Noel; Portsmouth, Daniel; Ehrlich, Hartmut J

    2010-02-01

    The growing prospect of avian influenza viruses achieving sustained interhuman transmission, combined with the recent emergence of a novel swine-origin A/H1N1 influenza strain, has brought the issue of influenza vaccine production capacity into sharp focus. It is becoming increasingly clear that traditional egg-based manufacturing processes may be insufficient to meet global vaccine demands in a pandemic situation that is caused by a highly pathogenic influenza virus. This review introduces the concepts of modern, cell culture-derived influenza vaccines and their manufacture, and explains the advantages of these vaccines in terms of both speed and efficiency of production as well as immunogenic efficacy. Vaccine production technologies using the mammalian cell lines Vero, MDCK and PER.C6, as well as the baculovirus/insect cell platform, are described in detail. Clinical data are provided from cell culture-derived vaccines that are at an advanced stage of development, and insights are provided into recent developments in the preclinical evaluation of more experimental technologies. PMID:20140813

  2. Adhesive forces in embryonic stem cell cultures

    PubMed Central

    Blancas, Alicia A; Chen, Chi-Shuo; Stolberg, Sarah

    2011-01-01

    Most cell culture systems grow and spread as contact-inhibited monolayers on flat culture dishes, but the embryonic stem cell (ESC) is one of the cell phenotypes that prefer to self-organize as tightly packed three-dimensional (3D) colonies. ESC also readily form 3D cell aggregates, called embryoid bodies (EB) that partially mimic the spatial and temporal processes of the developing embryo. Here, the rationale for ESC aggregation, rather than “spreading” on gelatin-coated or mouse embryonic fibroblast (MEF)-coated dishes, is examined through the quantification of the expression levels of adhesion molecules on ESC and the calculation of the adhesive forces on ESC. Modeling each ESC as a dodecahedron, the adhesive force for each ESC-ESC binding was found to be 9.1 × 105 pN, whereas, the adhesive force for ESC-MEF binding was found to be an order of magnitude smaller at 7.9 × 104 pN. We also show that E-cadherin is the dominating molecule in the ESC-ESC adhesion and blocking E-cadherin leads to a significant reduction in colony formation. Here, we mathematically describe the preference for ESC to self-assemble into ESC-ESC aggregates and 3D colonies, rather than to bind and spread on gelatin or MEF-coated dishes, and have shown that these interactions are predominantly due to E-cadherin expression on ESC. PMID:22274712

  3. Characterization of olfactory receptor neurons and other cell types in dissociated rat olfactory cell cultures

    Microsoft Academic Search

    S. K. Pixley

    1996-01-01

    In dissociated cell cultures, control over the cellular environment facilitates study of the differentiation of mature cellular phenotypes. Central to this approach is a rigorous characterization of the cells that reside in culture. Therefore, we have used a battery of cell type-specific antibody markers to identify the cell types present in dissociated cultures of olfactory mucosal cells (containing cells from

  4. Isoflavone daidzein possesses no antioxidant activities in cell-free assays but induces the antioxidant enzyme catalase.

    PubMed

    Kampkötter, Andreas; Chovolou, Yvonni; Kulawik, Andreas; Röhrdanz, Elke; Weber, Nadine; Proksch, Peter; Wätjen, Wim

    2008-09-01

    Epidemiologic studies have shown that dietary intake of isoflavonones is associated with several properties beneficial to human health. It has been suggested that at least some of these effects are related to the antioxidant activity of isoflavonoids. We analyzed the antioxidant activity of the major isoflavones found in soybeans, but none of these compounds showed prominent antioxidant effects in cell-free assay systems (trolox equivalent antioxidant capacity assay and 2,2-diphenyl-1-picrylhydrazyl assay). Therefore, we examined the hypothesis that the antioxidative effects of isoflavones are caused indirectly by up-regulation of antioxidative enzymes, thereby lowering intracellular concentration of reactive oxygene species. Daidzein shows a significant induction of catalase promoter activity at 100 micromol/L in a reporter gene assay and at 200 micromol/L in Northern blot experiments. Another hypothesis for antioxidant effects caused by isoflavones is due to metabolism by intestinal bacteria. Analyzing the daidzein metabolites 3'-OH-daidzein and 6-OH-daidzein in our cell culture model, we found strong antioxidant effects (2,2-diphenyl-1-picrylhydrazyl and trolox equivalent antioxidant capacity assay). We conclude that isoflavone daidzein up-regulates the antioxidant enzyme catalase but shows only little antioxidant capacity per se. Antioxidant effects of this dietary isoflavonone may also be due to formation of the antioxidant metabolites 6-OH-daidzein and 3'-OH-daidzein. PMID:19083468

  5. Survival of retinal ganglion cells in slice culture provides a rapid screen for olfactory ensheathing cell preparations.

    PubMed

    Dai, Chao; Qin Yin, Zheng; Li, Ying; Raisman, Geoffrey; Li, Daqing

    2010-10-01

    Transplants of olfactory ensheathing cells (OECs) cultured from the olfactory bulb are able to induce structural regeneration of severed central axons and return of function in rat models. For clinical purposes it would be preferable to obtain the cells from the more accessible olfactory mucosa in the nasal lining. However, in our laboratory preparations cultures from mucosal samples yielded around 5% of OECs compared with the 50% obtained from samples cultured from the bulb, and when transplanted these mucosal cell preparations were less effective at repair. There are a number of manipulations which may increase the OEC content and the effectiveness of mucosal preparations, but in vivo transplantation would be a highly labour intensive method for evaluating them. As a candidate for a high throughput assay to screen for beneficial effects of modifications to mucosal cells we here report the effects of co-culture of the cells with retinal explants. Both bulbar and mucosal cell preparations prolong the survival of the explants. Counts of the surviving retinal ganglion cells, identified by beta-III-tubulin immunohistochemistry and by their axon trajectory, show that the bulbar cell preparations have around twice the potency of those from the mucosa. This in vitro system, therefore, provides a bioassay that discriminates bulbar and mucosal cell preparations, and a useful tool for evaluating the functional effects of manipulations of cultured mucosal preparations. PMID:20682293

  6. Cell Co-culture Patterning Using Aqueous Two-phase Systems

    PubMed Central

    Frampton, John P.; White, Joshua B.; Abraham, Abin T.; Takayama, Shuichi

    2013-01-01

    Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type. PMID:23567187

  7. Perfluoroalkylated compounds induce cell death and formation of reactive oxygen species in cultured cerebellar granule cells.

    PubMed

    Reistad, Trine; Fonnum, Frode; Mariussen, Espen

    2013-03-27

    The present communication investigates the effects of different perfluoroalkylated compounds (PFCs) on formation of reactive oxygen species (ROS) and cell death in cultured cerebellar granule cells. This allows direct comparison with similar effects found for other environmental contaminants like polychlorinated biphenyls and brominated flame-retardants. The increase in ROS formation and cell death was assayed using the fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA) and the trypan blue exclusion assay. The effects of the PFCs were structure dependent. Cell death was induced at relatively low concentrations by perfluorooctyl sulfonate (PFOS), perfluorooctane sulfonylamide (PFOSA) and the fluorotelomer alcohol 1H, 1H, 2H, 2H-perfluorodecanol (FTOH 8:2) with EC(50)-values of 62 ± 7.6, 13 ± 1.8 and 15 ± 4.2 ?M (mean ± SD) respectively. PFOS, perfluorooctanoic acid (PFOA) and PFOSA induced a concentration dependent increase in ROS formation with EC(50)-values of 27 ± 9.0, 25 ± 11 and 57 ± 19?M respectively. Reduced cell viability and ROS formation were observed at concentration level close to what is found in serum of occupationally exposed workers. The effect of PFCs on ROS formation and cell viability was compared with other halogenated compounds and future investigations should emphasize effects of mixtures and how physical chemical properties of the compounds influence their toxicity. PMID:23340305

  8. Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line

    Microsoft Academic Search

    Stephanie K Bopp; Teresa Lettieri

    2008-01-01

    BACKGROUND: A broad spectrum of cytotoxicity assays is currently used in the fields of (eco)toxicology and pharmacology. To choose an appropriate assay, different parameters like test compounds, detection mechanism, specificity, and sensitivity have to be considered. Furthermore, tissue or cell line can influence test performance. For zebrafish (Danio rerio), as emerging model organism, cell lines are now increasingly used, but

  9. Rotating bio-reactor cell culture apparatus

    NASA Technical Reports Server (NTRS)

    Schwarz, Ray P. (inventor); Wolf, David A. (inventor)

    1991-01-01

    A bioreactor system is described in which a tubular housing contains an internal circularly disposed set of blade members and a central tubular filter all mounted for rotation about a common horizontal axis and each having independent rotational support and rotational drive mechanisms. The housing, blade members and filter preferably are driven at a constant slow speed for placing a fluid culture medium with discrete microbeads and cell cultures in a discrete spatial suspension in the housing. Replacement fluid medium is symmetrically input and fluid medium is symmetrically output from the housing where the input and the output are part of a loop providing a constant or intermittent flow of fluid medium in a closed loop.

  10. Microfluidic Probe for Single-Cell Lysis and Analysis in Adherent Tissue Culture

    PubMed Central

    Lauffenburger, Douglas A.; Han, Jongyoon

    2014-01-01

    Single-cell analysis provides information critical to understanding key disease processes that are characterized by significant cellular heterogeneity. Few current methods allow single-cell measurement without removing cells from the context of interest, which not only destroys contextual information but also may perturb the process under study. Here we present a microfluidic probe that lyses single adherent cells from standard tissue culture and captures the contents to perform single-cell biochemical assays. We use this probe to measure kinase and housekeeping protein activities, separately or simultaneously, from single human hepatocellular carcinoma cells in adherent culture. This tool has the valuable ability to perform measurements that clarify connections between extracellular context, signals and responses, especially in cases where only a few cells exhibit a characteristic of interest. PMID:24594667

  11. Erythrocytic malaria growth or invasion inhibition assays with emphasis on suspension culture GIA.

    PubMed

    Haynes, J David; Moch, J Kathleen; Smoot, Douglas S

    2002-01-01

    Erythrocytic cycle malaria parasite growth or invasion inhibition assays (GIA) compare the effects of various test and control substances on malaria parasite growth in erythrocytes or invasion into erythrocytes in vitro. Although inhibitions by antimalarial drugs in vitro correlate well with drug protective levels required in vivo, as yet there are too few data to know how well inhibitions by antibodies in vitro correlate with the types and degrees of immune protection in vivo. Antibody-mediated GIA is frequently complicated by parasite strain-specific inhibitions, as well as nonspecific inhibitory factors generated in sera collected or stored under nonoptimal conditions. In this chapter, we describe methods for collecting and processing sera, for using different strains of parasite, and a simplified method for staining parasite DNA with Hoechst dye 33342 before quantitating parasites using ultraviolet (UV)-excited flow cytometry. We also describe a new type of GIA using suspension cultures in a 48-well plate. Critical to this method is enclosing the plate in a gassed, heat-sealed plastic bag, which, being low mass, can easily be rested at a 13.5 degrees angle on a rotor platform (114 rpm with 1-in. displacement) to produce gentle pulsatile waves of media in each well. The suspension GIA, which, relative to the static GIA, increased inhibition by one antibody and decreased inhibition by another (Table 1), may better simulate in vivo blood flow and may thus better predict in vivo efficacy. PMID:12125152

  12. ASBESTOS AND GASTRO-INTESTINAL CANCER: CELL CULTURE STUDIES

    EPA Science Inventory

    Three forms of asbestos: amosite, crocidolite, and chrysotile, were assayed for their cytotoxicity and mutagenicity in cell clture. Using embjryonic human intestine derived and adult rat liver derived epitelial cells, the order of toxicity was chrysotile > amosite = crocidolite. ...

  13. Cell types in rat liver cultures: their identification and isolation

    Microsoft Academic Search

    J. W. Grisham

    1983-01-01

    This paper reviews the various types of cells in the liver in vivo and in hepatic cellular suspensions produced by perfusion of the liver with collagenase solutions. Methods to identify and isolate different types of hepatic cells are discussed. In vitro culture of various types of liver cells is reviewed and the identification of cultured cells is considered.

  14. Cardiac Cells Beating in Culture: A Laboratory Exercise

    ERIC Educational Resources Information Center

    Weaver, Debora

    2007-01-01

    This article describes how to establish a primary tissue culture, where cells are taken directly from an organ of a living animal. Cardiac cells are taken from chick embryos and transferred to culture dishes. These cells are not transformed and therefore have a limited life span. However, the unique characteristics of cardiac cells are maintained…

  15. Differentiated cultures of primary hamster tracheal airway epithelial cells.

    PubMed

    Rowe, Regina K; Brody, Steven L; Pekosz, Andrew

    2004-01-01

    Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating tracheal epithelial cultures from Syrian golden hamsters and characterized the cultures for cell composition and function. Soon after initial plating, the epithelial cells reached a high transepithelial resistance and formed tight junctions. The cells differentiated into a heterogeneous, multicellular culture containing ciliated, secretory, and basal cells after culture at an air-liquid interface (ALI). The secretory cell populations initially consisted of MUC5AC-positive goblet cells and MUC5AC/CCSP double-positive cells, but the makeup changed to predominantly Clara cell secretory protein (CCSP)-positive Clara cells after 14 d. The ciliated cell populations differentiated rapidly after ALI, as judged by the appearance of beta tubulin IV-positive cells. The cultures produced mucus, CCSP, and trypsin-like proteases and were capable of wound repair as judged by increased expression of matrilysin. Our method provides an efficient, high-yield protocol for producing differentiated hamster tracheal epithelial cells that can be used for a variety of in vitro studies including tracheal cell differentiation, airway disease mechanisms, and pathogen-host interactions. PMID:15780007

  16. Determination of the activity of pyrimethamine, trimethoprim, sulfonamides, and combinations of pyrimethamine and sulfonamides against Sarcocystis neurona in cell cultures

    Microsoft Academic Search

    David S Lindsay; J. P Dubey

    1999-01-01

    Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. The activities of pyrimethamine, trimethoprim, sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfamethazine, and sulfathiazole were examined against developing S. neurona merozoites in bovine turbinate cell cultures. A microtiter plate host cell lesion based assay was used to

  17. Detection of DNA damages and repair in human culture cells with simulated space radiation

    NASA Astrophysics Data System (ADS)

    Nagaoka, S.; Nakano, T.; Endo, S.; Onizuka, T.; Kagawa, Y.; Fujitaka, K.; Ohnishi, K.; Takahashi, A.; Ohnishi, T.

    1999-09-01

    DNA damages and its repair of cultured WI38 human fibroblast cells and T98G human glioblastoma cells were studied by exposing to carbon ion beams of HIMAC accelerator. The exposed cells were incubated at 37 °C for appropriate intervals and the damages were analyzed by alkaline comet assay and quantitative RT-PCR with p53 mRNA Highly inhomogeneous DNA damages were observed among the electrophoretic cell images of the comet assay. The degree of the damages was analyzed semi-quantitatively by using the Comet Index. The damaged fraction of WI38 cells was 85% immediately after 4 Gy (100 keV/?m) irradiation and decreased to 50% after 120 min. incubation indicating a repair of cell DNA. Time dependent p53 gene expression was also analyzed by the quantitative RT-PCR method.

  18. Equipment for large-scale mammalian cell culture.

    PubMed

    Ozturk, Sadettin S

    2014-01-01

    This chapter provides information on commonly used equipment in industrial mammalian cell culture, with an emphasis on bioreactors. The actual equipment used in the cell culture process can vary from one company to another, but the main steps remain the same. The process involves expansion of cells in seed train and inoculation train processes followed by cultivation of cells in a production bioreactor. Process and equipment options for each stage of the cell culture process are introduced and examples are provided. Finally, the use of disposables during seed train and cell culture production is discussed. PMID:24429549

  19. Neonatal rat heart cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Akins, R. E.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R.

    1997-01-01

    In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

  20. Neonatal rat heart cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Akins, Robert E.; Schroedl, Nancy A.; Gonda, Steve R.; Hartzell, Charles R.

    1994-01-01

    In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARV's adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARV's using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

  1. Epigenomic Consequences of Immortalized Plant Cell Suspension Culture

    Microsoft Academic Search

    Milos Tanurdzic; Matthew W Vaughn; Hongmei Jiang; Tae-Jin Lee; R. Keith Slotkin; Bryon Sosinski; William F Thompson; R. W Doerge; Robert A Martienssen

    2008-01-01

    Plant cells grown in culture exhibit genetic and epigenetic instability. Using a combination of chromatin immunoprecipitation and DNA methylation profiling on tiling microarrays, we have mapped the location and abundance of histone and DNA modifications in a continuously proliferating, dedifferentiated cell suspension culture of Arabidopsis. We have found that euchromatin becomes hypermethylated in culture and that a small percentage of

  2. Retrotransposition of marked SVA elements by human L1s in cultured cells.

    PubMed

    Hancks, Dustin C; Goodier, John L; Mandal, Prabhat K; Cheung, Ling E; Kazazian, Haig H

    2011-09-01

    Human retrotransposons generate structural variation and genomic diversity through ongoing retrotransposition and non-allelic homologous recombination. Cell culture retrotransposition assays have provided great insight into the genomic impact of retrotransposons, in particular, LINE-1(L1) and Alu elements; however, no such assay exists for the youngest active human retrotransposon, SINE-VNTR-Alu (SVA). Here we report the development of an SVA cell culture retrotransposition assay. We marked several SVAs with either neomycin or EGFP retrotransposition indicator cassettes. Engineered SVAs retrotranspose using L1 proteins supplemented in trans in multiple cell lines, including U2OS osteosarcoma cells where SVA retrotransposition is equal to that of an engineered L1. Engineered SVAs retrotranspose at 1-54 times the frequency of a marked pseudogene in HeLa HA cells. Furthermore, our data suggest a variable requirement for L1 ORF1p for SVA retrotransposition. Recovered engineered SVA insertions display all the hallmarks of LINE-1 retrotransposition and some contain 5' and 3' transductions, which are common for genomic SVAs. Of particular interest is the fact that four out of five insertions recovered from one SVA are full-length, with the 5' end of these insertions beginning within 5 nt of the CMV promoter transcriptional start site. This assay demonstrates that SVA elements are indeed mobilized in trans by L1. Previously intractable questions regarding SVA biology can now be addressed. PMID:21636526

  3. Establishment of a primary culture of Echinococcus multilocularis germinal cells.

    PubMed

    Yamashita, K; Uchino, J; Sato, N; Furuya, K; Namieno, T

    1997-06-01

    This study was designed to establish an in vitro primary culture of germinal cells of Echinococcus multilocularis, a parasite that causes alveolar echinococcosis of the liver (AEL). We also investigated the temperature-dependency of the cultured cells. The germinal cells, which originated from a human lesion, were cultured by an original fluid-suspension method at 25 degrees C or 37 degrees C for 4 weeks. Anchorage-dependent and -independent cells were observed by light microscopy, transmission electron microscopy, and immunocytochemistry to confirm their origin. Cell number and viability were examined by immunocytochemistry and mitochondrial exclusion test. The cultured cells were also inoculated into jirds (Meriones unguiculatus) to evaluate metacestode formation. Morphology and immunocytochemistry showed that the cultured cells were typically germinal cells. The cell number declined gradually over the 4-week culture period, but viability remained at 50% at 3 weeks. These findings were not associated with either of the two culture temperatures; moreover, host-associated cells were not noted in the cultured cells at 25 degrees C. The implanted cells formed metacestodes in the jird peritoneal cavity, and their histology demonstrated mature and typical alveolar-type echinococcal cysts. We successfully established an in vitro primary culture of germinal cells. This should contribute to future studies, and, hence, a better outcome for patients with AEL. PMID:9213248

  4. Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity.

    PubMed

    Chiu, Brian; Wan, Jim Z-M; Abley, Doris; Akabutu, John

    2005-01-01

    Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity (microg) may modulate the proliferation and differentiation. We investigated the application of microg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated microg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated microg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy. PMID:15835045

  5. Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

    NASA Astrophysics Data System (ADS)

    Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

    2005-05-01

    Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( ?g) may modulate the proliferation and differentiation. We investigated the application of ?g to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated ?g for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated ?g may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

  6. Salt tolerance in cultured cells of Spartina pectinata

    Microsoft Academic Search

    R. Scott Warren; Lisa M. Baird; Angela K. Thompson

    1985-01-01

    Suspension cultures with cell doubling times of ca. 2 days were developed from the halophytic grass Spartina pectinata. Maximum rates of exponential growth measured by direct cell counts and by total culture packed-cell-volume were not significantly reduced by NaCl up to 200 mM but dropped beyond this point. In contrast, total cell production over a one week culture cycle, by

  7. Recombinant Protein Production and Insect Cell Culture and Process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  8. Biology on a Chip: Microfabrication for Studying the Behavior of Cultured Cells

    PubMed Central

    Li, Nianzhen; Tourovskaia, Anna; Folch, Albert

    2013-01-01

    The ability to culture cells in vitro has revolutionized hypothesis testing in basic cell and molecular biology research and has become a standard methodology in drug screening and toxicology assays. However, the traditional cell culture methodology—consisting essentially of the immersion of a large population of cells in a homogeneous fluid medium—has become increasingly limiting, both from a fundamental point of view (cells in vivo are surrounded by complex spatiotemporal microenvironments) and from a practical perspective (scaling up the number of fluid handling steps and cell manipulations for high-throughput studies in vitro is prohibitively expensive). Micro fabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, the medium composition, as well as the type of neighboring cells surrounding the microenvironment of the cell. In addition, microtechnology is conceptually well suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. Here we review a variety of applications of microfabrication in cell culture studies, with an emphasis on the biology of various cell types. PMID:15139302

  9. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture

    PubMed Central

    Gilbert, PM; Havenstrite, KL; Magnusson, KEG; Sacco, A; Leonardi, NA; Kraft, P; Nguyen, NK; Thrun, S; Lutolf, MP; Blau, HM

    2010-01-01

    Freshly isolated muscle stem cells (MuSCs) exhibit robust regenerative capacity in vivo that is rapidly lost in culture. Using a bioengineered substrate to recapitulate key biophysical and biochemical niche features in conjunction with a novel highly automated single cell tracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. Unlike MuSCs on rigid plastic dishes (~106kPa), MuSCs cultured on soft hydrogel substrates that mimic the elasticity of muscle (12kPa) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice and assayed histologically and quantitatively by non-invasive bioluminescence imaging. Our studies provide novel evidence that by recapitulating physiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-based therapies for muscle wasting diseases. PMID:20647425

  10. Cell immunoblot assay study demonstrating the release of PACAP from individual anterior pituitary cells of rats and the effect of PACAP on LH release.

    PubMed

    Szabó, E; Nemeskéri, A; Heinzlmann, A; Suzuki, N; Arimura, A; Köves, K

    2002-11-15

    The presence of pituitary adenylate cyclase activating polypeptide (PACAP) was previously demonstrated in the anterior pituitary by radioimmunoassay, immunohistochemistry, and reverse transcript-polymerase chain reaction (RT-PCR). With the use of cell immunoblot assay (CIBA), when the pituitary cells were cultured on nitrocellulose membrane, the release of PACAP by individual anterior pituitary cells was observed. The released peptide, trapped by the nitrocellulose membrane forming a blot around the cells, was demonstrated by immunocytochemistry. Double labeling revealed that a part of PACAP-immunoreactive cells can release LH as well. With the use of sandwich enzyme immunoassay (S-EIA), it was found that the concentration of PACAP in the anterior pituitaries is 10(-10) M. In cell culture in a similar concentration, PACAP stimulated the LH release from female gonadotropes, but did not influence it from male ones. The stimulated release of LH was indicated by the enhancement in the diameter of LH blots compared to the untreated control cultures. We concluded that PACAP may be released from the anterior pituitary cells in a concentration which would be able to influence LH release not only in vitro but under in vivo conditions as well. The effect of PACAP on LH release was different in female and male pituitary cultures. PMID:12409218

  11. Analysis of Matrix-Dependent Cell Migration with a Barrier Migration Assay

    NSDL National Science Digital Library

    Sven Kroening (University Hospital Erlangen; Department of Nephrology and Hypertension REV)

    2010-06-15

    Cell migration plays a pivotal role in many biological processes and is modulated by cytokines and growth factors. In vivo, cells are embedded in an extracellular matrix (ECM). ECM proteins are linked to the cellular cytoskeleton by integrin adhesion receptors, which transmit extracellular signals into the cell, thereby affecting cell adhesion and migration as well as gene expression. We describe a cell migration assay that uses a barrier device to separate the cells. The assay enables quantification of the migration of adherent cells on defined matrix proteins and the ability to evaluate migration-associated characteristics of individual cells. Thus, the barrier cell migration assay is a useful tool for exploring matrix-dependent migration of adherent cells.

  12. [Isolation and culture of fetal bovine intestine-derived epithelial stem cells and the differentiation into hepatocyte-like cells].

    PubMed

    Sun, Tingting; Cai, Lianshun; Guan, Weijun

    2015-01-01

    Objective To establish the culture system of fetal bovine intestinal epithelial stem cells (IESCs) in vitro, identify specific markers of the cell lines and analyze the differentiation potential into hepatocyte-like cells. Methods IESCs were isolated from the 3- to 5-month fetal bovine intestine by the digestion of collagenase I, and cultured in the DMEM/F12 medium. The cell morphology was observed, and the proliferation ability and multiple differentiation potential were demonstrated by subculturing and its growth curve. The mRNA expressions of the surface markers Bmi1, Hes1, Lgr5 and cytokeratin 19 (CK19) were determined by reverse transcription PCR (RT-PCR), and the protein levels of Bmi1, LGR5 and CK19 were detected by immunofluorescence cytochemistry. Under the induction of fibroblast growth factor 4 (FGF-4) and hepatocyte growth factor (HGF), the cell differentiation into hepatocyte-like cells was assayed by the glycogen staining and RT-PCR. Results IESCs cultured in vitro expressed Bmi1, Hes1, Lgr5 and CK19 mRNAs, and CK19, Bmi1 and LGR5 proteins. The differentiated cells were positively stained by glycogen, and RT-PCR showed that the cells expressed ?-fetoprotein (AFP) and albumin (ALB) mRNAs. Conclusion The culture system of IESCs in vitro is successfully established, and the cells are differentiated into hepatocyte-like cells. PMID:25575060

  13. A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila

    NASA Astrophysics Data System (ADS)

    Apostol, Barbara L.; Kazantsev, Alexsey; Raffioni, Simona; Illes, Katalin; Pallos, Judit; Bodai, Laszlo; Slepko, Natalia; Bear, James E.; Gertler, Frank B.; Hersch, Steven; Housman, David E.; Marsh, J. Lawrence; Michels Thompson, Leslie

    2003-05-01

    The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds.

  14. Detection of Infectious Adenovirus in Cell Culture by mRNA Reverse Transcription-PCR

    PubMed Central

    Ko, Gwangpyo; Cromeans, Theresa L.; Sobsey, Mark D.

    2003-01-01

    We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 106 infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 106 IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples. PMID:14660388

  15. Detection of infectious adenovirus in cell culture by mRNA reverse transcription-PCR.

    PubMed

    Ko, Gwangpyo; Cromeans, Theresa L; Sobsey, Mark D

    2003-12-01

    We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 10(6) infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 10(6) IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples. PMID:14660388

  16. Extraction parameters for metabolomics from cultured cells.

    PubMed

    Ser, Zheng; Liu, Xiaojing; Tang, Ngoc Nu; Locasale, Jason W

    2015-04-15

    The successful extraction of metabolites is a critical step in metabolite profiling. By optimizing metabolite extraction, the range and quantitative capacity of metabolomics studies can be improved. We considered eight separate extraction protocols for the preparation of a metabolite extract from cultured mammalian cells. Parameters considered included temperature, pH, and cell washing before extraction. The effects on metabolite recovery were studied using a liquid chromatography high-resolution mass spectrometry (LC-HRMS) platform that measures metabolites of diverse chemical classes, including amino acids, lipids, and sugar derivatives. The temperature considered during the extraction or the presence of formic acid, a commonly used additive, was shown to have minimal effects on the measured ion intensities of metabolites. However, washing of samples before metabolite extraction, whether with water or phosphate-buffered saline, exhibited dramatic effects on measured intensities of both intracellular and extracellular metabolites. Together, these findings present a systematic assessment of extraction conditions for metabolite profiling. PMID:25613493

  17. Cellulose biosynthesis inhibitors: comparative effect on bean cell cultures.

    PubMed

    García-Angulo, Penélope; Alonso-Simón, Ana; Encina, Antonio; Alvarez, Jesús M; Acebes, José L

    2012-01-01

    The variety of bioassays developed to evaluate different inhibition responses for cellulose biosynthesis inhibitors makes it difficult to compare the results obtained. This work aims (i) to test a single inhibitory assay for comparing active concentrations of a set of putative cellulose biosynthesis inhibitors and (ii) to characterize their effect on cell wall polysaccharides biosynthesis following a short-term exposure. For the first aim, dose-response curves for inhibition of dry-weight increase following a 30 days exposure of bean callus-cultured cells to these inhibitors were obtained. The compound concentration capable of inhibiting dry weight increase by 50% compared to control (I(50)) ranged from subnanomolar (CGA 325'615) to nanomolar (AE F150944, flupoxam, triazofenamide and oxaziclomefone) and micromolar (dichlobenil, quinclorac and compound 1) concentrations. In order to gain a better understanding of the effect of the putative inhibitors on cell wall polysaccharides biosynthesis, the [(14)C]glucose incorporation into cell wall fractions was determined after a 20 h exposure of cell suspensions to each inhibitor at their I(50) value. All the inhibitors tested decreased glucose incorporation into cellulose with the exception of quinclorac, which increased it. In some herbicide treatments, reduction in the incorporation into cellulose was accompanied by an increase in the incorporation into other fractions. In order to appreciate the effect of the inhibitors on cell wall partitioning, a cluster and Principal Component Analysis (PCA) based on the relative contribution of [(14)C]glucose incorporation into the different cell wall fractions were performed, and three groups of compounds were identified. The first group included quinclorac, which increased glucose incorporation into cellulose; the second group consisted of compound 1, CGA 325'615, oxaziclomefone and AE F150944, which decreased the relative glucose incorporation into cellulose but increased it into tightly-bound cellulose fractions; and the third group, comprising flupoxam, triazofenamide and dichlobenil, decreased the relative glucose incorporation into cellulose and increased it into a pectin rich fraction. PMID:22489176

  18. Reduction of misleading ("false") positive results in mammalian cell genotoxicity assays. I. Choice of cell type.

    PubMed

    Fowler, Paul; Smith, Katie; Young, Jamie; Jeffrey, Laura; Kirkland, David; Pfuhler, Stefan; Carmichael, Paul

    2012-02-18

    Current in vitro mammalian cell genotoxicity assays show a high rate of positive results, many of which are misleading when compared with in vivo genotoxicity or rodent carcinogenicity data. P53-deficiency in many of the rodent cell lines may be a key factor in this poor predictivity. As part of an European Cosmetics Industry Association initiative for improvement of in vitro mammalian cell assays, we have compared several rodent cell lines (V79, CHL, CHO) with p53-competent human peripheral blood lymphocytes (HuLy), TK6 human lymphoblastoid cells, and the human liver cell line, HepG2. We have compared in vitro micronucleus (MN) induction following treatment with 19 compounds that were accepted as producing misleading or "false" positive results in in vitro mammalian cell assays [6]. Of these, six chemicals (2-ethyl-1,3-hexandiol, benzyl alcohol, urea, sodium saccharin, sulfisoxazole and isobutyraldehyde) were not toxic and did not induce any MN at concentrations up to 10mM. d,l-Menthol and ethionamide induced cytotoxicity, but did not induce MN. o-Anthranilic acid was not toxic and did not induce MN in V79, CHL, CHO, HuLy and HepG2 cells up to 10mM. Toxicity was induced in TK6 cells, although there were no increases in MN frequency up to and above the 55% toxicity level. The other 10 chemicals (1,3-dihydroxybenzene, curcumin, propyl gallate, p-nitrophenol, ethyl acrylate, eugenol, tert-butylhydroquinone, 2,4-dichlorophenol, sodium xylene sulfonate and phthalic anhydride) produced cytotoxicity in at least one cell type, and were evaluated further for MN induction in most or all of the cell types listed above. All these chemicals induced MN at concentrations <10mM, with levels of cytotoxicity below 60% (measured as the replication index) in at least one cell type. The rodent cell lines (V79, CHO and CHL) were consistently more susceptible to cytotoxicity and MN induction than p53-competent cells, and are therefore more susceptible to giving misleading positive results. These data suggest that a reduction in the frequency of misleading positive results can be achieved by careful selection of the mammalian cell type for genotoxicity testing. PMID:22138618

  19. Density gradient electrophoresis of cultured human embryonic kidney cells

    NASA Technical Reports Server (NTRS)

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

    1985-01-01

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

  20. Gravity, chromosomes, and organized development in aseptically cultured plant cells

    NASA Technical Reports Server (NTRS)

    Krikorian, Abraham D.

    1993-01-01

    The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

  1. Human Embryonic Stem Cells (hESCs) Cultured Under Distinctive Feeder-Free Culture Conditions Display Global Gene Expression Patterns Similar to hESCs from Feeder-Dependent Culture Conditions

    Microsoft Academic Search

    Tae-Min Yoon; Bomi Chang; Hyeung-Taek Kim; Joo-Hyun Jee; Dong-Wook Kim; Dong-Youn Hwang

    2010-01-01

    Human embryonic stem cell (hESC)–based assay systems and genetically modified hESCs are very useful tools for screening drugs\\u000a that regulate stemness and differentiation and for studying the molecular mechanisms involved in hESC fate determination.\\u000a For these types of studies, feeder cell–dependent cultures of hESCs are often problematic because the physiology of the feeder\\u000a cells is perturbed by the drug treatments

  2. Culture bag systems for clinical applications of adult human neural crest-derived stem cells

    PubMed Central

    2014-01-01

    Introduction Facing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. Here, neural crest-derived stem cells (NCSCs) offer many promising applications, although scale up of clinical-grade processes prior to potential transplantations is currently limiting. In this study, we aimed to establish a clinical-grade, cost-reducing cultivation system for NCSCs isolated from the adult human nose using cGMP-grade Afc-FEP bags. Methods We cultivated human neural crest-derived stem cells from inferior turbinate (ITSCs) in a cell culture bag system using Afc-FEP bags in human blood plasma-supplemented medium. Investigations of viability, proliferation and expression profile of bag-cultured ITSCs were followed by DNA-content and telomerase activity determination. Cultivated ITSCs were introduced to directed in vitro differentiation assays to assess their potential for mesodermal and ectodermal differentiation. Mesodermal differentiation was determined using an enzyme activity assay (alkaline phosphatase, ALP), respective stainings (Alizarin Red S, Von Kossa and Oil Red O), and RT-PCR, while immunocytochemistry and synaptic vesicle recycling were applied to assay neuroectodermal differentiation of ITSCs. Results When cultivated within Afc-FEP bags, ITSCs grew three-dimensionally in a human blood plasma-derived matrix, thereby showing unchanged morphology, proliferation capability, viability and expression profile in comparison to three dimensionally-cultured ITSCs growing in standard cell culture plastics. Genetic stability of bag-cultured ITSCs was further accompanied by unchanged telomerase activity. Importantly, ITSCs retained their potential to differentiate into mesodermal cell types, particularly including ALP-active, Alizarin Red S-, and Von Kossa-positive osteogenic cell types, as well as adipocytes positive in Oil Red O assays. Bag culture further did not affect the potential of ITSCs to undergo differentiation into neuroectodermal cell types coexpressing ?-III-tubulin and MAP2 and exhibiting the capability for synaptic vesicle recycling. Conclusions Here, we report for the first time the successful cultivation of human NCSCs within cGMP-grade Afc-FEP bags using a human blood plasma-supplemented medium. Our findings particularly demonstrate the unchanged differentiation capability and genetic stability of the cultivated NCSCs, suggesting the great potential of this culture system for future medical applications in the field of regenerative medicine. PMID:24629140

  3. Epigenetic disregulation induces cell growth retardation in primary cultured glial cells.

    PubMed

    Nagai, Kaoru; Natori, Takamitsu; Nishino, Toru; Kodaira, Fumiaki

    2008-05-01

    Some epigenetic mechanisms, including DNA methylation and histone deacetylation, act as transcriptional repression signals. In this study, we examined whether DNA methylation dependent transcriptional control regulates glial cell growth. Primary cultured mouse cortical glial cells were treated with the DNA methylation inhibitor 5-aza-deoxycytidine (5adC) or the histone deacetylase inhibitor sodium valproate (VPA), which inhibits DNA-methylation-dependent transcriptional repression. 5adC significantly reduced methylated C level determined by reversed-phase high-performance liquid chromatography (HPLC), while VPA did not. Treatments with these inhibitors significantly reduced cell number determined by MTT assay after 48 h. Both 5adC and VPA showed little cellular toxicity observed by live and dead cell staining. In contrast, both 5adC and VPA induced an abnormality in the cell cycle. Cells treated with the inhibitors represented a significantly higher ratio in the G2+M-phase and 5adC-treated cells showed a significantly lower ratio in the S-phase. Regarding the in vivo effect, prenatal treatment with VPA, which is an autistic model in rodents, significantly reduced the brain/body weight ratio in early postnatal days. Our data indicate that DNA-methylation- and histone-deacetylation-dependent transcriptional control is crucial for the regulation of glial cell growth. Our data suggest that abnormalities of epigenetic transcriptional regulatory mechanisms in glial cells cause an abnormal brain size, which may in turn cause mental diseases. PMID:18558336

  4. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture...Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture...a) Identification. Tissue culture media for human ex vivo tissue and cell...

  5. The radiosensitivity of a murine fibrosarcoma as measured by three cell survival assays.

    PubMed Central

    Rice, L.; Urano, M.; Suit, H. D.

    1980-01-01

    The radiation sensitivity of a weakly immunogenic spontaneous fibrosarcoma of the C3Hf/Sed mouse (designated FSa-II) was assessed by three in vivo cell survival methods: end-point dilution (TD50) assay, lung colony (LC) assay, and agar diffusion chamber (ADC) assay. The hypoxic fraction of this tumour was also determined by the ADC method. Although there was a good agreement of the cell survival data between the ADC and LC methods, the TD50 method yielded a considerably less steep cell survival curve. Beneficial aspects and limitations of each assay are discussed. In addition, the use of the ADC method for the growth of xenogeneic cell lines and a preliminary experiment with human tumour cells in non-immunosuppressed hosts suggest that this method may be a valuable adjunct for studying the growth and therapeutic responses of human tumour cells. PMID:6932931

  6. Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus.

    PubMed

    Andreazza, Nathalia Luiza; Abreu, Ilka Nacif; Sawaya, Alexandra Christine Helena Frankland; Mazzafera, Paulo

    2015-03-01

    Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results. PMID:25474486

  7. Resting-cell dehydrogenase assay measuring a novel water-soluble formazan detects catabolic differences among cells

    Microsoft Academic Search

    M. R. Leverone; T. C. Owen; F. S. Tieder; G. J. Stewart; D. V. Lim

    1996-01-01

    A resting-cell assay using the novel tetrazolium compound, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethyl-2-thiazolyl)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS), demonstrates the presence of dehydrogenases in cells. When used with the electron-coupling reagent phenazine methosulfate (PMS), MTS can be reduced by cells into a water-soluble formazan. This assay detects different catabolic activities of cells grown with various carbon sources. In this study, the effect of repression of metabolic

  8. A Cell Lysis and Protein Purification - Single Molecule Assay Devices for Evaluation of Genetically Engineered Proteins

    NASA Astrophysics Data System (ADS)

    Nakyama, Tetsuya; Tabata, Kazuhito; Noji, Hiroyuki; Yokokawa, Ryuji

    We have developed two devices applicable to evaluate genetically engineered proteins in single molecule assay: on-chip cell lysis device, and protein purification - assay device. A motor protein, F1-ATPase expressed in E.coli, was focused in this report as a target protein. Cell lysis was simply performed by applying pulse voltage between Au electrodes patterned by photolithography, and its efficiency was determined by absorptiometry. The subsequent processes, purification and assay of extracted proteins, were demonstrated in order to detect F1-ATPase and to evaluate its activity. The specific bonding between his-tag in F1-ATPase and Ni-NTA coated on a glass surface was utilized for the purification process. After immobilization of F1-ATPase, avidin-coated microspheres and adenosine tri-phosphate (ATP) solution were infused sequentially to assay the protein. Microsphere rotation was realized by activity of F1-ATPase corresponding to ATP hydrolysis. Results show that the cell lysis device, at the optimum condition, extracts enough amount of protein for single molecule assay. Once cell lysate was injected to the purification - assay device, proteins were diffused in the lateral direction in a Y-shape microchannel. The gradient of protein concentratioin provides an optimal concentration for the assay i.e. the highest density of rotating beads. Density of rotating beads is also affected by the initial concentration of protein injected to the device. The optimum concentration was achieved by our cell lysis device not by the conventional method by ultrasonic wave. Rotation speed was analyzed for several microspheres assayed in the purification - assay device, and the results were compatible to that of conventional assay in which F1-ATPase was purified in bulk scale. In conclusion, we have demonstrated on-chip cell lysis and assay appropriate for the sequential analysis without any pretreatment. On-chip devices replacing conventional bioanalytical methods will be integrated a total analysis system to evaluate engineered protein and DNA.

  9. Lipoprotein binding to cultured human hepatoma cells.

    PubMed Central

    Krempler, F; Kostner, G M; Friedl, W; Paulweber, B; Bauer, H; Sandhofer, F

    1987-01-01

    Binding of various 125I-lipoproteins to hepatic receptors was studied on cultured human hepatoma cells (Hep G2). Chylomicrons, isolated from a chylothorax, chylomicron remnants, hypertriglyceridemic very low-density lipoproteins, normotriglyceridemic very low-density lipoproteins (NTG-VLDL), their remnants, low-density lipoproteins (LDL), and HDL-E (an Apo E-rich high-density lipoprotein isolated from the plasma of a patient with primary biliary cirrhosis) were bound by high-affinity receptors. Chylomicron remnants and HDL-E were bound with the highest affinity. The results, obtained from competitive binding experiments, are consistent with the existence of two distinct receptors on Hep G2 cells: (a) a remnant receptor capable of high-affinity binding of triglyceride-rich lipoproteins and HDL-E, but not of Apo E free LDL, and (b) a LDL receptor capable of high-affinity binding of LDL, NTG-VLDL, and HDL-E. Specific binding of Apo E-free LDL was completely abolished in the presence of 3 mM EDTA, indicating that binding to the LDL receptor is calcium dependent. Specific binding of chylomicron remnants was not inhibited by the presence of even 10 mM EDTA. Preincubation of the Hep G2 cells in lipoprotein-containing medium resulted in complete suppression of LDL receptors but did not affect the remnant receptors. Hep G2 cells seem to be a suitable model for the study of hepatic receptors for lipoprotein in man. Images PMID:3038957

  10. Effect of LLLT on endothelial cells culture.

    PubMed

    Góralczyk, Krzysztof; Szyma?ska, Justyna; ?ukowicz, Ma?gorzata; Drela, Ewelina; Kotzbach, Roman; Dubiel, Mariusz; Michalska, Ma?gorzata; Góralczyk, Barbara; Zaj?c, Andrzej; Ro??, Danuta

    2015-01-01

    Growth factors as vascular endothelial growth factor (VEGF), produced by the endothelial cells, take an essential part in pathological and physiological angiogenesis. The possibility of angiogenesis modulation by application of laser radiation may contribute to the improvement of its use in this process. Thus, the aim of the study was to investigate the influence of low-level laser therapy (LLLT) on the proliferation of endothelial cells, secretion of VEGF-A and presence of soluble VEGF receptors (sVEGFR-1 and sVEGFR-2) in the medium after in vitro culture. Isolated human umbilical vein endothelial cells (HUVECs) were irradiated using a diode laser at a wavelength of 635 nm and power density of 1,875 mW/cm(2). Depending on radiation energy density, the experiment was conducted in four groups: I 0 J/cm(2) (control group), II 2 J/cm(2), III 4 J/cm(2), and IV 8 J/cm(2). The use of laser radiation wavelength of 635 nm, was associated with a statistically significant increase in proliferation of endothelial cells (p?=?0.0041). Moreover, at 635-nm wavelength, all doses of radiation significantly reduced the concentration of sVEGFR-1 (p?=?0.0197). PMID:25231826

  11. Ilex paraguariensis cell suspension culture characterization and response against ethanol

    Microsoft Academic Search

    Kátia H. Kraemer; Eloir P. Schenkel; Robert Verpoorte

    2002-01-01

    Cell suspension cultures of Ilex paraguariensis, a South American native tree known as the maté plant, were initiated in order to investigate plant defense. Cultures were characterized for their cell growth, chemical composition and sugar consumption. The present work quantified some effects of salicylic acid, methyl jasmonate, cellulase and ethanol on cell growth and sugar metabolism. Results suggest that salicylic

  12. Biolistic transformation of cotton embryogenic cell suspension cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic transformation of cotton is highly dependent on the ability to regenerate fertile plants from transgenic cells through somatic embryogenesis. Induction of embryogenic cell cultures is genotype-dependant. However, once embryogenic cell cultures are available, they can be effectively used fo...

  13. Interpretation of the Multinucleated Giant Cell in Human Trophoblast Cultures

    Microsoft Academic Search

    J. Foldes; Tehila Kehaty; Jeanna Schwartz

    1973-01-01

    Morphology of human trophoblast culture has been studied in relation to certain conditions such as duration of the pregnancy, age of the culture, and strength of the trypsinization. ‘Multinucleated giant cells’ appear to be actually fortuitous transversal sections of chorionic buds. Their presence and aspects depend on the degree of trypsinization. Origin of epithelioid polygonal cells and of fibroblast cells

  14. A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion

    Microsoft Academic Search

    Vera Brekhman; Gera Neufeld

    2009-01-01

    BACKGROUND: The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of in-vivo invasion assays, there is need for quantitative in-vitro invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled. METHODS: We have established a novel asymmetric 3D in-vitro invasion assay

  15. Comparison of culture and 2 real-time polymerase chain reaction assays to detect group B Streptococcus during antepartum screening

    Microsoft Academic Search

    Jennifer S. Goodrich; Melissa B. Miller

    2007-01-01

    Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates but is preventable if the mother is diagnosed before and treated at delivery. Using 200 vaginal–rectal swabs inoculated to enrichment (LIM) broths, we compared routine culture and 2 real-time polymerase chain reaction (PCR) assays for detection of GBS: the LightCycler (LC) Strep B analyte-specific reagents (ASRs)

  16. Increased hematopoietic progenitor cell maintenance in long-term bone marrow cultures containing minimal numbers of contaminating breast cancer cells

    Microsoft Academic Search

    S. L. Mann; S. S. Joshi; D. A. Crouse; J. O. Armitage; A. Kessinger; D. D. Weisenburger; W. P. Vaughan; J. G. Sharp

    1997-01-01

    The maintenance of hematopoietic progenitor cells as assayedin the mixed colony (CFU-GEMM) assay in humanlong-term bone marrow cultures was compared between normalallogeneic marrow transplantation donor collections and those fromcandidates for high-dose therapy and autologous bone marrowtransplantation (ABMT). To be eligible for ABMT, patientswere required to have a histologically normal appearingbone marrow and therefore any tumor contamination wasat minimal levels and

  17. Morphology, proliferation, and osteogenic differentiation of mesenchymal stem cells cultured on titanium, tantalum, and chromium surfaces.

    PubMed

    Stiehler, Maik; Lind, Martin; Mygind, Tina; Baatrup, Anette; Dolatshahi-Pirouz, Alireza; Li, Haisheng; Foss, Morten; Besenbacher, Flemming; Kassem, Moustapha; Bünger, Cody

    2008-08-01

    Metallic implants are widely used in orthopedic surgery and dentistry. Durable osseous fixation of an implant requires that osteoprogenitor cells attach and adhere to the implant, proliferate, differentiate into osteoblasts, and produce mineralized matrix. In the present study, we investigated the interactions between human mesenchymal stem cells (MSCs) and smooth surfaces of titanium (Ti), tantalum (Ta), and chromium (Cr). Mean cellular area was quantified using fluorescence microscopy (4 h). Cellular proliferation was assessed by (3)H-thymidine incorporation and methylene blue cell counting assays (4 days). Osteogenic differentiation response was quantified by cell-specific alkaline phosphatase activity (ALP) assay (4 days), expression analysis of bone-related genes (4 days), and mineralization assay (21 days). Undifferentiated and osteogenically stimulated MSCs cultured on the different surfaces showed the same tendencies for proliferation and differentiation. MSCs exposed to Ti surfaces demonstrated enhanced proliferation compared with Ta and Cr surfaces. Cultivation of MSCs on Ta surfaces resulted in significantly increased mean cellular area and cell-specific ALP activity compared with the other surfaces tested. Cells cultured on Cr demonstrated reduced spreading and proliferation. In conclusion, Ta metal, as an alternative for Ti, can be considered as a promising biocompatible material, whereas further studies are needed to fully understand the role of Cr and its alloys in bone implants. PMID:17975813

  18. Microfluidic devices for cell culture and handling in organ-on-a-chip applications

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Schulz, Ingo; Mosig, Alexander; Jahn, Tobias; Gärtner, Claudia

    2014-03-01

    For many problems in system biology or pharmacology, in-vivo-like models of cell-cell interactions or organ functions are highly sought after. Conventional stationary cell culture in 2D plates quickly reaches its limitations with respect to an in-vivo like expression and function of individual cell types. Microfabrication technologies and microfluidics offer an attractive solution to these problems. The ability to generate flow as well as geometrical conditions for cell culture and manipulation close to the in-vivo situation allows for an improved design of experiments and the modeling of organ-like functionalities. Furthermore, reduced internal volumes lead to a reduction in reagent volumes necessary as well as an increased assay sensitivity. In this paper we present a range of microfluidic devices designed for the co-culturing of a variety of cells. The influence of substrate materials and surface chemistry on the cell morphology and viability for long-term cell culture has been investigated as well as strategies and medium supply for on-chip cell cultivation.

  19. mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

    PubMed Central

    McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

  20. Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

    PubMed Central

    Liadi, Ivan; Roszik, Jason; Romain, Gabrielle; Cooper, Laurence J.N.; Varadarajan, Navin

    2013-01-01

    Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5. Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8. Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells. PMID:23407457

  1. Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

    PubMed Central

    2011-01-01

    Background We describe the development of an electrochemical sensor array for monitoring the proliferation effects of cissus populnea plant extracts on TM4 Sertoli cells. Methods The proliferation activities of the extracts on Sertoli cells were studied using a high-throughput electrochemical sensor array (DOX-96) and the analytical sensor characteristics were compared with conventional colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and fluorescence spectroscopy. Results This work shows that there is a definite positive trend in the proliferation effect of the extract of Cissus populnea on the TM4 Sertoli cells. All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm. At this concentration, the proliferation effect was established around 120% for both DOX-96 and MTT techniques, whereas fluorescence assays showed a higher level (120-150%). DOX-96 showed a lower limit of detection (1.25 × 10(4) cells/ml); whereas the LOD recorded for both MTT and fluorescence techniques was 2.5 × 10(4) cells/ml. Visual examination of the cells by means of confocal fluorescence microscopy confirmed the proliferation of Sertoli cells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm. Conclusions Overall, the DOX results compared well with the conventional methods of checking proliferation of cells. The fascinating feature of the sensor array is the ability to provide continuous proliferation experiments with no additional reagents including 96 simultaneous electrochemical experiments. The use of the DOX-96 could reduce a typical bioassay time by 20-fold. Thus the DOX-96 can be used as both a research tool and for practical cell culture monitoring. PMID:21575213

  2. An homogeneous assay for measuring the uptake and efflux of radiolabelled drugs in adherent cells.

    PubMed

    Graves, R; Davies, R; Owen, P; Clynes, M; Cleary, I; O'Beirne, G

    1997-06-01

    We have developed an homogeneous assay to measure the uptake and efflux of [14C]adriamycin (doxorubicin hydrochloride) in human squamous lung carcinoma cells (SKMES-1), using 96 well scintillating microplates. The assay was also used to examine the effect of inhibitors of multidrug resistance in adriamycin resistant cells (SKMES-1/ADR). The effect of adriamycin on cell growth and viability was examined by continuous monitoring of the uptake of [14C]thymidine. The non-invasive nature of these assays, and the ease of use of the microplates, suggests a role in screens for, and characterisation of, novel chemotherapeutic or chemosensitizing agents. PMID:9314096

  3. Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

    PubMed

    Radtke, Andrea L; Herbst-Kralovetz, Melissa M

    2012-01-01

    Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties. The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests. PMID:22491366

  4. An introductory undergraduate course covering animal cell culture techniques.

    PubMed

    Mozdziak, Paul E; Petitte, James N; Carson, Susan D

    2004-09-01

    Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when undertaking the first graduate student position, or instruction when hired for a specific industrial cell culture position. However, cell culture is an important candidate course for any biotechnology-related training program because it is a technique that must be performed by investigators before they perform many molecular procedures, and vertebrate cell culture is becoming increasingly important for biomanufacturing of therapeutic proteins. Therefore, a cell culture techniques course is an important offering for undergraduate students who aspire to graduate training, and also undergraduate students who will seek employment with biotechnology companies immediately after graduation. Recently, a cell culture techniques course was developed and delivered to students at North Carolina State University as a component of an undergraduate Biotechnology minor curricula. Currently, the instructors at North Carolina State University are seeking to provide students with the necessary technical and critical reasoning skills to successfully perform animal cell culture. PMID:21706746

  5. Particle Trajectories in Rotating Wall Cell Culture Devices

    NASA Technical Reports Server (NTRS)

    Ramachandran N.; Downey, J. P.

    1999-01-01

    Cell cultures are extremely important to the medical community since such cultures provide an opportunity to perform research on human tissue without the concerns inherent in experiments on individual humans. Development of cells in cultures has been found to be greatly influenced by the conditions of the culture. Much work has focused on the effect of the motions of cells in the culture relative to the solution. Recently rotating wall vessels have been used with success in achieving improved cellular cultures. Speculation and limited research have focused on the low shear environment and the ability of rotating vessels to keep cells suspended in solution rather than floating or sedimenting as the primary reasons for the improved cellular cultures using these devices. It is widely believed that the cultures obtained using a rotating wall vessel simulates to some degree the effect of microgravity on cultures. It has also been speculated that the microgravity environment may provide the ideal acceleration environment for culturing of cellular tissues due to the nearly negligible levels of sedimentation and shear possible. This work predicts particle trajectories of cells in rotating wall vessels of cylindrical and annular design consistent with the estimated properties of typical cellular cultures. Estimates of the shear encountered by cells in solution and the interactions with walls are studied. Comparisons of potential experiments in ground and microgravity environments are performed.

  6. Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays

    PubMed Central

    Reverté, Laia; Soliño, Lucía; Carnicer, Olga; Diogène, Jorge; Campàs, Mònica

    2014-01-01

    The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs. PMID:25431968

  7. Skeletal muscle satellite cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Molnar, Greg; Hartzell, Charles R.; Schroedl, Nancy A.; Gonda, Steve R.

    1993-01-01

    Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a three dimensional level of organization that could provide a more suitable model to study postnatal muscle development.

  8. Gap junctions in myometrial cell cultures: evidence for modulation by cyclic adenosine 3':5'-monophosphate.

    PubMed

    Dookwah, H D; Barhoumi, R; Narasimhan, T R; Safe, S H; Burghardt, R C

    1992-09-01

    Primary cultures of myometrial cells from juvenile rats, continuous cultures maintained by serial passage, and a pSV3neotransfected myometrial cell line were established and utilized for the study of development and modulation of gap junctional intercellular communication (GJIC) in vitro. The smooth muscle origin and homogeneity of the cultures were verified by immunofluorescence staining of alpha-smooth muscle actin and cellular desmin. Although gap junctions were not detected in thin sections of juvenile and adult myometrial tissues by transmission electron microscopy, they were detected in cultured myometrial cells derived from juvenile and adult animals. The presence of GJIC in cultured cells was confirmed using a fluorescence recovery after photo-bleaching assay. Administration of exogenous estradiol-17 beta (10(-7) M) resulted in an increase in GJIC in primary and passage 9 myometrial cultures, whereas pSV3neo-transfected myometrial cells were not significantly different from untreated controls. The lack of estrogen responsiveness in pSV3neo-transfected cultures correlated with lower levels of estrogen receptors than in primary cultures. Addition of 1 mM 8-bromo-cAMP resulted in rapid (within 2 min) increases in dye transfer in both control and estradiol-17 beta-primed primary cultures. Uncoupling of cells by treatment with 1 mM 1-octanol, followed by addition of 1 mM 8-bromo-cAMP, resulted in increased GJIC in control and estradiol-17 beta-primed cultures, although up-regulation of GJIC in estradiol-17 beta-primed cultures was much greater than in control cultures. Comparative experiments carried out on a spontaneously immortalized rat granulosa cell line (SIGC), which expresses the same connexin43 species as myometrial cells, exhibited similar responses to exogenous 8-bromo-cAMP following uncoupling of gap junctions with octanol. While the results of these investigations may not be extrapolated to myometrium in vivo, they suggest that myometrial cell culture may offer additional opportunities to explore the temporal expression and modulation of GJIC in myometrium. PMID:1324745

  9. Gentoxocity of blue rayon extracts from river waters using sister chromatid exchange in cultured mammalian cells

    Microsoft Academic Search

    Takeshi Ohe; Hisae Ito; Mika Kawabuti

    1993-01-01

    Using cultured Chinese hamster lung (CHL) cells, sister chromatid exchange (SCE) assays were carried out with blue rayon extracts\\u000a recovered at seven sampling locations from the Katsura, Nishitakase and Kamo Rivers, tributaries of the Yodo River, in Kyoto\\u000a City, Japan. The downstream extracts of wastewater treatment plants showed higher SCE frequencies than the upstream extracts\\u000a both with and without metabolic

  10. Effects of chocolate cyst fluid on endometrioma cell growth in culture

    Microsoft Academic Search

    Shawky Z. A Badawy; Violeta Cuenca; Shubhra Kumar; James Holland

    1998-01-01

    Objective: To evaluate the effect of chocolate cyst fluid on the proliferation of cultured human endometrioma cells and to assay the concentration of transforming growth factor-B1 in this fluid.Design: Controlled in vitro study.Setting: Department of Obstetrics and Gynecology, State University of New York Health Science Center.Patient(s): Five women with ovarian endometriomas.Intervention(s): Endometrioma tissue and chocolate fluid from five different patients

  11. Oxidized low density lipoprotein stimulates collagen production in cultured arterial smooth muscle cells

    Microsoft Academic Search

    Shiro Jimi; Keijiro Saku; Noriko Uesugi; Noriyuki Sakata; Shigeo Takebayashi

    1995-01-01

    We examined the interactive effect of oxidized low density lipoprotein (LDL) and ascorbic acid on collagen production in cultured smooth muscle cells (SMCs). Porcine aortic SMCs were incubated with 50–200 ?g\\/ml of human LDL with\\/without 5 ?M Cu2+ for 24 h. Collagen production was assayed by successive salt precipitation at acidic and neutral pH after pepsin digestion of 3H-proline-labeled collagenous

  12. Effects of solvents and dosing procedure on chemical toxicity in cell-based in vitro assays

    Microsoft Academic Search

    K. Tanneberger; A. Rico Rico; N. I. Kramer; F. J. M. Busser; J. L. M. Hermens; K. Schirmer

    2010-01-01

    Due to the implementation of new legislation, such as REACh, a dramatic increase of animal use for toxicity testing is expected and the search for alternatives is timely. Cell-based in vitro assays are promising alternatives. However, the behavior of chemicals in these assays is still poorly understood. We set out to quantify the exposure and associated toxicity of chemicals with

  13. Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane

    SciTech Connect

    Tao, W.; Wilkinson, J.; Stanbridge, E.J.; Berns, M.W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in media containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8x10-4-3x10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  14. Comparison of two cytotoxicity assays--tetrazolium derivative reduction (MTT) and tritiated thymidine uptake--on three malignant mouse cell lines using chemotherapeutic agents and investigational drugs.

    PubMed

    Arnould, R; Dubois, J; Abikhalil, F; Libert, A; Ghanem, G; Atassi, G; Hanocq, M; Lejeune, F J

    1990-01-01

    Two different techniques [reduction of a tetrazolium derivative (MTT) and 3HTdR uptake assays] were compared in order to evaluate the cytotoxic effects of different chemotherapeutic agents in vitro. The cytotoxicities of Melphalan, hexamethylmelamine and seven derivatives, and daunorubicin were measured on P388D1 mouse macrophage-like cell line, RC mouse renal carcinoma cell line, and B16 mouse melanoma cell lines. Growth inhibition was determined after one hour as well as after continuous (48 hours) exposure to drugs. The IC50 was calculated using an appropriate algorithm (FADHA) which allowed within and between run variabilities to be taken into account. Optimal conditions had to be elucidated for culture conditions before assay: number of cells/well and assay duration for each line. The MTT and 3HTdR uptake assays were found to be similar in terms of sensitivity and reproducibility, both for adherent and floating cell lines. However, the MTT assay has the advantages of low cost and time saving. It also avoids problems related to radioactivity manipulation and counting. Both techniques rank the same chemicals as active or inactive. The algorithm Fadha was found to be a very powerful mathematical tool for comparing the IC50 values obtained by both assays. PMID:2334120

  15. Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

    PubMed

    Raasch, Martin; Rennert, Knut; Jahn, Tobias; Peters, Sven; Henkel, Thomas; Huber, Otmar; Schulz, Ingo; Becker, Holger; Lorkowski, Stefan; Funke, Harald; Mosig, Alexander

    2015-01-01

    Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNF? as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNF?/IFN? treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under advanced culture conditions more closely resembling the in vivo situation. PMID:25727374

  16. Rapid, sensitive, and validated method for detection of Salmonella in food by an enrichment broth culture - nested PCR combination assay.

    PubMed

    Saroj, Sunil D; Shashidhar, R; Karani, Manisha; Bandekar, Jayant R

    2008-06-01

    A rapid nested PCR assay for detection of Salmonella from food was developed. The sensitivity of the assay developed was comparable to the traditional culture based methods with an advantage in reduction of assay time. The assay procedure with artificially contaminated samples was able to detect as low as 4CFU Salmonella/25g of food samples (sprout, carrot, cucumber and poultry meat). With two synthetic primers of 26 mer TS11 and 25 mer TS4, a 1.2kb fragment was amplified which served as a template for amplification of final 375bp product using TS11 and TS5 primers. No non-specific amplification from the native microbial flora of food samples was observed. The reaction generates a single band specific to Salmonella which allows the analyst to interpret data at ease and without any confusion. Enriched broth serves as template for the reaction which removes labour intensive DNA isolation procedures. In case of artificially contaminated samples, 6h enriched lactose broth can serve as template. However, for market samples where the organisms are under environmental stress, it is desirable to use template from Rappaport Vasiliadis medium. The assay also employes internal amplification control, which is amplified into a 300bp fragment and thus serves as positive control for the reaction and any possibility of false negative due to inhibitory action of food components on PCR reaction can be ruled out. PMID:18406104

  17. Vero cell assay validation of an alternative to the Ph. Eur. diphtheria potency tests.

    PubMed

    Gommer, A M

    1996-01-01

    In the framework of the Biological Standardisation Programme of the European Pharmacopoeia Commission, in 1993 a collaborative study was organised for the validation of an alternative to the diphtheria in vivo challenge tests required by the Ph.Eur. monograph V.2.2.7. The alternative assay is based on the detection of neutralising antibodies in the sera from mice immunised with the vaccines to be tested (Vero cell assay). In the study this assay method was validated against intradermal and lethal challenge in guinea-pigs, performed in conformity with Ph.Eur. Therefore the potency currently on the European market, was assayed in parallel by the different assay methods. Seventeen laboratories, from eleven different countries, participated in the study. Three laboratories performed the intradermal challenge assay, while three other laboratories performed the lethal challenge assay. All seventeen laboratories performed the Vero cell assay. The results of the study suggest that the potency of the diphtheria component of both monovalent diphtheria vaccines and combined diphtheria-tetanus vaccines can be estimated adequately by means of the Vero cell assay. It does not yet seem possible for all combined diphtheria-tetanus-pertussis vaccines to replace a potency assay based on the protective capacity of a vaccine in guinea-pigs by the Vero cell assay. This may be due to an adjuvant effect of the pertussis component of the vaccine, in combination with the adsorbent used, which may be more pronounced in mice than in guinea-pigs and may also differ between different strains of mice. PMID:8785952

  18. Psychoneuroimmunology and natural killer cells: the chromium release whole blood assay.

    PubMed

    Fletcher, Mary Ann; Barnes, Zachary; Broderick, Gordon; Klimas, Nancy G

    2012-01-01

    Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter will describe a chromium ((51)Cr) release bioassay designed to measure the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 eyrthroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4 h in vitro assay. PMID:22933153

  19. Assays to Examine Endothelial Cell Migration, Tube Formation, and Gene Expression Profiles

    PubMed Central

    Guo, Shuzhen; Lok, Josephine; Liu, Yi; Hayakawa, Kazuhide; Leung, Wendy; Xing, Changhong; Ji, Xunming; Lo, Eng H.

    2014-01-01

    Common methods for studying angiogenesis in vitro include the tube formation assay, the migration assay, and the study of the endothelial genome. The formation of capillary-like tubes in vitro on basement membrane matrix mimics many steps of the angiogenesis process in vivo and is used widely as a screening test for angiogenic or antiangiogenic factors. Other assays related to the study of angiogenesis include the cell migration assay, the study of gene expression changes during the process of angiogenesis, and the study of endothelial-derived microparticles. Protocols for these procedures will be described here. PMID:24510881

  20. A plasmacytoid dendritic cell (CD123+/CD11c-) based assay system to predict contact allergenicity of chemicals

    PubMed Central

    Ayehunie, Seyoum; Snell, Maureen; Child, Matthew; Klausner, Mitchell

    2009-01-01

    A predictive allergenicity test system for assessing the contact allergenicity of chemicals is needed by the cosmetic and pharmaceutical industry to monitor product safety in the marketplace. Development of such non-animal alternative assay systems for skin sensitization and hazard identification has been pursued by policy makers and regulatory agencies. We investigated whether phenotypic and functional changes to a subset of dendritic cells (DC), plasmacytoid DC (pDC), could be used to identify contact allergens. To achieve this goal, normal human DC were generated from CD34+ progenitor cells and cryopreserved. Frozen DC were thawed and the pDC fraction (CD123+/CD11c-) was harvested using FACS sorting. The pDC were cultured, expanded, and exposed to chemical allergens (N=26) or non-allergens (N=22). Concentrations of each chemical that resulted in >50% viability was determined using FACS analysis of propidium iodide stained cells using pDC from 2-5 donors. Expression of the surface marker, CD86, which has been implicated in dendritic cell maturation, was used as a marker of allergenicity. CD86 expression increased (? 1.5 fold) for 25 of 26 allergens (sensitivity = 96%) but did not increase for 19 of 22 non-allergens (specificity = 86%). In a direct comparison to historical data for the regulatory approved, mouse local lymph node assay (LLNA) for 23 allergens and 22 non-allergens, the pDC method had sensitivity and specificity of 96% and 86%, respectively, while the sensitivity and specificity of the LLNA assay was 83% and 82%, respectively. In conclusion, CD86 expression in pDC appears to be a sensitive and specific indicator to identify contact allergenicity. Such an assay method utilizing normal human cells will be useful for high throughput screening of chemicals for allergenicity. PMID:19665512

  1. Testing of nine different xeno-free culture media for human embryonic stem cell cultures

    Microsoft Academic Search

    Kristiina Rajala; Heidi Hakala; Sarita Panula; Suvi Aivio; Harri Pihlajamaki; Riitta Suuronen; Outi Hovatta; Heli Skottman

    2007-01-01

    BACKGROUND: Human embryonic stem cells (hESC) are excellent candidates for cell replacement therapies. However, currently used culture conditions contain animal-derived components that bear a risk of transmitting animal pathogens and incorporation of non-human immunogenic molecules to hESC. METHODS: Nine xeno-free culture media were compared with the conventional serum replacement (ko-SR) containing media in the culture of hESC on human feeder

  2. Tension, Free Space, and Cell Damage in a Microfluidic Wound Healing Assay

    E-print Network

    Murrell, Michael

    We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method ...

  3. Evaluation of the nanosphere verigene gram-positive blood culture assay with the VersaTREK blood culture system and assessment of possible impact on selected patients.

    PubMed

    Beal, Stacy G; Ciurca, Jane; Smith, Geremy; John, Jeffrey; Lee, Francesca; Doern, Christopher D; Gander, Rita M

    2013-12-01

    The Verigene Gram-positive blood culture (BC-GP) assay (Nanosphere, Northbrook, IL) is a molecular method for the rapid identification of Gram-positive organisms and resistance markers directly from blood culture bottles. A total of 148 VersaTREK REDOX 1 40-ml aerobic bottles demonstrating Gram-positive bacteria were tested. Results were compared with those from conventional biochemical and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) identifications. We obtained isolates of methicillin-resistant Staphylococcus aureus (MRSA) (24), methicillin-susceptible Staphylococcus aureus (MSSA) (14), methicillin-resistant Staphylococcus epidermidis (MRSE) (17), methicillin-susceptible Staphylococcus epidermidis (MSSE) (9), other coagulase-negative staphylococci (19), Streptococcus salivarius (5), Streptococcus parasanguinis (2), Streptococcus sanguinis (1), Streptococcus cristatus (1), the Streptococcus bovis group (5), Streptococcus agalactiae (9), the Streptococcus anginosus group (1), Streptococcus pneumoniae (6), vancomycin-resistant Enterococcus faecium (VRE FCM) (16), vancomycin-susceptible Enterococcus faecalis (3), Aerococcus viridans (2), Bacillus (6), Corynebacterium (8), Lactobacillus (2), Micrococcus (2), Neisseria mucosa (1), Escherichia coli (3), Candida tropicalis (1), Propionibacterium (1), and Rothia (1). Overall agreement with the culture results was 95%. A total of 137 of 138 (99%) monomicrobial cultures were concordant. We tested 9 polymicrobial samples and found 33% agreement. A chart review of 31 patients with MRSA, MSSA, or VRE demonstrated that the Nanosphere BC-GP assay might have led to more appropriate antibiotic selection for these patients an average of 42 h earlier. Additionally, contact isolation could have been initiated an average of 37 h earlier for patients with MRSA or VRE. The BC-GP assay may have a positive impact on patient care, health care costs, and antibiotic stewardship. PMID:24048531

  4. Microsystems platforms for array-based single-cell biological assays

    E-print Network

    Taff, Brian M., 1978-

    2008-01-01

    For much of the past century, plated cell cultures have served investigations regarding a variety of fundamental biological processes. Though this in vitro approach has been fruitful, for surveying topics including cell ...

  5. An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue

    Microsoft Academic Search

    C. Jacyn Baker; Norton M. Mock

    1994-01-01

    Cell viability or cell death is an important variable to monitor in many studies of host\\/pathogen interactions. However for studies that focus on events within the first few hours of the interaction, many of the viability assays currently being used are either too laborious and time consuming or measure the cell's temporary metabolic state rather than irreversible cell death. Evans

  6. Genotoxicity of alkene epoxides in human peripheral blood mononuclear cells and HL60 leukaemia cells evaluated with the comet assay.

    PubMed

    Fabiani, Roberto; Rosignoli, Patrizia; De Bartolomeo, Angelo; Fuccelli, Raffaela; Morozzi, Guido

    2012-08-30

    Volatile organic compounds (VOCs) exert their carcinogenic activity through the production of epoxide metabolites. Because of their high reactivity some epoxides are also produced in the chemical industry for the synthesis of other compounds. Therefore, human exposure to VOCs epoxides does occur and may be an important human health concern. In this study, the in vitro genotoxic potential of epoxides originating from 1,3-butadiene (3,4-epoxy-1-butene: EB; 1,2:3,4-diepoxybutane: DEB), isoprene (3,4-epoxy-2-methyl-1-butene: IO), styrene (styrene-7,8-oxide: SO), propylene (propylene oxide: PO) and 1-butene (1,2-epoxy-butane: BO) in human peripheral blood mononuclear cells (PBMCs) and promyelocytic leukaemia cells (HL60) was measured with the comet assay (single-cell gel electrophoresis, SCGE). The effect of inclusion of foetal calf serum (FCS, 5%) in the cell-culture medium and different durations of exposure (2h, 24h) were also investigated. All epoxides tested produced DNA damage in a concentration range that did not reduce cell viability. HL60 cells were more resistant than PBMCs to the DNA damage induced by the different epoxides. With the exception of IO, the treatment for 24h resulted in an increase of DNA damage. FCS slightly protected PBMCs from the genotoxic effects induced by IO and BO, whilst no such effect was noted for the other compounds. Overall, the dose-dependent effects that were seen allowed us to define a genotoxicity scale for the different epoxides as follows: SO>EB>DEB>IO>PO>BO, which is in partial agreement with the International Agency for Research on Cancer (IARC) classification of the carcinogenic hazards. PMID:22285587

  7. Ketoisophorone Transformation by Marchantia polymorpha and Nicotiana tabacum Cultured Cells

    E-print Network

    Paré, Paul W.

    Ketoisophorone Transformation by Marchantia polymorpha and Nicotiana tabacum Cultured Cells Mohamed ketoisophorone (2,2,6-trimethyl-2-cyclohexene-1,4-dione) (1) by Marchantia polymorpha and Nicotiana tabacum cell

  8. Co-culture of hepatocellular carcinoma cells and human umbilical endothelial cells damaged by SU11274

    PubMed Central

    TOMIZAWA, MINORU; SHINOZAKI, FUMINOBU; MOTOYOSHI, YASUFUMI; SUGIYAMA, TAKAO; YAMAMOTO, SHIGENORI; ISHIGE, NAOKI

    2014-01-01

    Mesenchymal-epithelial transition factor (c-Met) is a receptor that binds to the hepatocyte growth factor and is upregulated in hepatocellular carcinoma (HCC). The anti-tumor effects of (3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4- methyl-piperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-N-me- thyl-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (SU11274), a c-Met inhibitor, were investigated in the present study. HCC cells (HLE, HLF, PLC/PRL/5, Hep3B, Huh-6 and HepG2) and human umbilical vein endothelial cells (HUVECs) were used. Quantitative polymerase chain reaction was performed to detect the expression level of c-Met in HCC and HUVECs, and cyclin D1 in HCC. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed to assess the proliferation of the HCC cells and HUVECs cultured with SU11274. Co-culture of HLF or PLC/PRL/5 cells and HUVECs was established as an in vitro model of HCC tissues. The expression levels of c-Met in HLE, HLF, PLC/PRL/5, Hep3B, Huh-6 and HepG2, adult healthy liver and HUVECs were 4.43±0.50, 1.61±0.18, 3.70±0.08, 0.81±0.18, 6.60±1.29, 1.06±0.35, 1.00±0.09 and 88.8±17.3 (mean ± standard deviation), respectively. SU11274 (30 ?M) suppressed the proliferation of HLF, PLC/PRL/5 and HUVECs to 11.0±9.4, 46.5±30.7 and 29.4±5.0%, respectively. SU11274 (30 ?M) decreased the expression levels of cyclin D1 in HLF and PLC/PRL/5 cells to 45.1±11.6 and 30.1±10.3%, respectively. SU11274, at a concentration of 30 ?M damaged the morphology of the co-cultures of HLF or PLC/PRL/5 cells with HUVECs and all the cells died. c-Met is highly expressed in HUVECs and HCC cells, but not in Hep3B. At a 30-?M concentration, SU11274 suppresses the proliferation of HLF, PLC/PRL/5 and HUVECs. SU11274 (30 ?M) damages the co-cultures of HLF or PLC/PRL/5 cells with HUVECs. PMID:25279148

  9. Co-culture of hepatocellular carcinoma cells and human umbilical endothelial cells damaged by SU11274.

    PubMed

    Tomizawa, Minoru; Shinozaki, Fuminobu; Motoyoshi, Yasufumi; Sugiyama, Takao; Yamamoto, Shigenori; Ishige, Naoki

    2014-11-01

    Mesenchymal-epithelial transition factor (c-Met) is a receptor that binds to the hepatocyte growth factor and is upregulated in hepatocellular carcinoma (HCC). The anti-tumor effects of (3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4- methyl-piperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-N-me- thyl-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (SU11274), a c-Met inhibitor, were investigated in the present study. HCC cells (HLE, HLF, PLC/PRL/5, Hep3B, Huh-6 and HepG2) and human umbilical vein endothelial cells (HUVECs) were used. Quantitative polymerase chain reaction was performed to detect the expression level of c-Met in HCC and HUVECs, and cyclin D1 in HCC. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed to assess the proliferation of the HCC cells and HUVECs cultured with SU11274. Co-culture of HLF or PLC/PRL/5 cells and HUVECs was established as an in vitro model of HCC tissues. The expression levels of c-Met in HLE, HLF, PLC/PRL/5, Hep3B, Huh-6 and HepG2, adult healthy liver and HUVECs were 4.43±0.50, 1.61±0.18, 3.70±0.08, 0.81±0.18, 6.60±1.29, 1.06±0.35, 1.00±0.09 and 88.8±17.3 (mean ± standard deviation), respectively. SU11274 (30 ?M) suppressed the proliferation of HLF, PLC/PRL/5 and HUVECs to 11.0±9.4, 46.5±30.7 and 29.4±5.0%, respectively. SU11274 (30 ?M) decreased the expression levels of cyclin D1 in HLF and PLC/PRL/5 cells to 45.1±11.6 and 30.1±10.3%, respectively. SU11274, at a concentration of 30 ?M damaged the morphology of the co-cultures of HLF or PLC/PRL/5 cells with HUVECs and all the cells died. c-Met is highly expressed in HUVECs and HCC cells, but not in Hep3B. At a 30-?M concentration, SU11274 suppresses the proliferation of HLF, PLC/PRL/5 and HUVECs. SU11274 (30 ?M) damages the co-cultures of HLF or PLC/PRL/5 cells with HUVECs. PMID:25279148

  10. Two High Throughput Screen Assays for Measurement of TNF-? in THP-1 Cells

    PubMed Central

    Leister, Kristin P; Huang, Ruili; Goodwin, Bonnie L; Chen, Andrew; Austin, Christopher P; Xia, Menghang

    2011-01-01

    Tumor Necrosis Factor-? (TNF-?), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-?, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-? production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-? assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-? assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-? assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-? inhibitors. We also identified several novel inhibitors of TNF-?, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-? assays are robust and suitable for high throughput screening. PMID:21643507

  11. Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells.

    PubMed

    Seifertová, Daniela; Sk?pa, Petr; Rychtá?, Jan; La?ková, Martina; Pa?ezová, Markéta; Dobrev, Petre I; Hoyerová, Klára; Petrášek, Jan; Zažímalová, Eva

    2014-03-15

    Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells. PMID:24594395

  12. Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture

    SciTech Connect

    Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

    1985-05-01

    Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and /sup 14/C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of /sup 14/C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose.

  13. Convenient cell fusion assay for rapid screening for HIV entry inhibitors

    NASA Astrophysics Data System (ADS)

    Jiang, Shibo; Radigan, Lin; Zhang, Li

    2000-03-01

    Human immunodeficiency viruses (HIV)-induced cell fusion is a critical pathway of HIV spread from infected cells to uninfected cells. A rapid and simple assay was established to measure HIV-induce cell fusion. This study is particularly useful to rapid screen for HIV inhibitors that block HIV cell-to-cell transmission. Present study demonstrated that coculture of HIV-infected cells with uninfected cells at 37 degree(s)C for 2 hours resulted in the highest cell fusion rate. Using this cell fusion assay, we have identified several potent HIV inhibitors targeted to the HIV gp41 core. These antiviral agents can be potentially developed as antiviral drugs for chemotherapy and prophylaxis of HIV infection and AIDS.

  14. Cell-line Engineering of Chinese Hamster Ovary Cells for Low-temperature Culture

    E-print Network

    Kiat, Tan Hong

    Developments in mammalian cell culture and recombinant technology has allowed for the production of recombinant proteins for use as human therapeutics. Mammalian cell culture is typically operated at the physiological ...

  15. Osteogenic Differentiation of Mesenchymal Stem Cells on Pregenerated Extracellular Matrix Scaffolds in the Absence of Osteogenic Cell Culture Supplements

    PubMed Central

    Thibault, Richard A.; Scott Baggett, L.; Mikos, Antonios G.

    2010-01-01

    This study utilized a full-factorial design to investigate the effect of four factors: presence of whole bone marrow cells, presence of in vitro-generated mineralized extracellular matrix (ECM), presence of dexamethasone, and variations in culture duration, on the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) cultured on a polymer scaffold. Electrospun poly(?-caprolactone) (PCL) fiber mesh scaffolds were seeded with rat MSCs and cultured in complete osteogenic medium for 12 days to generate constructs containing mineralized ECM. MSCs or MSCs and whole bone marrow cells were seeded onto decellularized ECM constructs (PCL/ECM) or plain PCL scaffolds and cultured statically for 4, 8, and 16 days in medium either with or without dexamethasone. After each culture period, the cell number was determined by DNA analysis, and the osteogenic differentiation state of the cells was determined by alkaline phosphatase activity and calcium assays. MSCs seeded onto PCL/ECM constructs and cultured in medium either with or without dexamethasone demonstrated similar amounts of calcium deposition after 16 days. A significant increase in cell number over time compared with all other groups was observed when whole bone marrow cells were cocultured with MSCs on PCL scaffolds in medium without dexamethasone. This study establishes that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs is maintained even in the absence of dexamethasone and that the coculture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow. PMID:19863274

  16. Liver cells culture on three-dimensional micropatterned polydimethylsiloxane surfaces

    Microsoft Academic Search

    Fanny Evenou; Teruo Fujii; Yasuyuki Sakai

    Current in vitro cell culture technologies present some limitations as they can't simulate or mimic in vivo situations. Indeed, in vivo cells function in a three-dimensional (3D) structure where they have a close contact with adjacent cells. In this study, human hepatocarcinoma Hep G2 cells were cultured on 3D micropatterned polydimethylsiloxane (PDMS) substrates. Using soft-lithography techniques, arrays of octagonal macropillars

  17. A Robotic MCF-7:WS8 Cell Proliferation Assay to Detect Agonist and Antagonist Estrogenic Activity

    PubMed Central

    Casey, Warren

    2014-01-01

    Endocrine-disrupting chemicals with estrogenic activity (EA) or anti-EA (AEA) have been extensively reported to possibly have many adverse health effects. We have developed robotized assays using MCF-7:WS8 cell proliferation (or suppression) to detect EA (or AEA) of 78 test substances supplied by the Interagency Coordinating Committee on the Validation of Alternative Methods and the National Toxicology Program’s Interagency Center for the Evaluation of Alternative Toxicological Methods for validation studies. We also assayed ICI 182,780, a strong estrogen antagonist. Chemicals to be assayed were initially examined for solubility and volatility to determine optimal assay conditions. For both EA and AEA determinations, a Range-Finder assay was conducted to determine the concentration range for testing, followed by a Comprehensive assay. Test substances with potentially positive results from an EA Comprehensive assay were subjected to an EA Confirmation assay that evaluated the ability of ICI 182,780 to reverse chemically induced MCF-7 cell proliferation. The AEA assays examined the ability of chemicals to decrease MCF-7 cell proliferation induced by nonsaturating concentrations of 17?-estradiol (E2), relative to ICI or raloxifene, also a strong estrogen antagonist. To be classified as having AEA, a saturating concentration of E2 had to significantly reverse the decrease in cell proliferation produced by the test substance in nonsaturating E2. We conclude that our robotized MCF-7 EA and AEA assays have accuracy, sensitivity, and specificity values at least equivalent to validated test methods accepted by the U.S. Environmental Protection Agency and the Organisation for Economic Co-operation and Development. PMID:24213142

  18. Continuous cultures of fused cells secreting antibody of predefined specificity

    Microsoft Academic Search

    G. Köhler; C. Milstein

    1975-01-01

    THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe

  19. Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture

    PubMed Central

    Kalabis, Jiri; Wong, Gabrielle S; Vega, Maria E; Natsuizaka, Mitsuteru; Robertson, Erle S; Herlyn, Meenhard; Nakagawa, Hiroshi; Rustgi, Anil K

    2012-01-01

    This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform. PMID:22240585

  20. Genotoxic effects of sunlight-activated waste water in cultured mammalian cells.

    PubMed

    Strniste, G F; Chen, D J; Okinaka, R T

    1982-07-01

    Cultured Chinese hamster ovary cells were incubated with dilutions of an oil shale retort process water and exposed to nautral sunlight. An enhancement of sevenfold to ninefold was seen in photoinduced cytotoxicity (by a colony-forming assay) and mutagenicity [at the hypoxanthine phosphoribosyltransferase (HPRT) locus] for cells pretreated with the process water compared to effects seen in cells exposed to sunlight only. Significant photoinduced cytotoxicity was also observed in cultured human skin fibroblasts when exposed to the process water before being exposed to near UV (NUV) radiation. The mutation frequencies (determined for the HPRT locus) induced by the process water and NUV radiation were as great as those frequencies seen for far UV light alone. Increases in genotoxicity were observed in excision repair-deficient xeroderma pigmentosum skin fibroblasts when compared to the responses seen in normal cells. Risks to health resulting from the phototransformation of these oil shale retort process waste waters are unassessed at this time. PMID:6954312

  1. Embryoid formation in alfalfa cell suspension cultures from different plants

    Microsoft Academic Search

    K. N. Kao; M. R. Michayluk

    1981-01-01

    Summary  Cell suspension cultures were initiated from shoot tips of nine plants of alfalfa. Our results indicated that considerably\\u000a different nutritional conditions were required to induce embryogenesis in the cell cultures derived from these plants. By\\u000a proper adjustment of the hormone level and mineral salt concentration it was possible to induce embryogenesis in all of the\\u000a nine cultures tested. The embryoids

  2. Odontoblast Differentiation of Human Dental Pulp Cells in Explant Cultures

    Microsoft Academic Search

    M.-L. Couble; J.-C. Farges; F. Bleicher; B. Perrat-Mabillon; M. Boudeulle; H. Magloire

    2000-01-01

    .   In order to elucidate the mechanisms involved in human dentin formation, we developed a cell culture system to promote differentiation\\u000a of dental pulp cells into odontoblasts. Explants from human teeth were cultured in Eagle's basal medium supplemented with\\u000a 10% or 15% fetal calf serum, with or without ?-glycerophosphate (?GP). Addition of ?GP to the culture medium induced odontoblast\\u000a features

  3. Cell wall proteomics of sugarcane cell suspension cultures.

    PubMed

    Calderan-Rodrigues, Maria Juliana; Jamet, Elisabeth; Bonassi, Maria Beatriz Calderan Rodrigues; Guidetti-Gonzalez, Simone; Begossi, Amanda Carmanhanis; Setem, Laís Vaz; Franceschini, Livia Maria; Fonseca, Juliana Guimarães; Labate, Carlos Alberto

    2014-03-01

    The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl2 and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second-generation ethanol production. PMID:24436144

  4. A long-term flow cytometry assay to analyze the role of specific genes of Drosophila melanogaster S2 cells in surviving genotoxic stress.

    PubMed

    Yi, Xia; Lemstra, Willy; Vos, Michel J; Shang, Yongfeng; Kampinga, Harm H; Su, Tin Tin; Sibon, Ody C M

    2008-07-01

    Drosophila S2 cells are easy to manipulate and culture and are a versatile model system for high-throughput screens such as genome-wide siRNA screens to find genes involved in stress or therapy resistance or for screening through large compound libraries to identify cytotoxins. Clonogenic assays are considered the gold-standard to investigate the cytotoxicity of specific treatments or to compare the sensitivity of various cell types for a specific treatment. However, this assay cannot be used for Drosophila S2 cells as they are virtually unable to grow in distinct colonies. We designed a novel fluorescence-based flow cytometry assay to study long-term proliferation of S2 cells under various conditions and in the presence of specific gene products or after downregulation of specific gene products. Here we validate this assay and we used this novel method to investigate the role of checkpoint genes grapes/Dchk1 and DmChk2 in cell survival responses. Our data demonstrate that Grapes/Dchk1 but not DmChk2 is required to survive hydroxyurea. Our assay will be of use to investigate the long-term effects of various treatments in S2 cells and to evaluate the role of specific proteins therein. PMID:18496848

  5. Assessment of Cell Viability in Intact Glandular Tissue in Chironomus ramosus using Dye-exclusion and Colorimetric Assays.

    PubMed

    Nath, B B; Babrekar, A A; Parthasarathy, B

    2005-09-01

    Conventionally, dye-exclusion test for determining cell viability has been restricted only for cells in suspension in tissue culture. In this paper, salivary gland of Chironomus has been proposed as a simple tissue model system where dye-exclusion test can be reliably employed for the intact gland. We have compared suitability of commonly used vital dyes and nigrosin was found suitable for the salivary gland cells. Biochemical tests using tetrazolium salts are also commonly used for determining quantitative indices of cell viability in metabolically active cells. Ours is the first attempt to extend the same technique for the whole tissue. We standardized the conditions and prepared a protocol for MTT-based colorimetric assay suitable for the salivary gland of Chironomus. A strong correlation (r(2) = 0.9893) was obtained where increasing O.D. correlated linearly with the number of live glands. We concluded that nigrosin dye-exclusion and MTT metabolic inclusion assays are suitable methods for the viability test of metabolically active intact salivary gland of Chironomus which can serve as a potential model for the assessment of cytotoxicity in future. PMID:19003063

  6. Horizontally rotated cell culture system with a coaxial tubular oxygenator

    NASA Technical Reports Server (NTRS)

    Wolf, David A. (inventor); Schwarz, Ray P. (inventor); Trinh, Tinh T. (inventor)

    1991-01-01

    The present invention relates to a horizontally rotating bioreactor useful for carrying out cell and tissue culture. For processing of mammalian cells, the system is sterilized and fresh fluid medium, microcarrier beads, and cells are admitted to completely fill the cell culture vessel. An oxygen containing gas is admitted to the interior of the permeable membrane which prevents air bubbles from being introduced into the medium. The cylinder is rotated at a low speed within an incubator so that the circular motion of the fluid medium uniformly suspends the microbeads throughout the cylinder during the cell growth period. The unique design of this cell and tissue culture device was initially driven by two requirements imposed by its intended use for feasibility studies for three dimensional culture of living cells and tissues in space by JSC. They were compatible with microgravity and simulation of microgravity in one G. The vessels are designed to approximate the extremely quiescent low shear environment obtainable in space.

  7. The comet assay as an indicator test for germ cell genotoxicity.

    PubMed

    Speit, Günter; Vasquez, Marie; Hartmann, Andreas

    2009-01-01

    The in vivo comet assay is a well-established genotoxicity test. It is currently mainly performed with somatic cells from different organs to detect a genotoxic activity of potential carcinogens. It is regarded as a useful test for follow-up testing of positive or equivocal in vitro test results and for the evaluation of local genotoxicity. However, the comet assay also has the potential to detect germ cell genotoxicity and may be used for demonstrating the ability of a substance or its metabolite(s) to directly interact with the genetic material of gonadal and/or germ cells. Such results are important for the classification of germ cell mutagens, e.g. in the context of the "Globally Harmonized System of Classification and Labelling of Chemicals" (GHS). This review summarizes and discusses available information on the use of the comet assay with germ cells and cells from the gonads in genetic toxicology. The literature contains results from in vitro studies, ex vivo studies and in vivo studies. With regard to the assessment of germ cell genotoxicity, only in vivo studies are relevant but the other kind of studies provided important information on various aspects of the methodology. Many comet assay studies with human sperm have been performed in the context of male infertility and assisted fertilization. The results of these studies are not reviewed in detail here but various aspects of the assay modifications used are discussed. Measuring DNA effects by the comet assay in sperm requires additional steps for chromatin decondensation. Many different modifications of the alkaline and the neutral comet assay are in use but a standard protocol has not been established yet. High and variable background levels of DNA effects were reported and there is still need for standardization and validation of the comet assay with sperm. Some human biomonitoring studies with human sperm were published, but it seems to be premature to use these data for hazard identification and classification of chemicals. In contrast, the standard alkaline in vivo comet assay can easily be adapted to investigations with cells from reproductive organs. Tests with cells from the gonads (testis and ovary) seem to be most appropriate and a promising tool for demonstrating that a test compound reaches the gonads and is able to interact with the genetic material of germ cells. However, studies to standardize and validate these methods are necessary before the comet assay can be usefully applied in risk assessment of germ cell mutagens. PMID:18462987

  8. UDP-glucose:digitoxin 16'-O-glucosyltransferase from suspension-cultured Digitalis lanata cells.

    PubMed

    Kreis, W; May, U; Reinhard, E

    1986-12-01

    Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16'-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, ? -acetyldigitoxin, and ?-acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed. PMID:24248401

  9. Biona-C Cell Culture pH Monitoring System

    NASA Technical Reports Server (NTRS)

    Friedericks, C.

    1999-01-01

    Sensors 2000! is developing a system to demonstrate the ability to perform accurate, real-time measurements of pH and CO2 in a cell culture media in Space. The BIONA-C Cell Culture pH Monitoring System consists of S2K! developed ion selective sensors and control electronics integrated with the fluidics of a cell culture system. The integrated system comprises a "rail" in the Cell Culture Module (CCM) of WRAIR (Space Biosciences of Walter Read Army Institute of Research). The CCM is a Space Shuttle mid-deck locker experiment payload. The BIONA-C is displayed along with associated graphics and text explanations. The presentation will stimulate interest in development of sensor technology for real-time cell culture measurements. The transfer of this technology to other applications will also be of interest. Additional information is contained in the original document.

  10. In vitro male germ cell cultures of zebrafish.

    PubMed

    Sakai, Noriyoshi

    2006-07-01

    Transgenic modification of sperm before fertilization has the advantages of a much shorter timeline for the production of transgenic animals. A culture system using primary cultures of zebrafish male germ cells, in which the differentiation of spermatogonia to functional sperm can occur in vitro, allows us to introduce foreign DNA into the cultured sperm and to produce transgenics from the sperm. This chapter describes methods for the co-culture of male germ cells and a Sertoli cell feeder layer and the introduction of foreign DNA with retroviruses. This male germ cell culture system should prove useful not only in producing genetically modified sperm, but also in analyzing the regulatory function of Sertoli cells for spermatogenesis in vertebrates. PMID:16828310

  11. Uptake, accumulation, and egress of erythromycin by tissue culture cells of human origin.

    PubMed Central

    Martin, J R; Johnson, P; Miller, M F

    1985-01-01

    The ability of erythromycin A base to penetrate and accumulate in tissue culture cells of human origin was investigated. The antibiotic was highly concentrated by early passage cells of normal bronchus, kidney, liver, lung, and skin and by cancer cells derived from breast, liver, and lung. Intracellular levels 4 to 12 times that of the extracellular milieu were obtained in both early-passage and transformed cells. The total quantity of erythromycin accumulated depended on the extracellular concentration of antibiotic, but the cellular/extracellular ratios were, for the most part, independent of the initial extracellular drug concentration. In all cell types tested, the accumulated antibiotic rapidly egressed when cells were incubated in antibiotic-free medium. Bioactivity assays demonstrated that the expelled drug was unmetabolized, fully active antibiotic. The concentration of erythromycin by a variety of human cell types probably accounts, in part, for the effectiveness of the antibiotic against intracellular parasites such as Legionella and Chlamydia spp. PMID:3994346

  12. Minimally cultured tumor-infiltrating lymphocytes display optimal characteristics for adoptive cell therapy.

    PubMed

    Tran, Khoi Q; Zhou, Juhua; Durflinger, Katherine H; Langhan, Michelle M; Shelton, Thomas E; Wunderlich, John R; Robbins, Paul F; Rosenberg, Steven A; Dudley, Mark E

    2008-10-01

    Adoptive cell therapy (ACT) with tumor-reactive lymphocytes in patients with refractory melanoma can result in tumor regression and prolonged survival. Generating tumor-reactive lymphocyte cultures is technically difficult and resource intensive; these limitations have restricted the widespread application of ACT. Tumor-infiltrating lymphocytes (TIL) from melanoma contain tumor antigen-reactive cells. The "standard" method for producing TIL cultures for clinical administration requires extended in vitro expansion in interleukin-2, then identification of tumor-reactive cells by immunologic assays. We show here that limitations in reagents and methods during screening underrepresent the actual reactivity of TIL cultures. Furthermore, the extended culture times necessitated by the screening assays resulted in telomere shortening and reduced expression of CD27 and CD28 in the TIL cultures, properties that our prior studies showed are correlated with in vivo persistence and clinical response. We have thus developed an alternative "young" TIL method that demonstrated superior in vitro attributes compared with standard TIL. This approach uses the entire resected tumor to rapidly expand TIL for administration without in vitro testing for tumor recognition. Our observations suggest that younger TIL can have an undetermined but high level of antigen reactivity, and other advantageous attributes such as long telomeres and high levels of CD27 and CD28. We suggest that minimally cultured, unselected lymphocytes represent an alternative strategy for generating TIL cultures suitable for use in ACT that, if effective in vivo, may facilitate the widespread application of this approach to a broader population of patients with melanoma. PMID:18779745

  13. Single Cell Adhesion Assay Using Computer Controlled Micropipette

    PubMed Central

    Salánki, Rita; H?s, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

    2014-01-01

    Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today’s techniques typically have an extremely low throughput (5–10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (?30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes. PMID:25343359

  14. Optical Oxygen Sensors for Applications in Microfluidic Cell Culture

    PubMed Central

    Grist, Samantha M.; Chrostowski, Lukas; Cheung, Karen C.

    2010-01-01

    The presence and concentration of oxygen in biological systems has a large impact on the behavior and viability of many types of cells, including the differentiation of stem cells or the growth of tumor cells. As a result, the integration of oxygen sensors within cell culture environments presents a powerful tool for quantifying the effects of oxygen concentrations on cell behavior, cell viability, and drug effectiveness. Because microfluidic cell culture environments are a promising alternative to traditional cell culture platforms, there is recent interest in integrating oxygen-sensing mechanisms with microfluidics for cell culture applications. Optical, luminescence-based oxygen sensors, in particular, show great promise in their ability to be integrated with microfluidics and cell culture systems. These sensors can be highly sensitive and do not consume oxygen or generate toxic byproducts in their sensing process. This paper presents a review of previously proposed optical oxygen sensor types, materials and formats most applicable to microfluidic cell culture, and analyzes their suitability for this and other in vitro applications. PMID:22163408

  15. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned

    PubMed Central

    Maddox, Clinton B; Rasmussen, Lynn; White, E. Lucile

    2009-01-01

    In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high throughput screening arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the high throughput screening drug discovery platform, with special consideration being given to assay miniaturization, liquid handling complications and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry-over, and incubator induced artifacts. PMID:19492073

  16. High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells

    PubMed Central

    2013-01-01

    Background The kidney-specific NKCC cotransporter isoform NKCC2 is involved in the Na+ reabsorption in the Thich Ascending Limb (TAL) cells and in the regulation of body fluid volume. In contrast, the isoform NKCC1 represents the major pathway for Cl- entry in endothelial cells, playing a crucial role in cell volume regulation and vascular tone. Importantly, both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for the treatment of hypertension. Results Taking advantage of an existing Thallium (Tl+)-based kit, we set up a Tl+ influx-based fluorescent assay, that can accurately and rapidly measure NKCC transporter activity in adherent epithelial cells using the high-throughput Flex station device. We assessed the feasibility of this assay in the renal epithelial LLC-PK1 cells stably transfected with a previously characterized chimeric NKCC2 construct (c-NKCC2). We demonstrated that the assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, being a continuous assay, it offers improved sensitivity over previous endpoint NKCC functional assays. Conclusions So far the screening of NKCC transporters activity has been done by 86Rb+ influx assays. Indeed, a fluorescence-based high-throughput screening method for testing NKCC inhibitors would be extremely useful in the development and characterization of new anti-hypertensive drugs. PMID:23506056

  17. Incidence of Listeria species in bovine, ovine, caprine, camel and water buffalo milk using cultural method and the PCR assay

    PubMed Central

    Rahimi, Ebrahim; Momtaz, Hassan; Behzadnia, Asma; Baghbadorani, Zeinab Torki

    2014-01-01

    Objective To determine the prevalence rate of Listeria species in bovine, ovine, caprine, camel and water buffalo milk in Iran. Methods From September 2010 to December 2011 a total of 260 bulk milk samples including 85 bovine, 37 camel, 34 water buffalo, 56 ovine and 48 caprine bulk milk samples were collected from commercial dairy herds, in Fars and Khuzestan provinces, Iran and were evaluated for the presence of Listeria species using cultural method and the PCR assay. Results Using cultural method, 19 samples (7.3%) were positive for Listeria spp. The highest prevalence of Listeria was found in raw water buffalo milk (11.8%), followed by raw bovine milk (10.6%), raw ovine milk (7.1%), and raw caprine milk (4.2%) samples. All 37 camel milk samples from 20 camel breeding farms were negative for Listeria spp. The overall prevalence of Listeria was 7.3%, in which Listeria innocua was the most recovered species (4.2%); the remaining isolates were Listeria monocytogenes (1.9%), Listeria ivanovii (0.08%) and Listeria seeligari (0.04%). The PCR assay could identify 8 Listeria-contaminated milk samples that were negative using the cultural method. Conclusions The results presented in this study indicate the potential risk of infection with Listeria in people consuming raw and unpasteurized milk.

  18. Lymphokine-activated Killer Cells: Lysis of Fresh Syngeneic Natural Killer-resistant Murine Tumor Cells by Lymphocytes Cultured in Interleukin 2

    Microsoft Academic Search

    Maury Rosenstein; Yael Kaufmann; Steven A. Rosenberg

    1984-01-01

    Normal splenocytes that are cultured in the lymphokine, inter- leukin 2 (IL-2), for as short as 2 days develop lytic activity for fresh syngeneic natural killer-resistant tumor cells as well as natural killer-sensitive YAC cells in a 4-hr 51Cr release assay. Lymphokine-activated killer (LAK) cells do not lyse syngeneic fresh lymphocytes but do lyse syngeneic concanavalin A-induced lymphocyte blasts. Lysis

  19. Droplet optofluidic imaging for ?-bacteriophage detection via co-culture with host cell Escherichia coli.

    PubMed

    Yu, J Q; Huang, W; Chin, L K; Lei, L; Lin, Z P; Ser, W; Chen, H; Ayi, T C; Yap, P H; Chen, C H; Liu, A Q

    2014-09-21

    Bacteriophages are considered as attractive indicators for determining drinking water quality since its concentration is strongly correlated with virus concentrations in water samples. Previously, bacteriophage detection was based on a plague assay that required a complicated labelling technique and a time-consuming culture assay. Here, for the first time, a label-free bacteriophage detection is reported by using droplet optofluidic imaging, which uses host-cell-containing microdroplets as reaction carriers for bacteriophage infection due to a higher contact ratio. The optofluidic imaging is based on the effective refractive index changes in the microdroplet correlated with the growth rate of the infected host cells, which is highly sensitive, i.e. can detect one E. coli cell. The droplet optofluidic system is not only used in drinking water quality monitoring, but also has high potential applications for pathogenic bacteria detection in clinical diagnosis and food industry. PMID:25008551

  20. The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

    PubMed Central

    Azari, Hassan; Sharififar, Sharareh; Fortin, Jeff M.; Reynolds, Brent A.

    2012-01-01

    Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells. PMID:22546813

  1. Chromatographically purified immunoglobulin G of endemic and sporadic goiter patients stimulates FRTL5 cell growth in a mitotic arrest assay.

    PubMed

    Wilders-Truschnig, M M; Drexhage, H A; Leb, G; Eber, O; Brezinschek, H P; Dohr, G; Lanzer, G; Krejs, G J

    1990-02-01

    A strain of differentiated rat thyroid cells (FRTL5) in continuous culture was used to study the presence of thyroid growth-promoting immunoglobulins (TGI) in the serum of patients with endemic and sporadic euthyroid goiters. To identify true in vitro cell proliferation a microscopic mitotic arrest assay was used. Immunoglobulins G (IgGs) were prepared with QAE-Sephadex A-50 or protein-A-Sepharose. A positive growth stimulation index was found in IgG preparations of 65 of 71 patients with endemic goiter and in 9 of 14 IgG preparations of patients with sporadic goiter. IgG preparations of 15 control subjects from an area where endemic goiter due to iodine deficiency does not occur and of 18 subjects without iodine deficiency and without thyroid enlargement living in the endemic area did not stimulate FRTL5 cell growth. FRTL5 cell growth stimulation with IgGs of these euthyroid goiter patients could only be detected when IgG was tested in combination with a small dose of TSH. Immunoprecipitation with polyclonal and monoclonal antihuman IgG was able to abolish the growth-promoting effects. In 32 blinded samples the Feulgen cytobiochemical assay, formerly used to detect TGI, was compared with the FRTL5 mitotic arrest assay. The two methods showed similar results. Our observations of chromatographically purified IgG promoting thyroid cell proliferation in vitro provide good evidence that IgG was responsible for thyroid cell growth in vitro and suggest that autoimmune growth mechanisms may be involved in the pathogenesis of both endemic and sporadic goiters. PMID:1688867

  2. ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells.

    PubMed

    Hanson, Jodi; Sundararaman, Srividya; Caspell, Richard; Karacsony, Edith; Karulin, Alexey Y; Lehmann, Paul V

    2015-01-01

    Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor immunity, it is also advisable to account for determinant spreading by testing multiple epitopes. Efforts for comprehensive immune monitoring would require substantial numbers of PBMC to run the above tests systematically, which in most test cases is limiting. Immune monitoring with ELISPOT assays have been performed, thus far, in a 96-well format. In this study we show that one can increase cell utilization by performing the assay in 384-well plates whose membrane surface area is one third that of 96-well plates. Systematic testing of PBMC for antigen-specific T cell response in the two formats demonstrated that the 384-well assay corresponds to a one-in-three miniaturization of the 96-well assay. The lowest number of cells that can be used in the 384-well format, while allowing for sufficient contact with APC, is 33,000 PBMC/well. Therefore, with one million PBMC typically obtained from 1 mL of blood, a 30 well T cell ELISPOT assay can be performed in a 384-well format. PMID:25643292

  3. Detection of Brain Tumor Cells in the Peripheral Blood by a Telomerase Promoter-Based Assay

    PubMed Central

    MacArthur, Kelly M.; Kao, Gary D.; Chandrasekaran, Sanjay; Alonso-Basanta, Michelle; Chapman, Christina; Lustig, Robert A.; Wileyto, E. Paul; Hahn, Stephen M.; Dorsey, Jay F.

    2014-01-01

    Blood tests to detect circulating tumor cells (CTC) offer great potential to monitor disease status, gauge prognosis, and guide treatment decisions for patients with cancer. For patients with brain tumors, such as aggressive glioblastoma multiforme, CTC assays are needed that do not rely on expression of cancer cell surface biomarkers like epithelial cell adhesion molecules that brain tumors tend to lack. Here, we describe a strategy to detect CTC based on telomerase activity, which is elevated in nearly all tumor cells but not normal cells. This strategy uses an adenoviral detection system that is shown to successfully detect CTC in patients with brain tumors. Clinical data suggest that this assay might assist interpretation of treatment response in patients receiving radiotherapy, for example, to differentiate pseudoprogression from true tumor progression. These results support further development of this assay as a generalized method to detect CTC in patients with cancer. PMID:24525740

  4. 3D inverted colloidal crystals in realistic cell migration assays for drug screening applications.

    PubMed

    da Silva, Joakim; Lautenschläger, Franziska; Kuo, Cheng-Hwa R; Guck, Jochen; Sivaniah, Easan

    2011-12-01

    Screening drugs for their specific impact on cell mechanics, in addition to targeting adhesion and proteolysis, will be important for successfully moderating migration in infiltrative disorders including cancer metastasis. We present 3D inverted colloidal crystals made of hydrogel as a realistic cell migration assay, where the geometry and stiffness can be set independently to mimic the tissue requirements in question. We show the utility of this 3D assay for drug screening purposes, specifically in contrast to conventional 2D migration studies, by surveying the effects of commonly used cytoskeletal toxins that impact cell mechanics. This assay allows studying large cell numbers for good statistics but at single-cell resolution. PMID:22038190

  5. Effects of cadmium chloride on the cultured human lens epithelial cells

    PubMed Central

    Song, Nang-Hee

    2012-01-01

    Purpose To investigate cadmium chloride cytotoxicity in human lens epithelial cells as well as the mode of cell death and its mechanism. Methods Cultured human lens epithelial cells were challenged with cadmium chloride. Morphological changes of human lens epithelial cells caused by cadmium chloride exposure were evaluated by microscope. Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny tetrazolium bromice (MTT) assay. To explore the mechanism of cell death, p53 and caspase-8 levels were measured by western blotting. Results Microscopic examination indicated that cell death increased after cadmium chloride exposure compared to untreated cells. The MTT assay demonstrated that cadmium chloride significantly decreased cell viability in a dose dependent way. Western blot and quantitative analysis showed that both p53 and caspase-8 increased after cell exposure to cadmium chloride. p53 increased 210% and caspase-8 increased 30% in the experimental group as compared with the control group. Conclusions Cadmium chloride induced cytotoxicity and apoptosis in human lens epithelial cells and the mechanism of apoptosis involve an increased expression of p53 and caspase-8. PMID:22550391

  6. Comparison of the Binax NOW Flu A Enzyme Immunochromatographic Assay and R-Mix Shell Vial Culture for the 2003-2004 Influenza Season

    Microsoft Academic Search

    Robert C. Fader

    2005-01-01

    The Binax NOW Flu A enzyme immunochromatographic assay was compared to viral culture with R-Mix shell vials for 455 nasal-wash or nasal-aspirate specimens. The overall sensitivity, specificity, positive predic- tive value, and negative predictive value of the assay were 64.9%, 98.4%, 89.3%, and 93.2%, respectively. However, the assay sensitivity decreased significantly with increasing patient age. With the advent of antiviral

  7. Detection and Treatment of Mycoplasma Contamination in Cultured Cells

    Microsoft Academic Search

    Hsuan Jung; Shih-Yee Wang; I-Wen Yang; Ding-Wei Hsueh; Wei-Ju Yang; Tzu-Hao Wang; Hsin-Shih Wang

    Background: Mycoplasmas, the smallest and simplest prokaryotes that reside in endo- somes of mammalian cells, are widespread contaminants found in cell cul- tures. About 30% of all cell cultures, varying from 15 to 80%, are reportedly contaminated with mycoplasmas. Here, we present our experience in suc- cessfully detecting and treating mycoplasmal infection in various cell lines. Methods: The nested polymerase

  8. Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

    PubMed

    Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Sou?ek, Karel

    2013-05-01

    The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. PMID:23450810

  9. Video lensfree microscopy of 2D and 3D culture of cells

    NASA Astrophysics Data System (ADS)

    Allier, C. P.; Vinjimore Kesavan, S.; Coutard, J.-G.; Cioni, O.; Momey, F.; Navarro, F.; Menneteau, M.; Chalmond, B.; Obeid, P.; Haguet, V.; David-Watine, B.; Dubrulle, N.; Shorte, S.; van der Sanden, B.; Di Natale, C.; Hamard, L.; Wion, D.; Dolega, M. E.; Picollet-D'hahan, N.; Gidrol, X.; Dinten, J.-M.

    2014-03-01

    Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell