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Performance of Karyologic Studies on Primary Human Cell Cultures.  

National Technical Information Service (NTIS)

Primary cell cultures were established from lung, skin and kidney tissues from four aborted normal human fetuses. Karyologic studies were performed on these primary cell cultures up to 4th culture passage except for the first two fetal kidney cultures. Th...

L. Y. F. Hsu



Studies of ?-protein in human cell cultures  

Microsoft Academic Search

Summary  ?-Protein growth fraction (AGF) eliminates the 60- to 90-day adaptive phase required to establish actively growing cultures\\u000a of HeLa (Gey), human heart (Girardi), KB (Eagle) and other established cell lines in serum-free chemically defined medium\\u000a A3. AGF is effective at less than 0.4 ?g per ml. By using the procedures described in the text, it is possiblee to culture HeLa

Richard Holmes; Gretchen Mercer; Nalini Mohamed



Plant Cell Cultures in Space Biology Studies.  

National Technical Information Service (NTIS)

The rate of cell wall regeneration using rape protoplasts was studied in preparatory experiments for the Spacelab Biorack IML-1 mission. Under normal ground conditions the cell wall regenerates during the first 24 hr. Results from experiments using low te...

T. Iversen C. Baggerud K. Draget



Hepatotoxicity studies with primary cultures of rat liver cells  

Microsoft Academic Search

Summary  A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed in our laboratory. Growing\\u000a cultures in arginine-deficient medium inhibits fibroblast overgrowth, and relatively pure cultures of parenchymal hepatocytes\\u000a are obtained. This cell culture system has been used to study the cytotoxicity of two hepatotoxic agents, tetracycline and\\u000a norethindrone. Caffeine was evaluated as an agent thought to

David C. Anuforo; Daniel Acosta; Robert V. Smith



Microfibrous carriers for cell culture: a comparative study.  


Small patches of polyethylene terephthalate (PET) nonwoven microfibrous matrices have excellent properties and can be used as carriers for culturing cells in agitated bioreactors. The microfibrous carriers are highly porous and can provide large surface areas and three-dimensional space for high-density cell growth. In this work, the microfibrous carriers and several commercial microcarriers were used to study cell attachment kinetics, growth, and monoclonal antibody production with Chinese hamster ovary cells. Compared with commercial solid and macroporous microcarriers, the microfibrous carriers showed better or similar performances. In addition, the microfibrous carriers provided a wider operable range for agitation rate than commercial microcarriers, effectively protecting cells from shear stress and carrier collisions. In addition, the microfibrous carriers are available at a much lower cost than commercial microcarriers, providing an attractive alternative to microcarrier-based large-scale cell cultures. PMID:21608140

Wen, Yuan; Yang, Shang-Tian



Characterizing parameters of Jatropha curcas cell cultures for microgravity studies  

NASA Astrophysics Data System (ADS)

Jatropha (Jatropha curcas) is a tropical perennial species identified as a potential biofuel crop. The oil is of excellent quality and it has been successfully tested as biodiesel and in jet fuel mixes. However, studies on breeding and genetic improvement of jatropha are limited. Space offers a unique environment for experiments aiming at the assessment of mutations and differential gene expression of crops and in vitro cultures of plants are convenient for studies of genetic variation as affected by microgravity. However, before microgravity studies can be successfully performed, pre-flight experiments are necessary to characterize plant material and validate flight hardware environmental conditions. Such preliminary studies set the ground for subsequent spaceflight experiments. The objectives of this study were to compare the in vitro growth of cultures from three explant sources (cotyledon, leaf, and stem sections) of three jatropha accessions (Brazil, India, and Tanzania) outside and inside the petriGAP, a modified group activation pack (GAP) flight hardware to fit petri dishes. In vitro jatropha cell cultures were established in petri dishes containing a modified MS medium and maintained in a plant growth chamber at 25 ± 2 °C in the dark. Parameters evaluated were surface area of the explant tissue (A), fresh weight (FW), and dry weight (DW) for a period of 12 weeks. Growth was observed for cultures from all accessions at week 12, including subsequent plantlet regeneration. For all accessions differences in A, FW and DW were observed for inside vs. outside the PetriGAPs. Growth parameters were affected by accession (genotype), explant type, and environment. The type of explant influenced the type of cell growth and subsequent plantlet regeneration capacity. However, overall cell growth showed no abnormalities. The present study demonstrated that jatropha in vitro cell cultures are suitable for growth inside PetriGAPs for a period of 12 weeks. The parameters evaluated in this study provide the basic ground work and pre-flight assessment needed to justify a model for microgravity studies with jatropha in vitro cell cultures. Future studies should focus on results of experiments performed with jatropha in vitro cultures in microgravity.

Vendrame, Wagner A.; Pinares, Ania



Primary Cell Cultures for the Study of Myelination  

Microsoft Academic Search

\\u000a Tissue culture allows the separate establishment of neurons and glia under a variety of conditions of substrate and media.\\u000a By subsequent recombination of relatively pure cell populations, a number of questions of neuronal development and neuronal-glia\\u000a interactions, specifically myelination, may be studied. In vivo, axons of dorsal root ganglion (DRG) neurons course through\\u000a both the central nervous system (CNS) and

Mary I. Johnson; Richard P. Bunge; Patrick M. Wood


A study on mass culture of Panax quinquefolium cells.  


By omitting the component NH4NO3 and doubling the amount of KNO3 in MS medium, the Panax quinque folium cells cultured in such medium grew more rapidly and their saponin content was much higher than that cultured in regular MS medium. The growth rate and saponin content of the cells cultured in such medium (KNO33300 mg/l) increased 65.1% and 166.2% respectively as compared with that cultured in the regular medium. The application of oligosaccharins from Panax ginseng and Dendrobium candidum also increased their saponin content and growth rate. Especially, the content of Rg group saponins was apparently raised. It took more than 25 days for the cell suspension cultures to produce saponins in large amounts. The curve of saponin formation lagged slightly behind the growth curve in cell suspension culture and fermentation culture. The cell fermentation culture with a stabilized pH value was better than the culture with the pH value changing spontaneously on saponin content, growth rate and biomass. Finally, the culture patterns of P. quinque folium cells were compared and discussed. PMID:2132128

Zhou, L G; Zheng, G Z; Wang, S L



Electrophysiological studies of anion secretion in cultured human epididymal cells.  


1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/- 1.3 mV (mean +/- S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/- 0.9 microA cm-2 (mean +/- S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l-1 increased the ISC by 3.0 +/- 1.2 microA cm-2 (mean +/- S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol l-1). Forskolin (10 mumol l-1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l-1 KCl was found to be -30 +/- 14 mV (mean +/- S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l-1 adrenaline by an increase in inward current at -70 mV holding potential (delta I = -1600 +/- 900 pA, mean +/- S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 +/- 1 mV (mean +/- S.E.M., n = 15). 6. The adrenaline-induced inward current was found to be blockable by the Cl- channel blocker, DPC (0.25 mmol l-1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl-. 7. The effect of adrenaline on the whole-cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l-1 EGTA or 2 mmol l-1 BAPTA + 100 mumol l-1 TMB-8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca2+ concentration to 1 nmol l-1 or raising it to 10 mumol l-1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl- secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1362444

Huang, S J; Leung, A Y; Fu, W O; Chung, Y W; Zhou, T S; Chan, P S; Wong, P Y



Electrophysiological studies of anion secretion in cultured human epididymal cells.  

PubMed Central

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/- 1.3 mV (mean +/- S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/- 0.9 microA cm-2 (mean +/- S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l-1 increased the ISC by 3.0 +/- 1.2 microA cm-2 (mean +/- S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol l-1). Forskolin (10 mumol l-1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l-1 KCl was found to be -30 +/- 14 mV (mean +/- S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l-1 adrenaline by an increase in inward current at -70 mV holding potential (delta I = -1600 +/- 900 pA, mean +/- S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 +/- 1 mV (mean +/- S.E.M., n = 15). 6. The adrenaline-induced inward current was found to be blockable by the Cl- channel blocker, DPC (0.25 mmol l-1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl-. 7. The effect of adrenaline on the whole-cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l-1 EGTA or 2 mmol l-1 BAPTA + 100 mumol l-1 TMB-8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca2+ concentration to 1 nmol l-1 or raising it to 10 mumol l-1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl- secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Huang, S J; Leung, A Y; Fu, W O; Chung, Y W; Zhou, T S; Chan, P S; Wong, P Y



Performance of Karyologic Studies on Primary Human Cell Cultures.  

National Technical Information Service (NTIS)

Cells from skin, lung, and kidney biopsies from 6 fetuses were cultured in vitro. Photographs of original cultures and subcultures are on file. One hundred metaphase plates were sketched for each tissue type and of these twenty were karyotyped. Six hundre...

L. Y. F. Hsu




Microsoft Academic Search

A method for culturing non- or slowly growing, differentiated fetal rat liver cells is de- scribed. It involves the use of collagenase as a digesting agent and of a selective medium deficient in arginine which suppresses the growth of nonparenchymal liver cells . Evidence is presented that surviving cells (a) retain liver-specific urea cycle functions measured by their capacity to

H. L. Leffert; D. PAUL



Psyllid cell culture: System to study Candidatus Liberibacter replication  

Technology Transfer Automated Retrieval System (TEKTRAN)

A cell culture system was established for the potato psyllid, Bactericera cockerelli (Hemiptera: Psyllidae), a highly competent vector of the phloem-inhabiting bacterium Candidatus Liberibacter psyllaurous (CLp) associated with the zebra complex disease in potato. Commonly referred to as Zebra Chip ...


Bioconversion studies in cultured cells of Corydalis species.  


Structural analysis of the metabolites of dopamine and salsolinol in cultured cells of Corydalis species was carried out using the combination of LC-MS and LC-NMR techniques. Metabolic pathways were clarified without the need to isolate the individual metabolites. PMID:15248465

Iwasa, K; Kuribayashi, A; Sugiura, M; Nishiyama, Y; Ichimaru, M; Moriyasu, M; Lee, D U



Advances in cell culture  

SciTech Connect

This book presents papers on advances in cell culture. Topics covered include: Genetic changes in the influenza viruses during growth in cultured cells; The biochemistry and genetics of mosquito cells in culture; and Tree tissue culture applications.

Maramorosch, K. (Dept. of Entomology, Rutgers Univ., New Brunswick, NJ (US))



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

Microsoft Academic Search

BackgroundThis work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous

Ramaschandra G. Sharathchandra; Charmaine Stander; Dan Jacobson; Bongani Ndimba; Melané A. Vivier; Hany A. El-Shemy



Microbial contamination of cell cultures: A 2 years study  

Microsoft Academic Search

Cell line contamination is a major drawback of main cell banks of the world and it has cost of losing important biological products or valuable research. The causative agents are different chemicals, invertebrates, bacteria, fungi, parasites, viral species and even other cell lines. In this retrospective study, cell lines from various species such as human, fish, insect, animals either offered

A. Mirjalili; E. Parmoor; S. Moradi Bidhendi; B. Sarkari



A primary cell culture of Drosophila postembryonic larval neuroblasts to study cell cycle and asymmetric division  

Microsoft Academic Search

Drosophila melanogaster is a key model system that has greatly contributed to the advance of developmental biology through its extensive and sophisticated genetics. Nevertheless, only a few in vitro approaches are available in Drosophila to complement genetic studies in order to better elucidate developmental mechanisms at the cellular and molecular level. Here we present a dissociated cell culture system generated

Julian Ceron; Francisco J. Tejedor; Fernando Moya



Biosynthesis of cellulose: studies with tobacco protoplasts and cultured cells  

SciTech Connect

The cell wall of regenerating tobacco protoplasts was shown to be mainly composed of noncellulosic ..beta..-1,3- and ..beta..-1,4-linked glucans with a cellulose content of only about 5%. Some pectic and hemicellulosic material is released by these protoplasts into the culture medium. The DP distribution of the ..cap alpha..-cellulose in regenerating protoplasts as well as in suspension-cultured cells, callus, or tobacco mesophyll revealed the existence of mainly two DP fractions with low (DP<500) and higher (DP 2000-3000) molecular weight, both of which contribute to the cellulosic network of the primary cell wall. The alkali-soluble and alkali-insoluble products of glucan synthetase assays with particulate enzyme fractions were analyzed in detail. By prelabeling with (/sup 14/C)glucose, the existence of primer glucans, which are elongated in the appropriate in vitro assay, could be substantiated. Alkali-soluble glucans consisted of a very short, if any, primer glucan, to which about 40 glucose units were added in vitro. The glucans in the alkali-insoluble fraction have an average DP of 200-250 and are synthesized in vitro by chain elongation via addition of about 30 new glucose units to a 1,4-linked primer glucan of DPapprox.200. 27 references, 6 figures, 2 tables.

Franz, G.; Blaschek, W.; Haass, D.; Koehler, H.



The Use of Cultured Epithelial and Endothelial Cells for Drug Transport and Metabolism Studies  

Microsoft Academic Search

In an effort to develop novel strategies for delivery of drug candidates arising from rational drug design and recombinant DNA technology, pharmaceutical scientists have begun to employ the techniques of cell culture to study drug transport and metabolism at specific biological barriers. This review describes some of the general factors that should be considered in developing a cell culture model

Kenneth L. Audus; Ronnda L. Bartel; Ismael J. Hidalgo; Ronald T. Borchardt



A simple perfusion chamber for studying neurotransmitter release from cells maintained in monolayer culture.  


A perfusion chamber is described for studying the efflux of putative neurotransmitters from CNS cells maintained in monolayer culture. We have used this apparatus to investigate the efflux of newly accumulated [3H]GABA from cell cultures of the early postnatal rat cerebellum. PMID:6111627

Pearce, B R; Currie, D N; Dutton, G R; Hussey, R E; Beale, R; Pigott, R



The application of renal cells in culture in studying drug-induced nephrotoxicity  

Microsoft Academic Search

Summary  Kidney cells in culture represent one of many in vitro approaches for studying drug-induced nephrotoxicity. Pontential advantages\\u000a of cell culture systems compared to more traditional in vitro models include a) the ability to examine direct effects at the\\u000a cellular level, b) extended viability, c) ability for long-term storage, and d) capabilities for automation. Primary cultures\\u000a of kidney tubules as well

Patricia D. Williams



Use of liver cell cultures in mutagenesis studies  

SciTech Connect

A sensitive cell-mediated assay has been developed for testing the mutagenesis of liver carcinogens. Mutagenesis was detected in Chinese hamster V79 cells that were cocultivated with hepatocytes isolated after collagenase/hyaluronidase digestion of rat liver slices. Mutations were characterized by resistance to ouabain and 6-thioguanine. Seven of the nitrosamines, which are potent liver carcinogens, exhibited a mutagenic response. Mutagenesis with these carcinogens could be detected at doses. The polyaromatic hydrocarbon benzo(a)pyrene, which is not a liver carcinogen, but can cause fibrosarcomas, was not mutagenic in this assay, but was mutagenic in a fibroblast-mediated assay. The liver carcinogen, aflatoxin B/sub 1/, which usually does not induce fibrosarcomas, exhibited an inverse situation; it was mutagenic for V79 cells in the presence of liver cells but not in the presence of fibroblasts. We suggest that the use of various cell types, including hepatocytes prepared by the slicing method for carcinogen metabolism, and mutable V79 cells offers a sensitive assay for determining the mutagenic potential of chemical carcinogens, and may also allow a study of their organ specificity.

Huberman, E.; Jones, C.A.



Microspore-derived cell suspension cultures of oilseed rape as a system for studying gene expression  

Microsoft Academic Search

Abiotic stress, such as extreme temperature, drought, or excessive salinity, is one of the leading causes of crop loss worldwide.\\u000a Microspore-derived (MD) cell suspension cultures of Brassica napus L. cv. Jet Neuf have been shown to be a useful system for studying the biochemistry of developing oilseeds. In the present\\u000a study, we describe the application of MD cell suspension cultures

Yongzhong Shi; Gangbiao Xu; Timothy B. Warrington; Gordon K. Murdoch; E. Chris Kazala; Crystal L. Snyder; Randall J. Weselake



Study of colonic IgG Fc binding site in cultured epithelial cells  

Microsoft Academic Search

Previously, we identified a unique binding site for the Fc region of IgG in goblet cells of the human small intestine and colon. Understanding of the regulation and biological role of the binding site would be enhanced if it could be studied in cultured cells. Thus, we now have searched colonic carcinoma cell lines for presence of the site and

Kensuke Kobayashi; William R. Brown



Insect neuronal cultures: an experimental vehicle for studies of physiology, pharmacology and cell interactions.  


The current status of insect neuronal cultures is discussed and their contribution to our understanding of the insect nervous system is explored. Neuronal cultures have been developed from a wide range of insect species and from all developmental stages. These have been used to study the morphological development of insect neurones and some of the extrinsic factors that affect this process. In addition, they have been used to investigate the physiology of sodium, potassium and calcium channels and the pharmacology of acetylcholine and GABA receptors. Insect neurones have also been grown in culture with muscle and glial cells to study cell interactions. PMID:16874504

Beadle, D J




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. ...


Studies of the shear protective effects of Pluronic F-68 on wild carrot cell cultures  

Microsoft Academic Search

Numerous studies have confirmed that Pluronic F-68 can effectively protect mammalian cell cultures grown in intensely agitated bioreactors from the damaging effects of hydrodynamic shear. Like mammalian cells, plant cells exposed to intense shear conditions are also severely damaged. However, the possibility of shear-protection for plant cells by Pluronic F-68 (PF-68) is seldom addressed. This work addressed this deficiency and

D. D Sowana; D. R. G Williams; B. K O’Neill; E. H Dunlop



Effect of transferrin on amphibian limb regeneration: a blastema cell culture study  

Microsoft Academic Search

In order to study mitogenic control during axolotl limb regeneration, we have developed a primary blastema cell culture as a very sensitive bioassay for blastema mitogens. Transferrin, an iron-binding glycoprotein which has been shown to be the neurotrophic factor for muscle cells, is the mitogen which has been analysed in the present report. Addition of approximately 2 µg human transferrin\\/

Philippe Albert; Bénoni Boilly



Studies on Addiction and Withdrawal in Cell Culture.  

National Technical Information Service (NTIS)

After 415 days (70 subcultures) growth in the presence of slowly increased drug concentrations, KB cells with increased tolerance to amphetamine, benadryl, caffeine, codeine, and morphine have developed. No withdrawal symptoms have been observed upon the ...

F. R. Leach M. L. Higgins T. J. Shaw S. Stadnicki



Studies on Addiction and Withdrawal in Cell Cultures.  

National Technical Information Service (NTIS)

After 645 days growth in the presence of slowly increasing drug concentrations and maintaining at LD50 concentrations, KB cells with increased tolerance to amphetamine, benadryl, caffeine, codeine, meprobamate, and morphine have been developed. Complete w...

F. R. Leach M. L. Higgins T. J. Shaw S. Stadnicki



Study of gherkin lactase in cell culture and in seedlings.  


A synthetic substrate replacing lactose has facilitated application of a simple, rapid and sensitive method for the identification and determination of extracellular and intracellular gherkin lactase. The intracellular enzyme activity was estimated from the cell suspension, while the extracellular enzyme activity was established within the cell free cultivation medium. A suspension of gherkin cells was permeabilized by Tween 20, or Tween 80, or hexadecyltrimethyl ammonium bromide, or hexadecylpyridinium chloride or ethanol added one at a time and then immobilized by glutaraldehyde. The highest lactase activity was at pH 4.8 at a temperature of 55°C. The hydrolysis of substrate was linear for 4.5h and reached 60% conversion. The cells had high lactase activity and good stability. During long-term storage they demonstrated convenient physico-mechanical properties. PMID:20728921

Stano, Ján; Siekel, Peter; Neubert, Klaus; Mi?ieta, Karol



Qualitative and quantitative studies on mixed homologous chicken thymus cell cultures  

PubMed Central

The proliferative interaction of mixed homologous chicken thymus cells was studied in a serum-free culture system using thymus cell populations from thymus glands that had been experimentally manipulated to yield varying proportions of lymphocytes from the medulla and cortex of the gland. It could be demonstrated that the proliferative responsiveness of thymus cells to immuno-genetically different cells resided with the lymphocyte population located in the medulla of the thymus. The capability of medullary thymus cells to participate in a mixed lymphocyte interaction (MLI) was found to be nearly equal to that of splenic lymphocytes. The in vitro survival of medullary thymus cells was markedly superior to cortical thymus cells. Kinetic studies with medullary thymus cells revealed that the proliferative response in the MLI was initiated during the first 24–40 hr culture period and generally reached its peak 4 days after culture initiation. It could be demonstrated that responding cells were capable of several repeated divisions. In addition, previously nondividing cells entered the reaction for the first time up to the 3rd and possibly 4th day after culture initiation. Calculations revealed that 0·7–1·9% of the original viable thymus cell population participated in the response. It was also calculated that peripheral blood lymphocytes contaminating the thymus cell populations of both cell donors contributed less than 1% to the total number of responding thymus cells. When the response of mixed homologous thymus cells was compared in a chemically defined and serum containing medium, striking differences in the time course and magnitude of the reaction were seen. It could be shown that the viability and proliferation of thymus cells was adversely affected by serum and markedly enhanced by insulin supplementation to a chemically defined medium. ImagesFig. 2Fig. 3Fig. 4

Weber, W. T.



A system for culturing iris pigment epithelial cells to study lens regeneration in newt.  


Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw¹. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month²(,)³ . This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2)?(,)?. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration. PMID:21730940

Bhavsar, Rital B; Nakamura, Kenta; Tsonis, Panagiotis A



A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt  

PubMed Central

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

Tsonis, Panagiotis A.



A serum-free primary culture system for studying cell-substrate interactions during newt epidermal cell migration  

Microsoft Academic Search

Summary  To study the interaction of migrating newt epidermal cells with purified extracellular matrix (ECM) molecules we have developed\\u000a an in vitro migration assay using pieces of newt skin explanted onto culture dishes coated with various ECM molecules and\\u000a cultured for 18 h in defined serum-free medium. Newt epidermal cells migrate out from explants placed on dishes coated with\\u000a either collagen,

James T. Mahan; Donald J. Donaldson



Dynamic single cell culture array  

Microsoft Academic Search

We demonstrate perfusion culture of arrays of individually trapped cells with dynamic microfluidic control of cellular environment, high maintenance of cell isolation, and low cell death. Hydrodynamically trapped cells were shown to adhere and divide at a comparable rate to cells grown randomly on the same glass substrate. This technique will be useful in quantitative single cell studies that require

Dino Di Carlo; Liz Y. Wu; Luke P. Lee



Immunocytochemical and structural comparative study of committed versus multipotent stem cells cultured with different biomaterials.  


The aim of this work was the comparison of the behavior of committed (human osteoblast cells - hOB - from bone biopsies) versus multipotent (human dental pulp stem cells - hDPSC - from extracted teeth) cells, cultured on shot-peened titanium surfaces, since the kind of cell model considered has been shown to be relevant in techniques widely used in studies on composition/morphology of biomaterial surfaces. The titanium surface morphology, with different roughness, and the behavior of cells were analyzed by confocal microscope (CM), scanning electron microscope (SEM) and X-ray microanalysis. The best results, in terms of hOB adhesion/distribution, were highlighted by both CM and SEM in cultured plates having 20-?m-depth cavities. On the contrary, CM and SEM results highlighted the hDPSC growth regardless the different surface morphology, arranged in overlapped layers due to their high proliferation rate, showing their unfitness in biomaterial surface test. Nevertheless, hDPSC cultured inside 3D-matrices reproduced an osteocyte-like three-dimensional network, potentially useful in the repair of critical size bone defects. The behavior of the two cell models suggests a different use in biomaterial cell cultures: committed osteoblast cells could be appropriate in selecting the best surfaces to improve osseointegration, while multipotent cells could be suitable to obtain in vitro osteocyte-like network for regenerative medicine. The originality of the present work consists in studying for the first time two different cell models (committed versus multipotent) compared in parallel different biomaterial cultures, thus suggesting distinct targets for each cellular model. PMID:22440134

Palumbo, Carla; Baldini, Andrea; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Ferretti, Marzia; Zaffe, Davide



Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses  

PubMed Central

Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79–1683 and WSU 79–1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79–1683 still replicated significantly more efficient compared to FCoV WSU 79–1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats.



Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses.  


Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79-1683 and WSU 79-1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79-1683 still replicated significantly more efficient compared to FCoV WSU 79-1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats. PMID:23964891

Desmarets, Lowiese Mb; Theuns, Sebastiaan; Olyslaegers, Dominique Aj; Dedeurwaerder, Annelike; Vermeulen, Ben L; Roukaerts, Inge Dm; Nauwynck, Hans J



Study of the structure of canine mesenchymal stem cell osteogenic culture.  


This study was designed to investigate the morphological features of osteogenic cultures that were established from canine marrow derived-mesenchymal stem cells (MSCs). Tripotent canine MSCs were plated in osteogenic conditions for 3 weeks, at the end of which the cultures were observed by light and transmission electron microscopy. Alkaline phosphatase (ALP) activity of the culture was determined during the differentiation period. To assess whether endochondral or intramembranous ossification was involved in MSC bone differentiation, the cultures were explored for cartilage-related gene expression. Multiple nodule-like cell aggregates appeared to form in the osteogenic cultures. These nodules were covered by a periosteum-like layer and osteocyte-like cells of varying morphology were located in lacuna-like cavities within the nodule mass. Furthermore, the bone nodules possessed an abundant matrix in which clearly striated collagen I fibres were arranged in perpendicular bundles. Matrix vesicles involving in matrix mineralization were evident in the nodules. This was in accordance with increased ALP activity in the culture. No expression of cartilage-related genes was observed, which suggested that osteogenesis might occur by intramembranous ossification. In conclusion, canine MSCs could be an appropriate model for studying in vitro bone development. PMID:20594192

Eslaminejad, M B; Taghiyar, L



Cell Culture on Nanopillar Sheet: Study of HeLa Cells on Nanopillar Sheet  

NASA Astrophysics Data System (ADS)

This is the first report of the successful culturing of HeLa cells on nanopillar sheets—a new type of cell culture dish—in a different way from that on flat petri dishes. Nanopillar sheets were fabricated with a high-aspect ratio structure with a diameter of 80-1000 nm and a height of 1-3 ?m using nanoprint technology. Nanopillar structure with 500 nm diameter and 1 ?m height enabled easy subculture of the cells from the sheets without the conventional trypsinization method. Moreover, the HeLa cells divided and proliferated on the sheets in the same way as on the flat surfaces with different manner of adhesion.

Nomura, Shinobu; Kojima, Hiroko; Ohyabu, Yoshimi; Kuwabara, Kosuke; Miyauchi, Akihiro; Uemura, Toshimasa



Cell Culture Made Easy.  

ERIC Educational Resources Information Center

|Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

Dye, Frank J.



A differentiated porcine bronchial epithelial cell culture model for studying human adenovirus tropism and virulence  

Microsoft Academic Search

The species specificity of human adenoviruses (HAdV) almost precludes studying virulence and tropism in animal models, e.g. rodent models, or derived tissue and cell culture models. However, replication of HAdV type 5 (HAdV-C5) has been shown after intravenous injection in swine. In order to study adenovirus replication in airway tissue propagation of bronchial epithelial cells from porcine lungs was established.

E. Lam; M. Ramke; S. Groos; G. Warnecke; A. Heim



Optimizing stem cell culture  

PubMed Central

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.

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



Stem cell culture engineering  

Microsoft Academic Search

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,

Gargi Seth; Catherine M. Verfaillie



Cell senescence culturing methods.  


Development of therapeutic approaches that slow or ablate the adverse physiological and pathological changes associated with aging has been considered as an important goal for gerontological research. As cellular senescence is characterized as the basis for aging in organisms, culturing and subculturing of normal human diploid fibroblasts to mimic the in vivo aging processes have been developed as major methods to investigate molecular events involved in aging. It has been established that normal human diploid fibroblasts can proliferate in culture for only finite periods of time. There are many ways to study aging in vitro. In this chapter, we will discuss some of the basic laboratory procedures for cell senescence culturing methods. PMID:23929093

Chen, Huaping; Li, Yuanyuan; Tollefsbol, Trygve O



An enhanced chemically defined SILAC culture system for quantitative proteomics study of human embryonic stem cells.  


Stable isotope labeling by SILAC-based quantitative proteomics analysis provides an unprecedented tool for the study of mechanisms underlying the self-renewal and differentiation of human embryonic stem cells (hESCs). While we recently reported a chemically defined SILAC culture system specific for a rare cell proteomic reactor (R. Tian et al., Mol. Cell. Proteomics 2011, 10, M110.000679), total hESC yield, prolonged self-renewal capacity (i.e.<12 days), and laborious procedure remain substantial hurdles for its conventional application in hESC studies. Here, we devised an enhanced SILAC culture system consisting of a new chemically defined SILAC-medium and a novel culture protocol. As a result, with much less culture maneuvers, approximately 40-fold greater hESCs were produced than the system reported previously. Moreover, the enhanced SILAC culture system was sufficient to support the self-renewal of hESCs for >60 days and was also highly reproducible. As such, it provides a new platform that can be readily adapted by general laboratory for further comprehensive SILAC-based proteomics analysis of hESCs and induced pluripotent stem cells. PMID:21770031

Wang, Shuai; Tian, Ruijun; Li, Li; Figeys, Daniel; Wang, Lisheng



Plant cell suspension cultures.  


Plant cell suspension cultures are widely used in plant biology as a convenient tool for the investigation of a wide range of phenomena, bypassing the structural complexity of the plant organism in toto. The homogeneity of an in vitro cell population, the large availability of material, the high rate of cell growth and the good reproducibility of conditions make suspension-cultured cells suitable for the analysis of complex physiological processes at the cellular and molecular levels. Moreover, plant cell cultures provide a valuable platform for the production of high-value secondary metabolites and other substances of commercial interest. Here we describe how to initiate and maintain plant cell cultures starting from explants obtained from in vitro germinated seedlings. Isolation of protoplasts from plant cell suspension cultures and regeneration of plants via organogenesis and somatic embryogenesis are also presented. PMID:23073877

Moscatiello, Roberto; Baldan, Barbara; Navazio, Lorella



Progeria: A cell culture study and clinical report of familial incidence  

Microsoft Academic Search

This report relates the case histories of two sisters who demonstrated the typical symptoms of progeria at birth. One of these children had died previous to this study. The familial occurrence underlines the thesis that progeria is an autosomal-recessive disorder. The examination of the cultured skin fibroblasts from the younger child showed a clear decrease in cell growth. On the

Thomas Rautenstrauch; Friedemann Snigula; Thomas Krieg; Steffen Gay; P. K. Müller



Histochemical study of brown-fat cells in the golden hamster (Mesocricetus auratus) in cultures  

SciTech Connect

The authors undertake the task of studying the synthesis of certain hormones by brown-fat cells. The authors used brown-fat cells from the golden hamster. The metabolism of brown-fat cells was studied on precultured cells, which made it possible to detect the synthesis of the studied substances rather than their accumulation in the organ. The authors conducted three experiments. First, fragments of brown fat were cultivated in diffusion chambers in vivo. Pieces of brown fat were cultivated in parallel in vitro on agar (organotypic cultures) and on plasma (histotypic cultures). During cultivation in diffusion chambers, the chambers were implanted in the abdominal cavity of young white rats. For in vitro cultivation, TCM 199 plus 15-20% calf serum was used. A total of 36 cultures with 12 cultures in each series of experiments were performed. The auto-radiographic studies of brown-fat cells were conducted on 24-hour cultures and on brown-fat fragments taken from the intact animal. The cultures were incubated with isotopes for 1 h. Either (/sup 3/H)lysine (87.3 Ci/mM specific activity), (/sup 3/H)arginine (16.7 Ci/mM), (/sup 3/H)glycerol (43 Ci/mM), or (/sup 3/H)cholesterol (43 Ci/mM) were added to the medium. After incubation, the cultures were washed three times in pure medium, fixed in Sierra fluid, and embedded in paraffin. The paraffin sections were covered with Ilford K/sub 2/ emulsion, and the preparations were exposed for 20 days at 4/sup 0/C temperature. Radio-immunological methods were used to study the accumulation of estradiol-17-beta in the culture medium by the Dobson method and that of testerone. The culture medium was taken on cultivation days 2,4,6,8, and 10. The medium was changed during cultivation every third day, which made it possible to judge the rates of accumulation of material with increase in the cultivation times.

Sokolov, V.E.; Boyadzhieva-Mikhailova, A.; Koncheva, L.; Angelova, P.; Evgen'eva, T.P.



IV. Studies of Three Strains in Primary Bovine Foetal Kidney Cell Cultures  

PubMed Central

Three different strains of bluetongue virus were adapted to grow in primary bovine foetal kidney cell cultures. The cytopathic effects observed from the three strains were similar, and were characterized by shrinkage of cells and increased granularity. The specificity of the changes was confirmed by the fluorescent antibody technique. No significant immunological cross-reaction was detected by serum-virus neutralization tests from the strains studied.

Girard, A.; Ruckerbauer, G. M.; Gray, D. P.; Bannister, G. L.; Boulanger, P.



Basics of Cell Culture  

NSDL National Science Digital Library

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.

Afshar, Golnar



Culture of Goblet Cells.  

National Technical Information Service (NTIS)

The invention encompasses isolation, culture and characterization of goblet cells in vitro form mammalian conjuctiva. Goblet cells can be cultured from conjunctiva of such mammals as, e.g., humans, rats, mice, rabbits and the like. In another aspect of th...

D. A. Dartt J. D. Rios M. A. Shatos



[Studies on the cell suspension culture of Saussarea medusa in a stirred tank bioreactor].  


The cell suspension culture of Saussarea medusa in a 2L aerated and agitated bioreactor with a four-pitch-blade impeller was investigated. The effects of agitation speed, aeration and inoculum size on cell growth and flavonoids production were studied and it was found that cells had optimum growth and flavonoids production when cultivated at 75 r/min, 700-1000 L/min and an inoculum of 4.0-5.0 g/L. A high cell biomass of 13.8 g/L and flavonoids production of 416 mg/L were achieved after 12 days of cultivation. Time course study revealed that flavonoids biosynthesis was growth-associated. The studies on aggregates size distribution in the bioreactor showed that the aggregates break-up caused by hydrodynamic stress might adversely affect cell growth and lead to significant reduction of cell biomass and flavonoids production. PMID:11797222

Huang, Y; Zhao, D X; Lu, D P; Yan, F; Li, Z H; Chen, H Z; Zhao, Q



A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease.  


Over the past two decades, it has become increasingly apparent that Alzheimer's disease neuropathology is characterized by activated microglia (brain resident macrophages) as well as the classic features of amyloid plaques and neurofibrillary tangles. The intricacy of microglial biology has also become apparent, leading to a heightened research interest in this particular cell type. Over the years a number of different microglial cell culturing techniques have been developed to study either primary mammalian microglia, or immortalized cell lines. Each microglial system has advantages and disadvantages and should be selected for its appropriateness in a particular research context. This review summarizes several of the most common microglial cell culture systems currently being employed in Alzheimer's research including primary microglia; BV2 and N9 retroviral immortalized microglia; human immortalized microglia (HMO6); and spontaneously immortalized rodent microglial lines (EOC lines and HAPI cells). Particularities of cell culture requirements and characteristics of microglial behavior, especially in response to applied inflammogen stimuli, are compared and discussed across these cell types. PMID:22651808

Stansley, Branden; Post, Jan; Hensley, Kenneth



Mycoplasma pneumoniae host–pathogen studies in an air–liquid culture of differentiated human airway epithelial cells  

Microsoft Academic Search

The interaction between Mycoplasma pneumonaie and the airway epithelium in vivo is complex and multifaceted. While multiple in vitro studies have been conducted studying this interaction with cell lines and animal cell and organ culture models, the interactions between M. pneumoniae and fully differentiated human airway epithelium in air–liquid interface culture remains unexplored. In the present study we investigated M.

Thomas M. Krunkosky; Jarrat L. Jordan; Emily Chambers; Duncan C. Krause



Isolation and culture of term human cytotrophoblast cells and in vitro methods for studying human cytotrophoblast cells' calcium uptake.  


Human primary cytotrophoblast cell culture is a very useful model to study the endocrine and immunological functions of syncytiotrophoblasts, as well as the ion exchange between the mother and her fetus, like calcium. In this chapter, we expose the procedure to (1) isolate and purify the cytotrophoblast cells from human term placenta and (2) study syncytiotrophoblast calcium uptake. First, the methodology is based on the enzymatic dissociation of villous placental tissue, followed by Percoll gradient separation. Purity is assessed by flow cytometry using staining against cytokeratin-7, protein specific for trophoblast cells. Cell proliferation is evaluated by a Thiazolyl Blue Tetrazolium Bromide (MTT) assay, hormonal secretion is measured by enzyme-linked immunosorbent assay (ELISA), and fusion is estimated by immunofluorescence using staining against desmosomal proteins. Second, we describe the calcium uptake experiment using the cytotrophoblast cells in culture. PMID:19495697

Le Bellego, Frédérique; Vaillancourt, Cathy; Lafond, Julie



The animal cell culture collection  

Microsoft Academic Search

Summary  The Animal Cell Culture Collection established by the Advisory Committee and cooperating laboratories at the American Type\\u000a Culture Collection has been described. The description includes procedures and criteria for the acceptance and certification\\u000a of cells, guidelines for future studies, and policies for the selection of cells. The aim of this Collection is to fulfill\\u000a the needs of individual investigators for

C. S. Stulberg; L. L. Coriell; A. J. Kniazeff; J. E. Shannon



Cell lines and primary cell cultures in the study of bone cell biology  

Microsoft Academic Search

Bone is a metabolically active and highly organized tissue consisting of a mineral phase of hydroxyapatite and amorphous calcium phosphate crystals deposited in an organic matrix. Bone has two main functions. It forms a rigid skeleton and has a central role in calcium and phosphate homeostasis. The major cell types of bone are osteoblasts, osteoclasts and chondrocytes. In the laboratory,

Vicky Kartsogiannis; Kong Wah Ng




EPA Science Inventory

Methods were developed for aseptic maintenance of Cyprinodon variegatus fry for extended periods. Preliminary studies indicated that under optimum conditions sterile embryos develop normally for a sufficient time to function as carcinogenteratogen assay systems. An embryo-primary...


Mammalian Cell Culture Simplified.  

ERIC Educational Resources Information Center

|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)|

Moss, Robert; Solomon, Sondra



Biocompatibility studies on fibrin glue cultured with bone marrow mesenchymal stem cells in vitro  

Microsoft Academic Search

Summary  By culturing bone marrow mesenchymal stem cells of rabbits with fibrin gluein vitro, the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue\\u000a engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4–6 ml of bone marrow were aspirated from\\u000a rabbit femoral trochanter. The monocytes suspension was aspirated

Fang Huang; Peng Songlin; Chen Anmin; Li Fengfeng; Ren Kai; Hu Ning



Endosperm Cell and Organ Culture  

Microsoft Academic Search

This chapter describes efforts to culture maize endosperm to enable direct developmental and metabolic\\u000a studies of the cereal endosperm organ without the effects of the maternal plant. First, we describe production\\u000a of endosperm in culture through either central cell fertilization in vitro or through isolation of\\u000a embryo sacs following fertilization in vivo. These techniques have been used to culture cereal endosperm\\u000a but

D. Gruis; H. Guo; Q. Tian; O.-A. Olsen


[An experimental study on photosensitization of cultured human cells with hypocrellin A].  


The photosensitive effects of hypocrellin A on cultured HeLa cells were studied by the methods of immunofluorescence cytochemistry, flow cytometry and others. The results showed that, in the presence of 3.66 mumol/L hypocrellin A, illumination of HeLa cells with visible light, the morphology was changed and the microvilli on the cells were disappeared; the proliferation of the cells decreased with increasing irradiated dose and this effect seemed more sensitive in tumor cells than in normal cells; the increased population of cycle cells at S phase but decreased at G1 phase were observed by FCM; the photodamage effect of hypocrellin A induced the sparse distribution of cytoplasmic microtubles and almost disappearance of the fluorescence intensity of MTOC, they were observed in HeLa cells as shown by immunofluoresent microscopy. All the aforesaid results indicated that photosensitization of hypocrellin A inhibits cells growth, resulting from the damage in cell membrane and cytoskeleton and S phase delay of the cell cycle. PMID:7976055

Wang, J Z; Li, J F; Zhang, X Z



Development and validation of primary human myometrial cell culture models to study pregnancy and labour  

PubMed Central

Background The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated. Methods Myometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; ? smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1? was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni’s multiple comparisons test was performed. Results We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1?, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10. Conclusions Isolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain their ability to respond to an inflammatory stimulus. These distinct myometrial cell models will provide a useful tool to investigate mechanisms underlying the process of human labour and the concept of functional regionalization of the pregnant uterus.



[Studies of nucleic acid metabolism in cultured chick embryo cells infected with Mycoplasma gallisepticum].  


M. gallisepticum infection of cultured chick embryo cells led to a sharp reduction the rate of 3H-thymidine and 3H-uridine incorporation into DNA and RNA cells, and almost completely suppressed the transposition of uridine label from the nucleus into the cytoplasm, this pointing to the inhibition of escape of RNA synthesized de novo into the infected cells cytoplasm. As suggested, weak labeling of the cytoplasm after prolonged (about several hours) incubation of cultured cells with labeled urine could indicate infection of cell cultures with the mycoplasmae. PMID:566996

Zakharian, R A; Gasparian, G G



Perhexiline maleate-induced lipidosis in cultured human fibroblasts : Cell kinetics, ultrastructural and biochemical studies  

Microsoft Academic Search

Summary  Perhexiline maleate reduced the growth of human skin fibroblasts in cell culture at a concentration range of 0.3–3 micrograms\\/ml.\\u000a At the highest concentration, the cells survived only four days. Pleomorphic inclusions characteristic of drug-induced phospholipidoses\\u000a appeared in cultured cells. Analysis of the major lipid classes was performed on cells exposed to 3 micrograms\\/ml at four\\u000a days. Gangliosides, phospholipids and cholesterol

J.-J. Hauw; J.-M. Boutry; S. Albouz; M. L. Harpin; M. Baudrimont; R. Escourolle; N. Baumann



Poly(dimethylsiloxane) thin films as biocompatible coatings for microfluidic devices : cell culture and flow studies with glial cells.  

SciTech Connect

Oxygen plasma treatment of poly(dimethylsiloxane) (PDMS) thin films produced a hydrophilic surface that was biocompatible and resistant to biofouling in microfluidic studies. Thin film coatings of PDMS were previously developed to provide protection for semiconductor-based microoptical devices from rapid degradation by biofluids. However, the hydrophobic surface of native PDMS induced rapid clogging of microfluidic channels with glial cells. To evaluate the various issues of surface hydrophobicity and chemistry on material biocompatibility, we tested both native and oxidized PDMS (ox-PDMS) coatings as well as bare silicon and hydrophobic alkane and hydrophilic oligoethylene glycol silane monolayer coated under both cell culture and microfluidic studies. For the culture studies, the observed trend was that the hydrophilic surfaces supported cell adhesion and growth, whereas the hydrophobic ones were inhibitive. However, for the fluidic studies, a glass-silicon microfluidic device coated with the hydrophilic ox-PDMS had an unperturbed flow rate over 14 min of operation, whereas the uncoated device suffered a loss in rate of 12%, and the native PDMS coating showed a loss of nearly 40%. Possible protein modification of the surfaces from the culture medium also were examined with adsorbed films of albumin, collagen, and fibrinogen to evaluate their effect on cell adhesion.

Peterson, Sophie Louise; Sasaki, Darryl Yoshio; Gourley, Paul Lee; McDonald, Anthony Eugene



Development of intestinal cell culture models for drug transport and metabolism studies  

Microsoft Academic Search

Cell culture models offer many advantageous features for the analysis of drug transport and drug metabolism. From a basic research perspective, these systems offer the potential for manipulating the environment or cellular properties as a means to address mechanistic questions. From a drug discovery perspective, cell culture models can be used to expedite identification of compounds with favorable pharmacokinetic properties,

A. Quaroni; J. Hochman



Studies on primary cell cultures derived from ovarian tissue of Penaeus monodon  

Microsoft Academic Search

As part of a bioassay approach to investigate ovarian development and function, primary cell cultures were derived from Penaeus monodon ovaries at various stages of maturation. These cultures were established in modified Grace's or modified 2× L-15 media. Various supplements including growth factors, vitamins and minerals were trailed. Four morphologically different types of cells (epithelioid, fibroblastic, rounded, and epithelioid with

C. A. Fraser; M. R. Hall



Electron microscopic study of the African strain of malignant catarrhal fever virus in bovine cell cultures.  


Malignant catarrhal fever (African strain) is a viral disease of ruminants which is considered an exotic disease in the United States. Viral isolates obtained from one clinically ill gaur (Bos gaurus) and a greater kudu (Tragelaphus strepsiceros) located in a zoologic park in Oklahoma, and from one heifer (Bos taurus) and a domestic white-tailed deer (Odocoileus virginianus) experimentally inoculated with the isolated and identified African strain of malignant catarrhal fever virus (MCFV), were each studied in bovine cell cultures by electron microscopy. Certain of the viral isolated were previously characterized as MCFV by serologic and morphologic examinations, their cytopathic effect in cell cultures, and their ability to reproduce disease in a ruminant host. The virions of MCFV (African) examined by electron microscopy were icosahedral similar to herpes-virus, were between 98 and 194 nm, developed in the nucleus and matured in the cytoplasm of the cell, and exhibited budding. The virus in infected cells passed through the nuclear and plasma membranes and also into cytoplasmic vesicles from which it acquired one or more envelopes. Virions of malignant catarrhal fever were closely associated with the cellular endoplasmic reticulum, and aberrant morphologic forms of MCFV were observed in virus-infected cells. PMID:7073076

Castro, A E; Daley, G G



Growth, morphology and chemosensitivity studies on postconfluent cells cultured in 'V'-bottomed microtiter plates.  

PubMed Central

This study assessed the growth pattern, cellular organisation and chemosensitivity of established human tumour cell lines growing as postconfluent cultures in 'V'-bottomed, 96-well microtiter plates. Cross-sections of the colon (HT29, SW620, SW1116), ovarian (A2780) and head and neck (UM-SCC-22B) carcinoma microcultures allowed in situ evaluation of the cellular organisation in the wells. After 5 days of growth, every cell line had reached confluence, but each of them displayed a specific pattern of cell stacking which ranged from two to ten layers. Postconfluent HT29 cells displayed morphologic features suggestive of some degree of enterocytic differentiation. Growth and cytotoxicity could be studied reliably and reproducibly in this system with the sulforhodamine B protein assay. Against HT29 postconfluent cultures, the EC50's (drug concentrations producing absorbance readings 50% lower than those of non-treated wells) of 5-fluorouracil and of the ether lipid, hexadecylphosphocholine, were 1 mM and 50 microM respectively. The possibility to perform chemosensitivity tests using semiautomated microtiter plate technology supports further evaluation of this system as an alternative antitumour drug testing model. Images Figures 1a-1f

Pizao, P. E.; Lyaruu, D. M.; Peters, G. J.; van Ark-Otte, J.; Winograd, B.; Giaccone, G.; Pinedo, H. M.



Design and fabrication of a capillary cell culture chamber for the study of convective flow.  


The use of capillary culture chambers as artificial pancreas and artificial liver devices would be aided by an improved ability to control the movement of molecules through the capillary walls. The modeling and analysis of the flow and mass transfer in capillary cell culture chambers in which cultured mammalian cells are grown to form masses with tissue density in the extra-capillary spaces is desirable as a basis for scaleup and optimization of the particular microenvironment. The relative roles of diffusion and ultrafiltration with convection in enhancing mass transfer across the capillary membranes are poorly understood in real cell cultures and the effects on culture viability of flow conditions chosen to promote convective flow across the capillary membranes and through the cultured cell masses are also of interest. In this report, experience with materials and techniques for fabricating capillary culture chambers with a more readily analyzed fully defined regular geometric relationship between 90 to 100 capillaries in a parallel bundle is described. High and low pressure capillaries were interspersed in a regular array. Evidence is presented for retention of cell viability during flow conditions which were chosen to induce convective flow through the cultured cells in such a chamber. PMID:3957451

Wolf, C F; Lauffer, L L



Mycoplasma pneumoniae host-pathogen studies in an air-liquid culture of differentiated human airway epithelial cells.  


The interaction between Mycoplasma pneumonaie and the airway epithelium in vivo is complex and multifaceted. While multiple in vitro studies have been conducted studying this interaction with cell lines and animal cell and organ culture models, the interactions between M. pneumoniae and fully differentiated human airway epithelium in air-liquid interface culture remains unexplored. In the present study we investigated M. pneumoniae interactions with airway epithelium utilizing an air-liquid interface culture of differentiated normal human bronchial epithelial (NHBE) cells. Utilizing confocal microscopy we found that M. pneumoniae cells bound initially to ciliated epithelial cells, but colonization became more evenly distributed over the entire surface with time. M. pneumoniae infection resulted in stimulation of intercellular adhesion molecule 1 (ICAM-1) gene expression and soluble ICAM-1 production in this culture system. PMID:17261358

Krunkosky, Thomas M; Jordan, Jarrat L; Chambers, Emily; Krause, Duncan C



Combined cell culture-biosensing platform using vertically aligned patterned peptide nanofibers for cellular studies.  


This Article presents the development of a combined cell culture-biosensing platform using vertically aligned self-assembled peptide nanofibers. Peptide nanofibers were patterned on a microchip containing gold microelectrodes to provide the cells with a 3D environment enabling them to grow and proliferate. Gold microelectrodes were functionalized with conductive polymers for the electrochemical detection of dopamine released from PC12 cells. The combined cell culture-biosensing platform assured a close proximity of the release site, the cells and the active surface of the sensor, thereby rendering it possible to avoid a loss of sensitivity because of the diffusion of the sample. The obtained results showed that the peptide nanofibers were suitable as a cell culturing substrate for PC12 cells. The peptide nanofibers could be employed as an alternative biological material to increase the adherence properties of PC12 cells. Dopamine was amperometrically detected at a value of 168 fmole. PMID:23537161

Taskin, Mehmet B; Sasso, Luigi; Dimaki, Maria; Svendsen, Winnie E; Castillo-León, Jaime




PubMed Central

Due to the high solubility of oxygen in perfluorocarbons (PFCs), these compounds have been explored for improved cell and tissue oxygenation. The goal of this study is to investigate the effects of a PFC emulsion on cellular growth and function in a tissue engineered construct. A perfluorotributylamine (PFTBA) emulsion was co-encapsulated at 10 vol% with mouse ?TC-tet insulinoma cells in calcium alginate beads and cultured under normoxic and severely hypoxic conditions. The number of metabolically active cells and the induced insulin secretion rate were measured over time for up to 16 days. Results showed no significant effect of PFTBA relative to the PFTBA-free control. The alginate-PFC-cell system was also modeled mathematically, and simulations tracked the number of viable cells over time under the same conditions used experimentally. Simulations revealed only a small, likely experimentally undetectable difference in cell density between the PFC-containing and PFC-free control beads. It is concluded that PFTBA up to 10 vol% has no significant effect on the growth and function of encapsulated ?TC-tet cells under normoxic and hypoxic conditions.

Goh, Fernie; Gross, Jeffrey D.; Simpson, Nicholas E.; Sambanis, Athanassios



Electrical Impedance Study of Cultured Endothelial Cells Under Fluid Shear Stress  

Microsoft Academic Search

The lumen of blood vessels is lined with a monolayer of endothelial cells (EC). In this work, electric cell-substrate impedance sensing (ECIS) was used to monitor the effect of fluid shear stress (FSS) on the morphology and function of cultured EC layers. Confluent layers of bovine aortic endothelial cells (BAEC) were grown on small gold electrodes and exposed to different

Chunzhi Dong; Natacha Depaola; Charles R. Keese; Ivar Giaever



A study of irrigation fluids for neurosurgery on brain primary cell cultures  

Microsoft Academic Search

Summary Primary cell cultures from newborn rat brain hemispheres were exposed to different irrigation fluids used in neurosurgery. The cells died after incubation for 5 min with hydrogen peroxide, and the number of cells was drastically decreased after 10 sec of incubation. They shrank after incubation in Elliott's artificial cerebrospinal fluid for 3 h, but the viability as determined by

Elisabeth Hansson; B. Vällfors



Development and characterization of a primary culture of chicken embryonic tracheal epithelial cells and their use in avian studies  

Technology Transfer Automated Retrieval System (TEKTRAN)

A major route of infection of avian influenza is through cells of the airway epithelium. To study the molecular mechanism of infection and early host responses we created a primary chicken tracheal cell culture. Epithelial cells were isolated from the trachea of 18 day old chicken embryos and cult...


Culture, television, and opposition: Rethinking cultural studies  

Microsoft Academic Search

This essay critically addresses the issues of culture, cultural politics, social power, and the television audience in cultural studies. Although this approach has become a dominant one in media studies, its theoretical assumptions have not been sufficiently scrutinized. Our criticism focuses on two major themes. First, we argue that cultural studies tends to analyze all cultural interpretation in terms of

Ronald Lembo; Kenneth H. Tucker Jr



Studies of the neuronal transdifferentiation process in cultured human pheochromocytoma cells: effects of steroids with differing functional groups on catecholamine content and cell morphology  

Microsoft Academic Search

The neuronal differentiation of adrenal pheochromocytoma cells from human subjects was studied in vitro for periods of up to 65 days. Changes with time in culture were observed in both intracellular catecholamine content (progressive decreases in epinephrine, norepinephrine, and dopamine, except for a possible transient early increase in the latter) and in morphology (increases in neurite outgrowth) of cells cultured

JohnW Brown; LawrenceM Fishman; Andres Carballeira



Growth hormone effect on accumulation of arterial basement membrane-like material studied on rabbit aortic myomedial cell cultures  

Microsoft Academic Search

Summary  The effect of human growth hormone on arterial basement membrane-like (BM) material was studied. BM-like material was obtained from the cell layer of cultured aortic myomedial cells using a sonication-differential centrifugation technique. After the addition of small amounts of growth hormone (1 ng\\/ml) to the cultures, we observed a 26% increased incorporation of amino acids into BM-like material (2pp-N-acetyl-glucosaminidase were

T. Ledet; L. Heickendorff



Electrical Impedance Study of Cultured Endothelial Cells Under Fluid Shear Stress  

NASA Astrophysics Data System (ADS)

The lumen of blood vessels is lined with a monolayer of endothelial cells (EC). In this work, electric cell-substrate impedance sensing (ECIS) was used to monitor the effect of fluid shear stress (FSS) on the morphology and function of cultured EC layers. Confluent layers of bovine aortic endothelial cells (BAEC) were grown on small gold electrodes and exposed to different flow conditions, while the impedance of the system was monitored. When the cells are subjected to FSS, the impedance rapidly increases 5-10%, and if the FSS is removed after a few minutes duration, the impedance returns back to the initial level in about 10 minutes. If the FSS remains for a long duration, the impedance will decrease 20-30% over a 10-15 hour period but ultimately returns to the original value, as the cells apparently accommodate to the FSS condition. These results suggest that ECIS may provide a sensitive means to study the response of EC to shear stress in vitro.

Dong, Chunzhi; Depaola, Natacha; Keese, Charles R.; Giaever, Ivar



Embryos and culture cells: a model for studying the effect of progesterone.  


A positive association between P4 concentration and initial bovine embryo survival has been reported. The objective of this study was to establish two coculture systems as a model to study the influence of progesterone on the initial bovine embryo development. Granulosa cells (GC) or bovine oviduct epithelial cells (BOECs) were used at the base of embryo culture medium microdroplets (TCM199 and 10% of superovulated oestrus cow serum, (SOCS)) supplemented or not with progesterone (P4, 33.4 ng mL(-1)) and/or a progesterone receptor antagonist (onapristone, OP, 2.2x10(-5)M). Presumptive zygotes were transferred to monolayers after in vitro maturation and fertilization of bovine oocytes with thawed swim-up selected sperm. Embryo development was carried out according to the following groups: experiment 1, BOEC (n=378) and BOEC plus OP (n=325); experiment 2, GC (n=514); GC plus OP (n=509); BOEC (n=490); BOEC plus P4 (n=500); BOEC plus P4 and OP (n=502). Embryos were checked for cleavage at day 2 and for stage development between days 8 and 12 of culture. In experiment 1, no differences (P>0.05) were identified between BOEC and BOECOP groups for embryo rates of development, quality or developmental stages. Also in experiment 2, no differences were found in embryo rates of development, quality or developmental stages between embryos cultured under the two coculture systems when no supplementation was added. Embryo development rates were not affected by OP presence in GCOP group. However, P4 negatively affected Day 8 (D8) embryo development rates in BOEC system (BOECP4=16.8+/-2.6% vs. BOEC=23.7+/-1.7%, P=0.02). This negative effect was abolished when P4 antagonist (OP) was added to the culture medium. BOEC supplementation with P4 also induced a delay on embryo development at D8 as confirmed by a lower development score (BOECP4=3.0+/-1.4 vs. GC=3.4+/-0.1, GCOP=3.5+/-0.1, BOEC=3.4+/-0.1 and BOECP4OP=3.5+/-0.1; P<0.05). These results demonstrate that OP supplementation had no harmful effect on embryo development either in granulosa, where P4 is naturally synthesised, or in BOEC coculture systems. Also we can not confirm a direct association between high P4 concentrations and embryo survival during early stages, although P4 may influence early embryo development through different mechanisms mediated by the type of cells present. PMID:18374525

Pereira, R M; Marques, C C; Baptista, M C; Vasques, M I; Horta, A E M



Cellular differentiation in primary cell cultures from single zebrafish embryos as a model for the study of myogenesis.  


Culturing cells in vitro can produce a uniform population for the study of cellular differentiation, which is especially useful for the quantification of gene expression or the observation of subcellular structures. In zebrafish, a handful of immortalized cell lines have been used for these purposes, despite being heavily selected by passaging. Methods for primary cell culture of zebrafish embryonic blastomeres have been previously reported, but require combining a large number of genetically heterogeneous embryos, meaning that subsequent cell cultures are not clonal. Without genetically uniform cultures, this model system cannot exploit the wealth of available embryonic lethal mutants in zebrafish. We therefore describe methods for the generation of zebrafish embryonic blastomere cell cultures from single genetically characterized embryos. We examined myogenic differentiation and gene expression in single-embryo cultures from early wild-type embryos, as well as embryos containing an embryonic lethal mutation of unc45b, a myosin chaperone known to be required for sarcomere organization during myogenesis. We also demonstrated the practical usefulness of this technique by experimentally manipulating expression of specific genes in individual embryos before cell culture using standard tools of zebrafish biology such as morpholino-oligonucleotide gene knockdown and transgene-mediated gene expression. PMID:20936983

Myhre, J Layne; Pilgrim, David B



Perfusion Based Cell Culture Chips  

Microsoft Academic Search

\\u000a Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium\\u000a composition, long term unattended cultures and tissue like structuring of the cultures. However, as this chapter illustrates,\\u000a many issues remain to be identified regarding perfusion cell culture

A. Heiskanen; J. Emnéus; M. Dufva



Perfusion Based Cell Culture Chips  

NASA Astrophysics Data System (ADS)

Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers.

Heiskanen, A.; Emnéus, J.; Dufva, M.


A primary chicken tracheal cell culture system for the study of infection with avian respiratory viruses  

Technology Transfer Automated Retrieval System (TEKTRAN)

A major route of infection of avian influenza virus (AIV) and Newcastle disease virus (NDV) in chickens is through cells of the airway epithelium. Here we describe the development and optimization of conditions for culture of tracheal epithelial cells from chicken embryos as well as their use in st...


Scale-up study on suspension cultures of Taxus chinensis cells for production of taxane diterpene  

Microsoft Academic Search

Suspension cells of Taxus chinensis were cultivated in both shake flasks and bioreactors. The production of taxuyunnanine C (TC) was greatly reduced when the cell cultures were transferred from shake flasks to bioreactors. Oxygen supply, shear stress and stripping-off of gaseous metabolites were considered as potential factors affecting the taxane accumulation in bioreactors. The effects of oxygen supply on the

Zhi-Wei Pan; Hong-Qiang Wang; Jian-Jiang Zhong



Use of plant cell cultures to study graminicide effects on lipid metabolism.  


Graminicides belonging to the cyclohexanedione and aryloxyphenoxypropionate classes are well established to act by disrupting acyl lipid biosynthesis via specific inhibition of acetyl-CoA carboxylase. Species of grass inherently resistant to such herbicides, or biotypes of grassy weed species which display acquired resistance to recommended rates of graminicide application, are known to possess an altered plastidic multifunctional acetyl-CoA carboxylase showing reduced sensitivity to these herbicides in vitro. Studies reported here demonstrate that cell suspension cultures of maize, a graminicide-sensitive species and Poa annua, a graminicide-insensitive species, display a similar differential sensitivity of acyl lipid biosynthesis as tissue from corresponding intact plants. Acyl lipid biosynthesis in P. annua can be inhibited if sufficiently high concentrations of graminicide are used. The major plastidic form and the minor cytosolic forms of acetyl-CoA carboxylase were successfully purified from maize cell suspensions, were compared to those from leaf tissue and were shown to be differentially inhibited by graminicides in a similar manner to their counterparts from leaf tissue. These studies demonstrate that cell suspensions are useful for studying the mode of action of graminicides, especially in view of the limited amount of material obtainable from many grassy species which are very fine-growing. PMID:12809713

Price, Lindsey J; Herbert, Derek; Cole, David J; Harwood, John L



Use of Cell Separation and Short-Term Culture Techniques to Study Erythroid Cell Development  

Microsoft Academic Search

Cell populations highly enriched for the different stages of erythroid cell matura- tion were obtained by three sequential operations: harvesting of erythroid cells after induction of erythroid hyperplasia in the spleens of mice, elimination of the more mature erythrocytes by immunologic techniques, and separation of the residual nucleated erythroid cells as a function of size by the velocity sedimentation tech-

Jonathan Glass; Linda M. Lavidor; Stephen H. Robinson



Preparation of primary cell cultures from lamprey  

Microsoft Academic Search

The lamprey is an important model for studies of evolution and comparative biology. The ability to culture cells from lamprey tissues makes it possible to employ an in vitro approach to address basic questions in these areas. Methods are described for the initiation of cell cultures derived from tissues of adult and larval sea lamprey (Petromyzon marinus). Primary cultures initiated

Chunguang Ma; Paul Collodi



3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies.  


Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity. PMID:23377618

Gunness, Patrina; Mueller, Daniel; Shevchenko, Valery; Heinzle, Elmar; Ingelman-Sundberg, Magnus; Noor, Fozia



Steroid production, cell proliferation, and apoptosis in cultured bovine antral and mural granulosa cells: development of an in vitro model to study estradiol production.  


This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH-induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [14C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P < 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH-induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells. PMID:9590533

Rouillier, P; Matton, P; Dufour, M; Sirard, M A; Guilbault, L A



A microfluidic system for automatic cell culture  

NASA Astrophysics Data System (ADS)

This study presents a new chip capable of automating the cell culture process by using microfluidic technology. This microfluidic cell culture system comprising microheaters, a micro temperature sensor, micropumps, microvalves, microchannels, a cell culture area and several reservoirs was fabricated by using micro-electro-mechanical-systems' fabrication processes. Traditional manual cell culture processes can be performed on this chip. A uni-directional pneumatic micropump was developed to transport the culture reagents and constraint the solutions to flow only in one direction, safeguarding the entire culture process from contamination. A new micro check valve was also used to prevent the culture solutions from flowing back into the microchannels. The microheaters and the micro temperature sensor were used to maintain a constant temperature during the cell culturing process. The pH value suitable for cell growth was also regulated during the cell culture process. A typical cell culturing process for human lung cancer cells (A549) was successfully performed to demonstrate the capability of the developed microfluidic system. This automatic cell culturing system can be eventually integrated with subsequent microfluidic modules for cell purification, collection, counting and lysis to form a cell-based micro-total-analysis system. Preliminary results have been presented in The Asia-Pacific Conference of Transducers and Micro-Nano Technology (APCOT), 25-28 June 2006

Huang, Chun-Wei; Lee, Gwo-Bin



Effect of pulse duration on KrF laser treatment of a polyethersulfone film: cell culture study  

NASA Astrophysics Data System (ADS)

KrF laser processing of polyethersulfone (PES) films at three different pulse durations regarding to the change in biocompatibility is presented with cell culture study. Various microstructure and chemistry results were obtained on the surface upon laser irradiation. It is shown that nanoscale hydrophilic ripples significantly affect the adhesion of L929 cells on the surface.

Pazokian, Hedieh; Mollabashi, Mahmoud; Selimis, Alexandros; Stratakis, Emmanuel; Barzin, Jalal; Jelvani, Saeid



Phenol Red in Tissue Culture Media is a Weak Estrogen: Implications concerning the Study of Estrogen-Responsive Cells in Culture  

Microsoft Academic Search

Although much attention has been paid to the removal of hormones from sera and to the development of serum-free media for studies on hormone-responsive cells in culture, little consideration has been given to the possibility that the media components themselves may have hormonal activity. We have found that phenol red, which bears a structural resemblance to some nonsteroidal estrogens and

Yolande Berthois; John A. Katzenellenbogen; Benita S. Katzenellenbogen



Principles of cancer cell culture.  


The basics of cell culture are now relatively common, though it was not always so. The pioneers of cell culture would envy our simple access to manufactured plastics, media and equipment for such studies. The prerequisites for cell culture are a well lit and suitably ventilated laboratory with a laminar flow hood (Class II), CO(2) incubator, benchtop centrifuge, microscope, plasticware (flasks and plates) and a supply of media with or without serum supplements. Not only can all of this be ordered easily over the internet, but large numbers of well-characterised cell lines are available from libraries maintained to a very high standard allowing the researcher to commence experiments rapidly and economically. Attention to safety and disposal is important, and maintenance of equipment remains essential. This chapter should enable researchers with little prior knowledge to set up a suitable laboratory to do basic cell culture, but there is still no substitute for experience within an existing well-run laboratory. PMID:21516394

Cree, Ian A



A comparative study of L-type voltage sensitive Ca 2+ channels in rat brain regions and cultured neuronal cells  

Microsoft Academic Search

Radioligand binding studies using the L-type voltage sensitive Ca2+ channel (VSCC) antagonist (+)-[3H]PN200–110 revealed the following rank order channel density in rat brain and cultured neuronal cell homogenates: striatum?cerebrocortex>>cerebellum=brainstem>SH-SY5Y cell line>NG108–15 cell line>1321N1 cell line>PC12 cell line. There were no significant differences in the equilibrium dissociation constant, Kd for (+)-[3H]PN200–110 or pK50 for nifedipine. K+ depolarization in SH-SY5Y cells and

Kazuyoshi Hirota; David G Lambert



Perhexiline maleate-induced lipidosis in cultured human fibroblasts: cell kinetics, ultrastructural and biochemical studies.  


Perhexiline maleate reduced the growth of human skin fibroblasts in cell culture at a concentration range of 0.3-3 micrograms/ml. At the highest concentration, the cells survived only four days. Pleomorphic inclusions characteristic of drug-induced phospholipidoses appeared in cultured cells. Analysis of the major lipid classes was performed on cells exposed to 3 micrograms/ml at four days. Gangliosides, phospholipids and cholesterol levels four to six times above controls were found. No major qualitative abnormalities were detected in phospholipids. On the contrary, an abnormal pattern of gangliosides was seen by densitometry of silica gel thin-layer plates with increases of GD3 and of an unknown ganglioside. Drug induced lipidosis may involve other lipids than phospholipids, particularly gangliosides. PMID:6108643

Hauw, J J; Boutry, J M; Albouz, S; Harpin, M L; Baudrimont, M; Escourolle, R; Baumann, N



Experimental Model for Studying the Primary Cilia in Tissue Culture Cells  

Microsoft Academic Search

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tobulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number

I. B. Aliev; L. A. Gorgidze; A. Komarova; O. A. Chernobelskaya; I. A. Vorobjev


Ultrastructural studies of the replication of fowl adenoviruses in primary cell cultures  

Microsoft Academic Search

The replication of four fowl adenovirus strains (FAV?1, strain Phelps; FAV?5, strains 340 and TR?22; and FAV?7, strain YR?36), in primary chick kidney cell cultures, is described. Differences were found in the distribution of virus particles and virus associated inclusions between viruses belonging to different cytopathology subgroups. Thus in cells infected with FAV?1 (Phelps) and FAV?5 (340) (i.e. subgroup 1)

B. M. Adair; W. L. Curran; J. B. McFerran



UVA and visible light-induced reactive oxygen species (ROS) formation in cell cultures: an electron paramagnetic resonance (EPR) study  

NASA Astrophysics Data System (ADS)

The stimulatory effect of low energy light (LEL) has been attributed to irradiation-induced ROS formation. In the present study we demonstrate that irradiating various cell cultures such as fibroblasts, cardiac and sperm cells with UVA or various light sources in the visible range results in singlet oxygen and OH radical formation. These radicals were monitored by using the EPR technique. We believe that the light induced ROS could mediate previously documented effects of LEL on these cells.

Lubart, Rachel; Lavie, R.; Friedmann, Harry; Sinyakov, M.; Shainberg, Asher; Breitbart, Haim; Grossman, Nili



Photodynamic laser therapy for rheumatoid arthritis. Cell culture studies and animal experiments.  


The introduction of arthroscopic techniques has improved the surgical therapy of rheumatoid arthritis. The additional application of the holmium:yttrium aluminum garnet (Ho:YAG) laser likewise holds great promise by providing complete hemorrhagic control. Unfortunately, a minimally invasive solution for use in smaller joints has not yet emerged. The present study describes the possible treatment of these joints by means of photodynamic laser therapy. Cell culture studies with human synovial fibroblasts obtained from patients with rheumatoid arthritis have demonstrated a cytotoxic effect after administration of Photosan-3 as a photosensitizer and subsequent laser irradiation at 630 nm. for the in vivo studies, IgG-induced arthritis in rabbits, which is histologically consistent with the proliferative phase of rheumatoid arthritis, was used as the animal model. The histologic picture following photodynamic laser therapy with Photosan-3 revealed complete synovial destruction which also extended to the border of the subjacent joint capsule. In contrast, bradytrophic structures, e.g. cartilage. menisci, and ligaments, remained unchanged at both the macroscopic and microscopic levels. Therefore, photodynamic laser therapy can be considered a new method in the surgical treatment of inflammatory disease of the synovial membrane. It has the advantage of being minimally invasive, while offering a high degree of efficacy and selectivity. PMID:9127856

Hendrich, C; Hüttmann, G; Lehnert, C; Diddens, H; Siebert, W E



Cell Culture Models and Methods  

Microsoft Academic Search

\\u000a This chapter reviews methods to harvest and culture several different cell systems commonly employed in cardiac electrophysiology\\u000a research. The cell systems covered fall into three main categories: (1) primary cell culture; (2) cell lines; and (3) stem\\u000a cell-derived cardiomyocytes. Each cell system has its own set of advantages and disadvantages. Often the best choice depends\\u000a on the question an investigator

Alena Nikolskaya; Vinod Sharma


Cocoa bean cell and embryo culture  

Microsoft Academic Search

Callus culture of cocoa bean was initiated from immature cotyledons on agar medium. By dispersing these callus cells, a liquid\\u000a suspension culture was established. The lipid composition of cocoa suspension culture was investigated and compared with those\\u000a of cocoa beans of different maturities. Factors affecting fatty acid and triglyceride synthesis in cocoa suspension cultures\\u000a also were studied. In parallel studies,

Ming-Che Wen; Bruce German; John E. Kinsella



Design Study Conducted of a Stirred and Perfused Specimen Chamber for Culturing Suspended Cells on the International Space Station.  

National Technical Information Service (NTIS)

A tightly knit numerical/experimental collaboration among the NASA Ames Research Center, NASA Glenn Research Center, and Payload Systems, Inc., was formed to analyze cell culturing systems for the International Space Station. The Cell Culture Unit is a fa...

E. S. Nelson J. P. Kizito



Study on application of high doses plasmodium berghei in cell culture  

NASA Astrophysics Data System (ADS)

Malaria, one of the most important infection disease problems in the world, is caused by protozoan parasites of the genus Plasmodium. This disease is responsible for hundreds of the millions of clinical cases and more than one million deaths per year, for this reason, malaria is a priority and the WHO estimates that half of the world population is at risk. In this work we study how the absorbed dose inactivates the parasite (Plasmodium berghei) in rodent model (BALB/c mice), by applying X-ray irradiation. The dose was increased from 10 to 50 Gy in parasitized red blood cells (PRBC) with merozoite stage using in vitro short cultures. Also the reduction of the irradiation effect was determined by intra-peritoneal inoculations of irradiated parasites. Afterwards, the parasitaemia was assessed daily on smears made from tail blood and stained with Giemsa's reagent. Besides, the effect of irradiation was evaluated using an immunological test as indirect immunofluorescence assay (IFA). The results of this study showed that the most effective radiation for inactivation of parasites is about 50 Gy and the immunofluorescence pattern showed a different distribution of the fluorescence on parasites. These results showed direct correlation between the effect of irradiated parasites and parasitaemia in the group of mice infected with RBC after 50 Gy irradiation. Our results indicated that the threshold is between 30 to 50 Gy to inactivate the parasites.

Spencer, L. M.; de Santis, M.; Davila, J.; Foinquinos, A.; Salcedo, E.; Sajo-Bohus, L.



Comparative genomic study of gastric epithelial cells co-cultured with Helicobacter pylori  

PubMed Central

AIM: To identify genes potentially involved in Helicobacter pylori (H. pylori)-induced gastric carcinogenesis. METHODS: GES-1 cells were co-cultured with H. pylori strains isolated from patients with gastric carcinoma (GC, n = 10) or chronic gastritis (CG, n = 10) for in vitro proliferation and apoptosis assays to identify the most and least virulent strains. These two strains were cagA-genotyped and used for further in vivo carcinogenic virulence assays by infecting Mongolian gerbils for 52 wk, respectively; a broth free of H. pylori was lavaged as control. Genomic profiles of GES-1 cells co-cultured with the most and least virulent strains were determined by microarray analysis. The most differentially expressed genes were further verified using quantitative real-time polymerase chain reaction in GES-1 cells infected with the most and least virulent strains, and by immunohistochemistry in H. pylori positive CG, precancerous diseases, and GC biopsy specimens in an independent experiment. RESULTS: GC-derived H. pylori strains induced a potent proliferative effect in GES-1 cells in co-culture, whereas CG-derived strains did not. The most (from a GC patient) and least (from a CG patient) virulent strains were cagA-positive and negative, respectively. At week 52, CG, atrophy, metaplasia, dysplasia, and GC were observed in 90.0%, 80.0%, 80.0%, 90%, and 60.0%, respectively, of the animals lavaged with the most virulent strain. However, only mild CG was observed in 90% of the animals lavaged with the least virulent strain. On microarray analysis, 800 differentially expressed genes (49 up- and 751 down-regulated), involving those associated with cell cycle regulation, cell apoptosis, cytoskeleton, immune response, and substance and energy metabolisms, were identified in cells co-cultured with the most virulent strain as compared with those co-cultured with the least virulent strain. The six most differentially expressed genes (with a betweenness centrality of 0.1-0.2) were identified among the significant differential gene profile network, including JUN, KRAS, BRCA1, SMAD2, TRAF1, and HDAC6. Quantitative real-time polymerase chain reaction analyses verified that HDAC6 and TRFA1 mRNA expressions were significantly more up-regulated in GES-1 cells co-cultured with the most virulent strain than in those co-cultured with the least virulent strain. Immunohistochemistry of gastric mucosal specimens from H. pylori-positive patients with CG, intestinal metaplasia (IM), dysplasia, and GC showed that moderately positive and strongly positive HDAC6 expression was detected in 21.7% of CG patients, 30.0% of IM patients, 54.5% of dysplasia patients, and 77.8% of GC patients (P < 0.001). The up-regulation of TRAF1 expressions was detected in 34.8%, 53.3%, 72.7%, and 88.9% specimens of CG, IM, dysplasia, and GC, respectively (P < 0.001). CONCLUSION: The overexpression of HDAC6 and TRAF1 in GES-1 cells co-cultured with the GC-derived strain and in H. pylori-positive dysplasia and GC suggests that HDAC6 and TRAF1 may be involved in H. pylori-induced gastric carcinogenesis.

Wang, Fen; Luo, Li-Dan; Pan, Jian-Hua; Huang, Li-Hua; Lv, Hong-Wei; Guo, Qin; Xu, Can-Xia; Shen, Shou-Rong



Recent advances in crayfish hematopoietic stem cell culture: a model for studies of hemocyte differentiation and immunity.  


Hematopoiesis is the process by which blood cells (hemocytes) mature and subsequently enter the circulation and we have developed a new technique to culture the hematopoietic progenitor cells in vitro. The reason for the successful culture was the isolation of a plasma protein that turned out to be a novel cytokine, astakine 1 (Ast1) containing a domain present in several vertebrates, so-called prokineticins. Now we have detected several astakines from other invertebrate species. Depending on our discovery of the cytokine Ast1 we have an opportunity to study in detail the differentiation of cells in the hematopoietic tissue of a crustacean, a tissue of evolutionary interest for studies of the connection between the vascular system and the nervous system. We have been able to isolate the entire hematopoietic tissue and for the first time detected a link between this tissue and the brain. We have further localized a proliferation center in the tissue and characterized its different parts. We have also used this system to isolate a new hematopoietic factor CHF that is important in the crossroad between apoptosis and hemocyte differentiation. Our technique for culture of crayfish hematopoietic stem cells provides a simple tool for studying the mechanism of hematopoiesis, but also enables detailed studies of immune defense reactions. Further, the culture system has been used for studies of viral defense and the system is suitable for gene silencing which allows functional characterization of different molecules involved in host defense as well as in hemocyte differentiation. PMID:23686548

Söderhäll, Irene



Antigenic and immunogenic studies on cell culture-derived Babesia canis.  


Babesia canis antigens derived from cell culture reacted specifically with immune serum from dogs convalescing from babesiosis. The antigens were heterogenous as compared to antigens elaborated in vivo. The major antigenic moiety from cell culture eluted in the first peak of Sephadex G-200 is indicative of a molecular weight around 900 000. In contrast, in vivo-derived antigen coeluted with albumin and hemoglobin suggesting a molecular weight of 67 000. The major antigenic mass is proteinacious and contains disulfide bonds as indicated by thermolability and sensitivity to 2-mercaptoethanol. Both particulate and soluble B. canis antigens were immunogenic, particularly when emulsified in Saponin as an adjuvant. Such antigens conferred a considerable degree of protection in Saponin as an adjuvant. Such antigens conferred a considerable degree of protection in susceptible dogs and it suggested that immunoprophylaxis to B. canis may be feasible. PMID:6179286

Molinar, E; James, M A; Kakoma, I; Holland, C; Ristic, M



Serum-Free Production of Poliovirus: A Comparative Study Using Microcarriers, Roller Bottles and Stationary Cell Culture  

Microsoft Academic Search

Serum has been a necessity for many years, and continues to be important for the culture of attachment-dependant cell lines. The lack of serum in an adherent cell culture medium poses difficulties such as the attachment, spreading, and formation of a cell monolayer; proteolytic damage during monolayer dissociation; and limitations to the use of some culture systems. As the use

Cory J. Card; Thomas Smith; Brent Hunsaker; Bill Barnett


Fabrication of polymer fiber scaffolds by centrifugal spinning for cell culture studies  

Microsoft Academic Search

We demonstrate a mass production amenable technology for the fabrication of polymer fibers that can be used as 3D scaffolds for cell culture and tissue engineering. As the first attempt, we used centrifugal melt spinning technique to fabricate fiber matrix of poly-lactic-co-glycolic acid (PLGA) which is a well-known biodegradable co-polymer. We then developed a solvent assisted centrifugal spinning technique to

Li Wang; Jian Shi; Li Liu; Emilie Secret; Yong Chen



Photosensitization of experimental hepatocellular carcinoma with protoporphyrin synthesized from administered ?-aminolevulinic acid: studies with cultured cells and implanted tumors  

Microsoft Academic Search

Background\\/Aims: Photodynamic therapy using porphyrins or related compounds and laser light is an investigational treatment for neoplasms. The aim of this study was to establish wether this might be applicable for hepatocellular carcinoma using protoporphyrin synthesized in the tissue from administered ?-aminolevulinic acid.Methods: We measured porphyrin accumulation in normal rat hepatocytes and Morris hepatoma cells in culture, and in subcutaneously

Norman G. Egger; James A. Schoenecker; William K. Gourley; Massoud Motamedi; Karl E. Anderson; Steven A. Weinman



Regulation of cardiac myosin synthesis: Studies of RNA content in cultured heart cells  

SciTech Connect

Contraction regulates the myosin content and the rate of myosin synthesis in cultured neonatal rat heart cells. To further explore the mechanism for this regulation the authors examined various parameters of RNA content and RNA synthesis in contracting versus noncontracting myocytes. While contraction stimulated myosin heavy chain (MHC) synthesis by 72% compared to that of KCl-arrested cells, simultaneous analyses of polysome profiles were no different under the two culture conditions. Incorporation of ({sup 3}H) uridine monophosphate into cellular RNA revealed no change in the rate of total RNA or ribosomal subunits synthesis. In vitro translation of cellular RNA yielded similar incorporation of ({sup 35}S) methionine not trichloroacetic acid precipitable protein. Specific transcription of the MHC gene was examined by dot-blot analysis and was unaltered by contraction. Northern blot analysis of the MHC sequences detected by a cDNA probe revealed an mRNA sequence corresponding to a molecular weight of approximately 30 S. These data suggest that RNA synthesis and RNA content are unaltered by contraction in cultured heart cells and therefore the changes in myosin synthesis may be mediated at a post-transcriptional control level.

McDermott, P.; Whitaker-Dowling, P.; Klein, I. (Univ. of Pittsburgh, PA (United States) Cornell Univ., New York, NY (United States))



A Multifunctional 3D Co-Culture System for Studies of Mammary Tissue Morphogenesis and Stem Cell Biology  

PubMed Central

Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

Campbell, Jonathan J.; Davidenko, Natalia; Caffarel, Maria M.; Cameron, Ruth E.; Watson, Christine J.



Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells  

SciTech Connect

The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatic exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.

Mason, J.M.; Zeiger, E.; Haworth, S.; Ivett, J.; Valencia, R.



Primary cell culture of meningothelial cells—a new model to study the arachnoid in glaucomatous optic neuropathy  

Microsoft Academic Search

Background  In a previous report, we found that the occurrence and amount of meningothelial cell nests in the subarachnoid space are significantly\\u000a increased in glaucomatous optic nerves compared to normals. In order to allow research into the role of meningothelial cells\\u000a during diseases of the optic nerve, an in vitro model is necessary. For this purpose, we developed a culture method

Xiaorong Xin; Bin Fan; Hanspeter E. Killer; Albert Neutzner; Josef Flammer; Peter Meyer



Stromal cells in long-term murine bone marrow culture: FACS studies and origin of stromal cells in radiation chimeras  

SciTech Connect

Adherent layers from hematopoietically active long-term bone marrow cultures (LTBMC), incubated with fluorescent beads, were analyzed for autofluorescence and phagocytic ability, using a fluorescence-activated cell sorter (FACS). Four groups of cells were separated from the adherent layers, including a group of large polygonal fibroblastoid stromal cells. Long-term chimeras were made by lethal irradiation of CBA/Ca (CBA) and C57Bl6/J (B6) mice and repopulation with phosphoglycerate kinase (PGK-1) alloenzyme-congenic bone marrow cells. Hematopoietically active LTBMC were established from such chimeras, and donor and host contributions of FACS-sorted adherent-layer cells were measured. While macrophages and other hematopoietic cells were of donor origin, the fibroblastoid stromal cells were mainly or entirely host derived.

Lennon, J.E.; Micklem, H.S.



Automatic monitoring of biochemical parameters in tissue culture. Studies on synchronously growing mouse myeloma cells  

PubMed Central

An instrument is described that will maintain a population of mammalian cells at constant cell density while automatically monitoring the growth rate of the culture and the extent of precursor incorporation into a variety of cell products. The apparatus was used in an investigation of cyclic changes in the incorporation of labelled precursors into the DNA, RNA, total protein and myeloma protein synthesized in synchronous cultures of a mouse myeloma line. The incorporation of [3H]uridine into trichloroacetic acid-insoluble material reveals a slight periodicity, with maxima and minima corresponding to late S phase and the mitotic phases respectively. The incorporation of [3H]lysine into total intracellular protein also shows a slight oscillation, with maxima and minima occurring during the respective G2 and mitotic phases. Cyclical changes in the synthesis of serologically precipitable myeloma protein were found to vary somewhat according to the conditions used to synchronize the cells. In experiments conducted with 4.0mm-thymidine, maximal incorporation of label took place during S phase or early G2 phase. Experiments with 1.0mm-thymidine revealed a significantly less marked periodicity of myeloma protein synthesis.

Cowan, N. J.; Milstein, C.



Maize black Mexican sweet suspension cultured cells are a convenient tool for studying aquaporin activity and regulation.  


Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black Mexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P(f)) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers. PMID:19847101

Cavez, Damien; Hachez, Charles; Chaumont, François



CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies  

PubMed Central

Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development; however, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude this system has broad applications for in vitro preclinical development for B-cell malignancies.

Ito, Daisuke; Frantz, Aric M.; Williams, Christina; Thomas, Rachael; Burnett, Robert C.; Avery, Anne C.; Breen, Matthew; Mason, Nicola J.; O'Brien, Timothy D.; Modiano, Jaime F.



FTIR microscopic studies on normal and H-Ras transfected cultured mouse fibroblast cells  

NASA Astrophysics Data System (ADS)

Infrared (IR) absorption spectra are well known for their selectivity and minutiae fingerprint of molecular structure. The biochemical changes in the sub-cellular levels developing in abnormal cells, including a majority of cancer forms, manifest themselves in different optical signatures, which can be detected in infrared spectroscopy. The molecular vibrational modes which are responsible for IR absorption spectra, are characteristic of the biochemistry of the cells and their sub-cellular components. We measured the infrared absorption spectra of monolayers of cultured normal and ras gene transformed mouse fibroblasts, using microscopic infrared system (micro-FTIR) technique. The absorption for normal cells was higher than the malignant ones in the spectral range 1000 - 1500 and 2800 - 3000 cm-1. The effect on phospholipid metabolism due to ras gene incorporation is also discussed.

Salman, Ahmad; Ramesh, Jagannathan; Grossman, Nili; Hammody, Ziad; Cohen, Beny; Mordechai, Shaul



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...


Keratin 19 in the adult human prostate: tissue and cell culture studies  

Microsoft Academic Search

Keratin 19 is found primarily in simple epithelia. In mammary epithelia, keratin 19 was localized to a subset of luminal cells, suggesting that keratin 19-negative cells may be the proliferative compartment of the secretory cell lineage. The structural and functional similarities of prostate and breast led us to examine keratin 19 expression in the prostate. Immunohistochemical studies revealed that keratin

Donna M. Peehl; Robert G. Sellers; John E. McNeal



Characterization of Bradykinin Receptors in Canine Cultured Corneal Epithelial Cells: Pharmacological and Functional Studies  

Microsoft Academic Search

The pharmacological properties of bradykinin (BK) receptors were characterized in canine cultured corneal epithelial cells (CECs) using [3H]-BK as a radioligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant of 0.34 ± 0.07 nM and a maximum receptor density of 179 ± 23 fmol\\/mg protein. Neither a B1 receptor-selective agonist (des-Arg9-BK) nor antagonist ([Leu8, des-Arg9]-BK) significantly inhibited [3H]-BK

Samuel C. M. Huang; Li-Der Hsiao; Chin-Sung Chien; Chuan-Chwan Wang; Chi-Tso Chiu; Ray J. F. Tsai; Chuen-Mao Yang



Characterization of bradykinin receptors in canine cultured corneal epithelial cells: Pharmacological and functional studies  

Microsoft Academic Search

The pharmacological properties of bradykinin (BK) receptors were characterized in canine cultured corneal epithelial cells (CECs) using [3H]-BK as a radioligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant of 0.34 ± 0.07 nM and a maximum receptor density of 179 ± 23 fmol\\/mg protein. Neither a B1 receptor-selective agonist (des-Arg9-BK) nor antagonist ([Leu8, des-Arg9]-BK) significantly inhibited [3H]-BK

Samuel C. M. Huang; Li-Der Hsiao; Chin-Sung Chien; Chuan-Chwan Wang; Chi-Tso Chiu; Ray J. F. Tsai; Chuen-Mao Yang



An alternate protocol for establishment of primary caprine fetal myoblast cell culture: an in vitro model for muscle growth study.  


Cultured myoblasts have been used extensively as an in vitro model in understanding the underlying mechanisms of myogenesis. Various protocols for establishing a pure myoblast culture have been reported which involve the use of special procedures like flow cytometry and density gradient centrifugation. In goat, only a few protocols for establishing a myogenic cell culture are available and these protocols use adult muscle tissues which often does not yield sufficient numbers of precursor cells with adequate proliferative capacity. Considering the disadvantages of adult myoblasts, we are proposing an alternate protocol using caprine fetus which does not require any special procedures. In the present study, more than 90-95% fetal-derived cell populations had the typical spindle to polyhedral shape of myoblast cell and stained positive for desmin, hence confirming their myogenic origin. These cells attained the maximum confluency as early as 3-4 d against 3 wk by adult myoblasts indicating a better growth potential. Further, quantitative real-time PCR analysis revealed a higher expression (p?

Singh, Satyendra Pal; Kumar, Rohit; Kumari, Priya; Mitra, Abhijit



Rabbit renal proximal tubule cells in primary culture: A new in vitro model for studying chemical-induced nephrotoxicity  

SciTech Connect

The goal of these studies was to use rabbit proximal tubule cells in primary culture as in in vitro model for studying nephrotoxicity by documenting the growth, biochemical properties, and the effects of two direct acting nephrotoxins. During culture alkaline phosphatase and {gamma}-glutamyltranspeptidase activity decreased while lactate dehydrogenase and N-acetyl-glucosaminidase activity increased. Enzyme systems responsible for xenobiotic metabolism like cytochrome P-450 associated mixed-function oxidase and glutathione S-transferase activity decreased while cellular glutathione levels and redox state were maintained in culture. As measured by vital dye exclusion, exposure of confluent cultures to HgCl{sub 2} or CH{sub 3}HgCl resulted in a concentration-dependent increase in cell mortality with 24 hour LC{sub 50} values of 34.2 and 6.1 {mu}M, respectively. Uptake studies using {sup 203}Hg-tagged HgCl{sub 2} or CH{sub 3}HgCl revealed steady-state cellular uptake of CH{sub 3}HgCl within 3 hrs compared to 24 hrs for HgCl{sub 2}. Although the time course for accumulation was 8-times faster for CH{sub 3}HgCl than HgCl{sub 2}, the intracellular concentration, which elicited cytotoxicity, was 3-times less.

Aleo, M.D.



Evaluation of drug toxicity with hepatocytes cultured in a micro-space cell culture system  

Microsoft Academic Search

A micro-space cell culture system was recently developed in which cells such as hepatocytes can be cultured and formed into a multicellular three-dimensional (3D) architecture. In this study, we assessed the performance of HepG2 cells cultured in this micro-space cell culture system in a drug toxicity test, and evaluated the effects of micro-space culture on their hepatocyte-specific functions. The micro-space

Kazuaki Nakamura; Reiko Mizutani; Atsushi Sanbe; Shin Enosawa; Mureo Kasahara; Atsuko Nakagawa; Yoko Ejiri; Norie Murayama; Yuki Miyamoto; Tomohiro Torii; Shinji Kusakawa; Junji Yamauchi; Motohiro Fukuda; Hiroshi Yamazaki; Akito Tanoue



Cell cycle analysis of primary sponge cell cultures.  


Proliferation of sponge cells is generally measured via cell counts or viability assays. However, more insight into the proliferative state of a sponge cell population can be obtained from the distribution of the cells over the different phases of the cell cycle. Cell cycle distribution of sponge cells was measured via flow cytometry after staining the DNA with propidium iodide. The five sponges studied in this paper all showed a large fraction of cells in G1/G0 compared to G2/M and S, indicating that cells were not actively dividing. In addition, some sponges also showed a large apoptotic fraction, indicating cell death. Additional apoptosis measurements, based on caspase activity, showed that harvesting and dissociation of sponge tissue to initiate a primary cell culture was directly correlated with an increase in apoptotic cells. This indicates that for the development of cell cultures, more attention should be given to harvesting, dissociation, and quality of starting material. Finally, cultivation conditions used were ineffective for proliferation, since after 2 d of cultivating Haliclona oculata cells, most cells shifted towards the apoptotic fraction, indicating that cells were dying. For development of in vitro sponge cell cultures, flow cytometric cell cycle analysis is a useful method to assess the proliferative state of a sponge cell culture and can be used to validate improvements in harvesting and dissociation, to select sponges with good proliferative capacities and to study the influence of culture conditions for stimulating cell growth. PMID:21416188

Schippers, Klaske J; Martens, Dirk E; Pomponi, Shirley A; Wijffels, René H



Development of an in vitro cell culture model to study milk to plasma ratios of therapeutic drugs  

PubMed Central

Objective: To create an in vitro cell culture model to predict the M/P (concentration of drug in milk/concentration in maternal plasma) ratios of therapeutic drugs viz. rifampicin, theophylline, paracetamol, and aspirin. Materials and Methods: An in vitro cell culture model using CIT3 cells (mouse mammary epithelial cells) was created by culturing the cells on transwells. The cells formed an integral monolayer, allowing only transcellular transport as it happens in vivo. Functionality of the cells was confirmed through scanning electron microscopy. Time wise transfer of the study drugs from plasma to milk was studied and compared with actual (in vivo) M/P ratios obtained at reported tmax for the respective drugs. Results: The developed model mimicked two important intrinsic factors of mammary epithelial cells viz. secretory and tight-junction properties and also the passive route of drug transport. The in vitro M/P ratios at reported tmax were 0.23, 0.61, 0.87, and 0.03 respectively, for rifampicin, theophylline, paracetamol, and salicylic acid as compared to 0.29, 0.65, 0.65, and 0.22, respectively, in vitro. Conclusion: Our preliminary effort to develop an in vitro physiological model showed promising results. Transfer rate of the drugs using the developed model compared well with the transfer potential seen in vivo except for salicylic acid, which was transferred in far lower concentration in vitro. The model has a potential to be developed as a non-invasive alternative to the in vitro technique for determining the transfer of therapeutic drugs into breast milk.

Athavale, Maithili A.; Maitra, Anurupa; Patel, Shahnaz; Bhate, Vijay R.; Toddywalla, Villi S.



Some properties of Bomirski Ab amelanotic melanoma cells, which underwent spontaneous melanization in primary cell culture  

Microsoft Academic Search

Summary Four types of the Bomirski Ab amelanotic melanoma primary cell culture, differing in the presence of calf serum in the medium and in the cell number used for starting the culture, were employed in the study. In all types of cell culture, rapid melanization occurred in the cytoplasm of the cultured cells. Calf serum in the culture medium stimulated

Andrzej Stomifiski



Rat hepatocyte primary cell cultures  

Microsoft Academic Search

Summary  The conditions for obtaining representative, adult rat hepatocyte primary cell cultures were improved such that viable yields\\u000a of 50% of the liver were produced which gave rise to cultures representing 30% of the liver. The survival of the cultures\\u000a in various media was compared revealing that in complex media, particularly containing galactose, survival was improved.

Gary M. Williams; Edilberto Bermudez; Dominick Scaramuzzino




Microsoft Academic Search

At the present time it is practically impossible to demonstrate the various chemical substances of the living cell, owing to the fact that most means of analysis cause the death of the cell. A few of the differential stains, such as Sudan III. and Nile blue, have been applied to studies of living tissues, but these do not behave in



Molecular studies of human connexin 43 (Cx43) expression in isolated corporal tissue strips and cultured corporal smooth muscle cells  

Microsoft Academic Search

Intercellular communication plays an important role in erectile function. The goal of this study, therefore, was two-fold. Firstly, to determine if cultured corporal smooth muscle cells provide a valid model system for evaluating the role of junctional communication to erectile physiology, and secondly, to explore the possibility that there may be age-related alterations in Cx43 mRNA expression. Human corpus cavernosum

S Serels; NS Day; YP Wen; A Giraldi; SW Lee; A Melman; GJ Christ



Genetic variation of hexosaminidase A and arylsulfatase A activity. Correlation study in amnio-maternal paris of cultured cells  

Microsoft Academic Search

Pairs of cultured amniotic cells and maternal fibroblasts (“feto-maternal pairs”) were studied for hexosaminidase A (HXA) and arylsulfatase A (ASA) activity. These lysosomal enzyme activities are genetically deficient in Tay-Sachs disease and metachromatic leukodystrophy, respectively. After HXA was standardized by relating it to hexosaminidase B (HXB) activity, a feto-maternal correlation coefficient of r=0.51 (n=32; 95% confidence limits 0.197–0.73) was found

K. Harzer; K. Hayashi



Aflatoxins in Mammalian Cell Cultures.  

National Technical Information Service (NTIS)

KB and HeLa mammalian tissue culture cells were cultured in the presence of 0.4, 1, and 4 ppm of aflatoxin. The incorporation of labeled uridine into the different RNA components separated by sucrose gradient ultracentrifugation and by methylated albumin ...

R. A. Chung



Monoclonal antibody to intermediate filaments of cytokeratin type. I. Drug studies and reactivity with cultured cells and tissue sections.  


Establishment of a mouse-mouse hybridoma and partial characterization of IgM monoclonal antibody (M-04) identifying cytoplasmic filamentous structures is described. Immunofluorescence performed on a panel of various cultured human cell types as well as on frozen sections of normal and tumour tissues revealed specificity of M-04 antibody for cells of epithelial origin. Using MCF-7 cell line as a model, staining patterns of microtubules, microfilaments and M-04-target filaments in untreated cells were compared with those pretreated with Colcemid and Cytochalasin B. From both differential staining of various cell types and the results of drug studies it is concluded that monoclonal antibody M-04 binds to intermediate filaments of cytokeratin type. Furthermore, restricted expression of M-04 target determinant among epithelial tissues is suggested from the lack of reaction in stratified skin epithelium. PMID:2578996

Bártek, J; Kovarík, J; Lauerová, L; Munzarová, M



Cell Culture Models for Neurotoxicology  

Microsoft Academic Search

A range of in vitro cell culture methods are available for neurotoxicology which typically represent one of the two predominant cell types present in the brain, neurons and glial cells. These systems can be used in a two tiered approach, whereby simple cytotoxic models reveal the gross effects of a drug or compound and, subsequently, more complex and subtle assays

Glyn Stacey; Barbara Viviani



Biocompatibility studies of human fetal osteoblast cells cultured on gamma titanium aluminide.  


Ti-48Al-2Cr-2Nb (at. %) (gammaTiAl), a gamma titanium aluminide alloy originally designed for aerospace applications, appears to have excellent potential for bone repair and replacement. The biological response to gammaTiAl implant is expected to be similar to other titanium-based biomaterials. Human fetal osteoblast cells were cultured on the surface of gammaTiAl and Ti-6Al-4V disks with variable surface roughness for both SEM and immunofluorescent analysis to detect the presence of collagen type I and osteonectin, proteins of the bone extracellular matrix. Qualitative results show that cell growth and attachment on gammaTiAl was normal compared to that of Ti-6Al-4V, suggesting that gammaTiAl is not toxic to osteoblasts. The presence of collagen type I and osteonectin was observed on both gammaTiAl and Ti-6Al-4V. The results obtained suggest gammaTiAl is biocompatible with the osteoblast cells. PMID:17597368

Rivera-Denizard, Omayra; Diffoot-Carlo, Nannette; Navas, Vivian; Sundaram, Paul A



Interaction of phospholipid vesicles with cultured mammalian cells. II. Studies of mechanism  

PubMed Central

The mechanism of interaction of artificially generated lipid vesicles (approximately 500 A diameter) with Chinese hamster V79 cells bathed in a simple balanced salt solution was investigated. The major pathways of exogenous lipid incorporation in vesicle-treated cells are vesicle-cell fusion and vesicle-cell lipid exchange. At 37 degrees C, the fusion process is dominant, while at 2 degrees C or with energy depleted cells, exchange of lipids between vesicles and cells is important. The fusion mechanism was demonstrated using vesicles of [14C]lecithin containing trapped [13H]inulin. Consistent with a fusion hypothesis, both components became cell associated at 37 degrees C in nearly the same proportions as they were present in the applied vesicles. Additional arguments in favor of vesicle-cell fusion and against phagocytosis or adsorption of intact vesicles are presented. At 2 degrees C or with inhibitor-treated cells, the [3H]inulin uptake was largely suppressed, while the lipid uptake was reduced to a lesser extent. Evidence for vesicle-cell lipid exchange was obtained using V79 cells grown on 3H precursors for cellular lipids. [14C]lecithin vesicles, incubated with such cells, showed no change in their elution properties when subjected to molecular sieve chromatography on Sepharose 4B. However, radioactivity and thin-layer chromatographic analyses revealed that a variety of cell lipiids had been exchanged into the uniamellar vesicles. Further evidence for the fusion and exchange processes was obtained using vesicles prepared from mixtures of [3H]lecithin and [14C]cholesterol. A two-step fusion mechanism consistent with the present findings is proposed as a working model for other fusion studies.



Studies on the purification of thrombopoietin from kidney cell culture medium  

SciTech Connect

A thrombocytopoiesis-stimulating factor (TSF) has been purified from human embryonic kidney (HEK) cell culture medium. In the initial purification step, crude HEK cell culture medium was fractionated with saturated ammonium sulfate (step I). The proteins precipitated by 40% to 60% and 60% to 80% ammonium sulfate saturation increased the percent of sulfur /sup 35/ incorporation into platelets of assay mice (P less than 0.01). The ammonium sulfate-precipitated proteins that contained significant TSF activity were further refined on Sephadex G-75 columns (step II). The fraction containing the highest specific activity (greatest 35S incorporation into platelets of assay mice per milligram of protein) was further purified by diethylaminoethyl (DEAE)-cellulose column chromatography (step III). TSF activity was eluted from the columns between 0.3 and 1.0 mol/L NaCl. Additional Sephadex chromatography of post-DEAE-chromatographic preparations further increased the purity of the TSF (step IV). TSF from this four-step procedure was further processed on a DEAE-high-performance liquid chromatography (HPLC) column (step Va) or size exclusion (SE)-HPLC columns (step Vb). After HPLC, the activity was localized in a region corresponding to a retention time of 6 to 8 minutes for the DEAE-HPLC, but longer times were found after SE-HPLC. TSF was further purified by additional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and SE-HPLC (step VI). The final product had significant TSF activity and represented a purification of approximately 500,000-fold. It was also shown that the isoelectric pH of partially purified TSF was 4.7 and the molecular weight of the more highly purified preparation was approximately 32,000. After extraction by a combination of chromatographic procedures, a single homogeneous product was obtained.

McDonald, T.P.; Cottrell, M.; Clift, R.; Khouri, J.A.; Long, M.D.



Comparative Evaluation of Different Cell Lysis and Extraction Methods for Studying Benzo(a)pyrene Metabolism in HT-29 Colon Cancer Cell Cultures  

PubMed Central

Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies.

Myers, Jeremy N.; Rekhadevi, Perumalla V.; Ramesh, Aramandla



A Three-Dimensional Cell Culture Model to Study the Mechano-Biological Behavior in Periodontal Ligament Regeneration  

PubMed Central

Periodontitis is a disease affecting the supporting structures of the teeth, which can eventually result in tooth loss. A three-dimensional (3D) tissue culture model was developed that may serve to grow a 3D construct that not only transplants into defective periodontal sites, but also allows to examine the effect of mechanical load in vitro. In the current in vitro study, green fluorescent protein labeled periodontal ligament (PDL) cells form rat incisors were embedded in a 3D matrix and exposed to mechanical loading alone, to a chemical stimulus (Emdogain; enamel matrix derivative [EMD]) alone, or a combination of both. Loading consisted of unilateral stretching (8%, 1?Hz) and was applied for 1, 3, or 5 days. Results showed that PDL cells were distributed and randomly oriented within the artificial PDL space in static culture. On mechanical loading, the cells showed higher cell numbers. Moreover, cells realigned perpendicular to the stretching force depending on time and position, with great analogy to natural PDL tissue. EMD application gave a significant effect on growth and upregulated bone sialoprotein (BSP) and collagen type-I (Col-I), whereas Runx-2 was downregulated. This implies that PDL cells under loading might tend to act similar to bone-like cells (BSP and Col-I) but at the same time, react tendon like (Runx-2). The combination of chemical and mechanical stimulation seems possible, but does not show synergistic effects. In this study, a new model was successfully introduced in the field of PDL-related regenerative research. Besides validating the 3D model to mimic an authentic PDL space, it also provided a useful and well-controlled approach to study cell response to mechanical loading and other stimuli.

Oortgiesen, Daniel A.W.; Yu, Na; Bronckers, Antonius L.J.J.; Yang, Fang; Walboomers, X. Frank



Cold response of dedifferentiated barley cells at the gene expression, hormone composition, and freezing tolerance levels: studies on callus cultures.  


In this study, data is presented how dark-grown, embryogenic barley callus cells respond to cold without any light-dependent, chloroplast-related mechanism, independently of the systemic signals. The expression of HvCBF9, HvCBF14, and HvCOR14b genes, members of one of the most important cold-inducible regulatory system, was measured by real-time PCR. Characteristic of the cold response was similar in the crowns of seedlings and in dark-grown callus cultures, however, gene expression levels were lower in calli. Endogenous concentration of auxins, abscisic acid, and salicylic acid did not change, but phaseic acid and neophaseic acid showed robust accumulation after cold acclimation. Freezing tolerance of the cultures was also higher after 7 days of cold-hardening. The results suggest the presence of a basal, light-independent, cold-responsive activation of the CBF-COR14b pathway in barley cultures. The effects of Dicamba, the exogenous auxin analog used for maintaining tissue cultures were also studied. Dicamba seems to be a general enhancer of the gene expression and physiological responses to cold stress, but has no specific effect on the activation. Our data along with previous findings show that this system might be a suitable model for studying certain basic cellular mechanisms involved in the cold acclimation process in cereals. PMID:22669585

Vashegyi, Ildikó; Marozsán-Tóth, Zsuzsa; Galiba, Gábor; Dobrev, Petre I; Vankova, Radomira; Tóth, Balázs



Cryopreservation of Dedifferentiated Cell Cultures  

Microsoft Academic Search

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

Elke Heine-Dobbernack; Heiko Kiesecker; Heinz Martin Schumacher



Microsoft Academic Search

We have studied the processes which are elaborated by hippocampal neurons in dissociated cell culture. Nerve cells, which were obtained from fetal rats at 18 to 20 days of gestation, were plated at very low density onto polylysine-treated coverslips and were maintained in serum-free medium. Under such conditions, some cells develop without contacting any neighboring neurons or glial cells. Examples



Substrate Micropatterning as a New in Vitro Cell Culture System to Study Myelination.  


Myelination is a highly regulated developmental process whereby oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system ensheathe axons with a multilayered concentric membrane. Axonal myelination increases the velocity of nerve impulse propagation. In this work, we present a novel in vitro system for coculturing primary dorsal root ganglia neurons along with myelinating cells on a highly restrictive and micropatterned substrate. In this new coculture system, neurons survive for several weeks, extending long axons on defined Matrigel tracks. On these axons, myelinating cells can achieve robust myelination, as demonstrated by the distribution of compact myelin and nodal markers. Under these conditions, neurites and associated myelinating cells are easily accessible for studies on the mechanisms of myelin formation and on the effects of axonal damage on the myelin sheath. PMID:22348182

Liazoghli, Dalinda; Roth, Alejandro D; Thostrup, Peter; Colman, David R



Substrate Micropatterning as a New in Vitro Cell Culture System to Study Myelination  

PubMed Central

Myelination is a highly regulated developmental process whereby oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system ensheathe axons with a multilayered concentric membrane. Axonal myelination increases the velocity of nerve impulse propagation. In this work, we present a novel in vitro system for coculturing primary dorsal root ganglia neurons along with myelinating cells on a highly restrictive and micropatterned substrate. In this new coculture system, neurons survive for several weeks, extending long axons on defined Matrigel tracks. On these axons, myelinating cells can achieve robust myelination, as demonstrated by the distribution of compact myelin and nodal markers. Under these conditions, neurites and associated myelinating cells are easily accessible for studies on the mechanisms of myelin formation and on the effects of axonal damage on the myelin sheath.



In vitro culture studies of Sutherlandia frutescens on human tumor cell lines.  


Sutherlandia frutescens is a South African herb used traditionally by the natives to treat cancer, and more recently to improve the overall health in HIV/AIDS patients. Gas chromatography/mass spectrometer profiling and liquid chromatographic/mass spectral investigation confirmed and quantified the presence of canavanine, GABA and arginine in the herbal preparation used in this study. In vitro study demonstrated a concentration dependent effect of Sutherlandia on several tumor cell lines, with 50% inhibition (IC50) of proliferation of MCF7, MDA-MB-468, Jurkat and HL60 cells at 1/250, 1/200, 1/150 and 1/200 dilutions, respectively. Sutherlandia treatment did not induce HL60 differentiation along the macrophage/monocyte or granulocyte lineage. It demonstrated antioxidant activity in reducing free radical cations with an estimated activity of 0.5 microl of Sutherlandia extract equivalent to that of 10 microM of Trolox. However, it did not significantly suppress lipopolysaccharide stimulated nitric oxide production by murine macrophage/monocyte RAW 264.7 cells, nor did it significantly inhibit IL-1beta and TNF-alpha mRNA expression in RAW 264.7 cells. In conclusion, Sutherlandia ethanolic extract showed a concentration dependent antiproliferative effect on several human tumor cell lines but did not show significant antioxidant effects. Further studies are needed to explore the activities of this multipurpose South African herbal preparation. PMID:15182898

Tai, Joseph; Cheung, Susan; Chan, Edwin; Hasman, David



Cell culture processes for monoclonal antibody production  

PubMed Central

Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; (2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; (3) appropriate on-line and off-line sensors capable of providing information that enhances process control; and (4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation and ensure compliance with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., generation of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development.

Li, Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert




Microsoft Academic Search

Epithelial cells from the human mammary gland were propagated up to 40 days in vitro as primary cell cultures. Large and medium-sized ducts, dissected from 4 different glands, served as a specific source of the epithelial cells. Ducts were opened, cut in small pieces, and were treated with trypsin-EDTA. The loosened epithelial cells were obtained in suspension and inoculated into

B. Allen Flaxman



Phagosomal pH and glass fiber dissolution in cultured nasal epithelial cells and alveolar macrophages: a preliminary study.  

PubMed Central

The dissolution rate of glass fibers has been shown to be pH sensitive using in vitro lung fluid simulant models. The current study investigated whether there is a difference in phagosomal pH (ppH) between rat alveolar macrophages (AM) and rat nasal epithelial cells (RNEC) and whether such a difference would influence the dissolution of glass fibers. The ppH was measured in cultured AM and RNEC using flow cytometric, fluorescence-emission rationing techniques with fluorescein-labeled, amorphous silica particles. Glass fiber dissolution was determined in AM and RNEC cultured for 3 weeks with fast dissolving glass fibers (GF-A) or slow dissolving ones (GF-B). The mean diameters of GF-A were 2.7 microns and of GF-B, 2.6 microns, the average length of both fibers was approximately 22 to 25 microns. Dissolution was monitored by measuring the length and diameter of intracellular fibers and estimating the volume, assuming a cylindrical morphology. The ppH of AM was 5.2 to 5.8, and the ppH of RNEC was 7.0 to 7.5. The GF-A dissolved more slowly in RNEC than in AM, and no dissolution was evident in either cell type with GF-B. The volume loss with GF-A after a 3-week culture with AM was 66% compared to 45% for cultured RNEC. These results are different from those obtained using in vitro lung fluid-simulant models where dissolution is faster at higher pH. This difference suggests that dissolution rates of glass fibers in AM should not be applied to the dissolution of fibers in epithelial cells. Images Figure 1. a Figure 1. b Figure 2. a Figure 2. b Figure 3. a Figure 3. b

Johnson, N F



An endothelial-cell-enriched primary culture system to study vascular endothelial growth factor (VEGF A) expression in a teleost, the Japanese eel ( Anguilla japonica)  

Microsoft Academic Search

A partial gene for eel (Anguilla japonica) vascular endothelial growth factor (VEGF) has been cloned and an endothelial-cell-enriched primary culture derived from rete mirabile established to study regulation of the expression of the eel VEGF gene. Cells were cultured in M199 medium containing 0.1% fetal calf serum (FCS) and serum-free M199 medium for long-and short-term experiments, respectively. Cells were separately

Yung-Sen Huang; Wen-Lian Huang; Wei-Fan Lin; Ming-Chyuan Chen; Shan-Ru Jeng




Microsoft Academic Search

The scqucntial transformation of chickcn monocytcs into macrophages, cpithelioid cells, and multinucleatcd giant cells in vitro was studied by electron microscopy after fixation and cmbcdment in situ. The following changes occur. In the nucleus, margination of chro- matin, cvidcnt in monocytes, decreases in later forms. Nucleoli become more complcx and nuclear pores increase in number. In cytoplasm, a progressive increase




Gland formation of human colon cancer cells combined with foetal rat mesenchyme in organ culture: an ultrastructural study  

Microsoft Academic Search

Summary The morphogenetic potential of human colon cancer cells was examined by combining cells with foetal rat mesenchyme in organ culture. Out of four cell lines examined, LS174T cells formed glandular structures composed of a simple col- umnar or cuboidal epithelium with a lumen in the centre of the cell mass. Ultrastructurally, these gland-forming LS174T cells exhibited po- larity, with



Glial cell growth in culture: Influence of living cell substrata  

Microsoft Academic Search

The role of the microenvironment in the growth of glial cells in culture has been the topic of ongoing research in this laboratory. Recently, we reported a study on the contribution of fibroblast cell substratum and extracellular matrix in glial cell growth. In the present study we report data concerning a) the influence of a neuronal-enriched living substratum from chick

N. Sakellaridis; D. Mangoura; A. Vernadakis



Organotypic 3D cell culture models: using the rotating wall vessel to study host-pathogen interactions.  


Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host-pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed. PMID:20948552

Barrila, Jennifer; Radtke, Andrea L; Crabbé, Aurélie; Sarker, Shameema F; Herbst-Kralovetz, Melissa M; Ott, C Mark; Nickerson, Cheryl A



Development of a mouse model for studying the effect of embryo culture on embryonic stem cell derivation.  


For most of the derived human embryonic stem cell (ESC) lines thus far, the majority of human embryos used have been frozen in liquid nitrogen at or prior to the compacting stage for up to 10 years before human ESC derivation. As such they were grown in media that were relatively simple in their formulation compared with those used today. Here we report that culture of mouse embryos in media similar to these produces blastocysts in which both the inner cell mass cell number and the number of ESC progenitor cells (epiblast cells) in the inner cell mass are reduced compared with blastocysts cultured in a purpose-designed sequential (G1/G2) system commonly used today. Embryos cultured in a simple medium were less likely to attach and generate outgrowths. Further, these outgrowths had increased metabolic activity, which has been linked to differentiation, and altered gene expression. Culture of embryos in a simple medium to the compacting stage followed by culture in G2 to the blastocyst stage reduced some of these effects. However, none were improved to the level seen for culture in G1/G2. These results highlight the influence of embryo culture on embryo quality and pluripotency, which is a key factor in determining ESC isolation efficiencies. PMID:21105769

Campbell, Jared M; Mitchell, Megan; Nottle, Mark B; Lane, Michelle



Development in primary cell culture of demosponges  

Microsoft Academic Search

We have established primary cell culture of the marine demosponge Dysidea avara and Suberites domuncula. Microbial contamination was controlled by the use of a pool of antibiotics confirming the goodness of this procedure. Effect of pH, temperature and light was studied to establish the better growth conditions. The comparison of lipid composition of sponge and cells suggested a series of

Salvatore De Rosa; Salvatore De Caro; Carmine Iodice; Giuseppina Tommonaro; Kamen Stefanov; Simeon Popov



Marine invertebrate cell cultures: new millennium trends.  


This review analyzes activities in the field of marine invertebrate cell culture during the years 1999 to 2004 and compares the outcomes with those of the preceding decade (1988 to 1998). During the last 5 years, 90 reports of primary cell culture studies of marine organisms belonging to only 6 taxa (Porifera, Cnidaria, Crustacea, Mollusca, Echinodermata, and Urochordata) have been published. This figure represents a 2-fold increase in the annual number of publications over the decade 1988 to 1998. Three other trends distinguish the two reviewed periods. First, in recent years studies attempting to improve cell culture methodologies have decreased, while interest in applications of already existing methodologies has increased. This reflects the effects of short-term cultures in attracting new researchers and scientific disciplines to the field. Second, only 17.8% of the recent publications used long-term cultures, compared with 30.0% of the publications in the previous decade. Third, during recent years research in cell cultures has studied fewer model species more extensively (mainly, Botryllus schlosseri, Crassostrea, Mytilus, Penaeus, and Suberites domuncula), signifying a shift from previous investigations that had studied a more diverse range of organisms. From 1988 to 1998 the phylum Mollusca was the most studied taxon (34.4%), but recent years have seen more studies of Porifera and Crustacea (30.0% and 32.2% of publications) than of Mollusca (21.1%). Still, not even a single established cell line from any marine invertebrate has yet been made available. However, the use of new cellular, genomic, and proteomic tools may fundamentally change our strategy for the development of cell cultures from marine invertebrates. PMID:16132466

Rinkevich, Baruch



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


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

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



Biosynthesis of the D2 cell adhesion molecule: pulse-chase studies in cultured fetal rat neuronal cells  

PubMed Central

D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing chase times the Mr of both molecules increased to 187,000-201,000 (A) and 137,000-158,000 (B). These were similar to the sizes of D2-CAM labeled with [14C]glucosamine, [3H]fucose and [14C]mannosamine, indicating that the higher Mr species are glycoproteins. In the presence of tunicamycin, which specifically blocks the synthesis of high mannose cores, Mr were reduced to 175,000 (A) and 124,000 (B). Newly synthesized A and B are susceptible to degradation by endo-beta-N-acetyl-glucosaminidase H, which specifically degrades high mannose cores, but they are resistant to such degradation after 150 min of posttranslational processing. Hence, we deduce that A and B are initially synthesized with four to five high mannose cores which are later converted into N-linked complex oligosaccharides attached to asparagine residues. However, no shift of [35S]methionine radioactivity between A and B was detected with different pulse or chase times, showing that these molecules are not interconverted. Thus, our data indicate that the neuronal D2-CAM glycoproteins are derived from two mRNAs.



Cell Culture in 3Dimensional Microfluidic Structure of PDMS (polydimethylsiloxane)  

Microsoft Academic Search

In this paper, a device with 3-dimensional microfluidic structure composed of two stacked layers of PDMS (polydimethylsiloxane) is fabricated for mammalian cell culture. This microdevice is tested with Hepatocarcinoma liver cells (Hep G2 cells). The purpose of this study is to understand to what extent cell culture in a PDMS microdevice is available. The experimental protocols for Hep G2 cell

Eric Leclerc; Yasuyuki Sakai; Teruo Fujii



A Comparative Study on Morphochemical Properties and Osteogenic Cell Differentiation within Bone Graft and Coral Graft Culture Systems.  


The objective of this study was to compare the morphological and chemical composition of bone graft (BG) and coral graft (CG) as well as their osteogenic differentiation potential using rabbit mesenchymal stem cells (rMSCs) in vitro. SEM analysis of BG and CG revealed that the pores in these grafts were interconnected, and their micro-CT confirmed pore sizes in the range of 107-315 µm and 103-514 µm with a total porosity of 92% and 94%, respectively. EDS analysis indicated that the level of calcium in CG was relatively higher than that in BG. FTIR of BG and CG confirmed the presence of functional groups corresponding to carbonyl, aromatic, alkyl, and alkane groups. XRD results revealed that the phase content of the inorganic layer comprised highly crystalline form of calcium carbonate and carbon. Atomic force microscopy analysis showed CG had better surface roughness compared to BG. In addition, significantly higher levels of osteogenic differentiation markers, namely, alkaline phosphatase (ALP), Osteocalcin (OC) levels, and Osteonectin and Runx2, Integrin gene expression were detected in the CG cultures, when compared with those in the BG cultures. In conclusion, our results demonstrate that the osteogenic differentiation of rMSCs is relatively superior in coral graft than in bone graft culture system. PMID:24151432

Puvaneswary, Subramaniam; Balaji Raghavendran, Hanumantha Rao; Ibrahim, Nurul Syuhada; Murali, Malliga Raman; Merican, Azhar Mahmood; Kamarul, T



A Comparative Study on Morphochemical Properties and Osteogenic Cell Differentiation within Bone Graft and Coral Graft Culture Systems  

PubMed Central

The objective of this study was to compare the morphological and chemical composition of bone graft (BG) and coral graft (CG) as well as their osteogenic differentiation potential using rabbit mesenchymal stem cells (rMSCs) in vitro. SEM analysis of BG and CG revealed that the pores in these grafts were interconnected, and their micro-CT confirmed pore sizes in the range of 107-315 µm and 103-514 µm with a total porosity of 92% and 94%, respectively. EDS analysis indicated that the level of calcium in CG was relatively higher than that in BG. FTIR of BG and CG confirmed the presence of functional groups corresponding to carbonyl, aromatic, alkyl, and alkane groups. XRD results revealed that the phase content of the inorganic layer comprised highly crystalline form of calcium carbonate and carbon. Atomic force microscopy analysis showed CG had better surface roughness compared to BG. In addition, significantly higher levels of osteogenic differentiation markers, namely, alkaline phosphatase (ALP), Osteocalcin (OC) levels, and Osteonectin and Runx2, Integrin gene expression were detected in the CG cultures, when compared with those in the BG cultures. In conclusion, our results demonstrate that the osteogenic differentiation of rMSCs is relatively superior in coral graft than in bone graft culture system.

Puvaneswary, Subramaniam; Balaji Raghavendran, Hanumantha Rao; Ibrahim, Nurul Syuhada; Murali, Malliga Raman; Merican, Azhar Mahmood; Kamarul, T.



Hofstede never studied culture  

Microsoft Academic Search

The continuation of accounting research utilising Hofstede's cultural indices suggests an absence of sufficient consideration for the reasons behind the rejection of such a universalist approach in anthropology and sociology. These reasons include the assumption of equating nation with culture and the difficulty, and limitations on an understanding of culture by means of numeric indices and matrices. Alternative approaches for

Rachel F. Baskerville



Cell death pattern of a varicose vein organ culture model.  


The study aimed to investigate the viability of a varicose vein (VV) organ culture model by assessing cell death pattern. To assess pattern of cell death with time, VV organ cultures were incubated for up to 14 days with regular medium changed. To assess viability, cell death of VV organ cultures treated with sodium azide and their untreated counterparts was assayed. Increased cell death was measured in VV organ cultures from day 0 to 2. Cell death decreased gradually after day 2 and plateaued from day 8 to 14.VV organ cultures treated with sodium azide demonstrated significantly more cell death in tissue (P = 0.001). Cell death measured in cultures treated with sodium azide continued to increase until day 7. In conclusion, this study demonstrated the viability of a VV organ culture model with most cell death occurred within the first two days and then declined to a relatively low level. PMID:23526103

Lim, Chung S; Kiriakidis, Serafim; Paleolog, Ewa M; Davies, Alun H



The role of endogenous noradrenaline in the beta-blocker withdrawal phenomenon — studies with culture heart cells  

Microsoft Academic Search

Summary An in vitro model to evaluate the role of endogenous noradrenaline in the beta-blocker withdrawal phenomenon is described: Beating chicken heart muscle cells (5000 beta1-adrenoceptors\\/cell) and heart nonmuscle cells (3000 beta2-adrenoceptors\\/cell) were cultured in serum-free, hormone-supplemented medium. Basal state, subtype selective down-regulation of beta-adrenoceptors by endogenous noradrenaline (decrease in receptor number, beta1 more than beta2) was simulated by addition

C. Reithmann; A. Thomschke; K. Werdan



Isoniazid Proliposome Powders for Inhalation--Preparation, Characterization and Cell Culture Studies  

PubMed Central

The aims of this study were to develop proliposome powders containing isoniazid (INH) in a dry powder aerosol form. INH-proliposome powders were prepared by a spray drying method. Proliposome physicochemical properties were determined using cascade impactor, X-ray diffraction and differential scanning calorimetry. The toxicity of proliposomes to respiratory-associated cell lines and its potential to provoke immunological responses from alveolar macrophages (AM) were determined. Free INH and INH-proliposome bioactivities were tested in vitro and in AM infected with Mycobacterium bovis (M. bovis). Aerosolization properties of INH-proliposome powders at 60 L/min, the powders showed mass median aerodynamic diameters of 2.99–4.92 ?m, with fine particle fractions (aerosolized particles less than 4.4 ?m) of 15–35%. Encapsulation of INH was 18–30%. Proliposome formulations containing INH to mannitol ratios of 4:6 and 6:4 exhibited the greatest overlapping peak between the drug and mannitol. INH-proliposomes were evidently nontoxic to respiratory-associated cells, and did not activate AM to produce inflammatory mediators—including interleukin-1? (IL-1?), tumor necrosis factor-? (TNF-?), and nitric oxide—at a toxic level. The efficacy of INH-proliposome against AM infected with M. bovis was significantly higher than that of free INH (p < 0.05). INH-proliposomes are potential candidates for an alternative tuberculosis treatment.

Rojanarat, Wipaporn; Changsan, Narumon; Tawithong, Ekawat; Pinsuwan, Sirirat; Chan, Hak-Kim; Srichana, Teerapol



Study on the Structure of the Lungs, Separation of the Lung Cells and Culture.  

National Technical Information Service (NTIS)

The research consists of two major divisions: lung cell separation and lung morphology. The first 11 months of the contract period was devoted to cell separation. The laboratory was successful in isolating alveolar type II cells from the lungs in good pur...

Y. Kikkakawa



Photoreceptor-like cells from reprogramming cultured mammalian RPE cells  

PubMed Central

Purpose Previous studies showed that chick retinal pigment epithelium (RPE) cells can be reprogrammed by a specific gene to take on the path of photoreceptor differentiation. In this study, we tested whether this reprogramming scheme could be applied to mammalian RPE cells. Methods Human RPE cell lines ARPE-19, a spontaneously transformed line of RPE cells derived from a 19-year-old person, and hTERT-RPE1, a telomerase-immortalized RPE cell line derived from a 1-year-old person, were commercially obtained and cultured as recommended. Primary RPE cell cultures were established using RPE isolated from 3- to 6-month-old pig and postnatal day 5 mouse. Cultured cells were transduced with a virus expressing neuroD, neurogenin1 (ngn1), or ngn3, basic helix-loop-helix (bHLH) genes previously identified as capable of inducing RPE-to-photoreceptor reprogramming in the chick system. Alternatively, cells in the culture were transfected chemically or physically through electroporation with vector DNA expressing one of the three genes. The cultures were then analyzed for RPE-to-photoreceptor reprogramming with in situ hybridization and/or immunostaining for photoreceptor gene expression. Results Both hTERT-RPE1 and ARPE-19 cultures gave rise to cells bearing markers of photoreceptors after transduction or transfection with vehicles expressing neuroD or ngn1. The new cells expressed genes encoding photoreceptor proteins, including interphotoreceptor retinoid-binding protein IRBP), recoverin, retinal cone arrestin 3, transducin ?-subunit, Cone-rod homeobox protein (Crx), and red opsin. They displayed morphologies resembling differentiating photoreceptor cells. In primary porcine and mouse RPE cell cultures, transduction with lenti virus (Lvx-IRES-ZsGreen1) expressing ngn1 or ngn3 resulted in the emergence of ZsGreen1+ cells that exhibited morphologies reminiscent of differentiating photoreceptor cells. Immunochemistry showed that some ZsGreen1+ cells were positive for neural marker microtubule-associated protein 2 (Map2) and photoreceptor hallmark proteins red opsin and rhodopsin. Conclusions The results suggest that cells in human RPE cell lines and in primary cultures of porcine and mouse RPE respond to gene-induced reprogramming by giving rise to photoreceptor-like cells. The responsiveness of primary RPE cells, especially those from porcine cells, enhances the biologic feasibility of exploring RPE-to-photoreceptor reprogramming for in situ mammalian photoreceptor replacement without cell transplantation.

Yan, Run-Tao; Huang, Jian; Guidry, Clyde; Wang, Shu-Zhen



Wound Coverage by Cultured Skin Cells.  

National Technical Information Service (NTIS)

The aim was to confirm and extend in vitro and in vivo studies reported last year and develop new means for wound treatment. For the studies in vitro we used different tissue culture techniques. Quantitative data were obtained by cell counts and incorpora...

M. Eisinger



Isolation and culture of mouse intestinal cells.  


Complex cell signal transduction mechanisms regulate intestinal epithelial shape, polarity, motility, organelles, cell membrane components as well as physical and mechanical properties to influence alimentary digestion, absorption, secretion, detoxification and fluid balance. Interactions between the epithelial cells and adjacent mesenchyme are central to intestinal homeostasis although the key regulatory molecules of specific differentiation steps remain unclear. Isolation and primary culture of heterotypic murine intestinal cells provides a model system for elucidation of essential molecular cross-talk between epithelium and mesenchyme that may provide several biological and practical advantages over transformed cell lines. An in vitro primary culture system for neonatal rat or mouse intestinal cells has been established that forms monolayers, expresses intestine-specific epithelial features including intestinal brush borders and appropriate hydrolase enzymes. Our studies confirm the promise of this method which may advance our understanding of heterotypic cellular interactions implicated in intestinal function and may provide important insights into the pathobiology of disease. PMID:20204629

Campbell, Charles Frederick



Tissue Cell Culture Studies Comparative Cytotoxicity of Catalyst M and Catalyst I, with Cover Letter dated 04/20/1994.  

National Technical Information Service (NTIS)

The tissue cell culture tests are designed to determine the cytopathic effects of a material or its extracts in contact with monclayers of diploid human cells. Since both test materials are liquids, each was applied to sterile filter pads for evaluation. ...



DNA damage and repair measurements from cryopreserved lymphocytes without cell culture--a reproducible assay for intervention studies.  


Single-cell gel electrophoresis (the Comet assay) can be used to measure DNA damage and DNA repair capacity (DRC). However, to test DRC of cryopreserved lymphocytes, published methods include steps for cell culturing and phytohemagglutinin stimulation, which may limit use of this assay in intervention studies. We developed a modified Comet assay protocol that allows us to measure DRC from cryopreserved lymphocytes without these in vitro manipulations. Assay reproducibility was evaluated by performing the assay six times on different dates using six aliquots from one blood draw of one individual. The interindividual variation was assessed by performing the assay using one aliquot from six individuals. When gamma-irradiation was used as the mutagen, intra-assay coefficients of variation (CVs.) for baseline DNA damage, damage after gamma-irradiation exposure, and DRC--measured as tail moment--were 8, 31, and 10%, respectively. Interindividual CVs. were higher. When H(2)O(2) was used as the mutagen, intra-assay CVs. for damage measurements were lower for a protocol modification that included damage and repair at 37 degrees C (CVs. ranging from 8 to 35%) than for the more standard 4 degrees C protocol. Analyzing moment arm--the average distance of DNA migration within the tail--yielded similar results. DNA repair was successfully detected in each experiment. Comparing freshly isolated lymphocytes to cryopreserved lymphocytes from the same individuals' blood draw indicated that DRC was highly correlated when determined using moment arm values. This modified protocol extends the use of the Comet assay to measuring DRC in intervention studies (e.g., dietary interventions) in that it assesses cellular response after cryopreservation without cell culture or other extensive manipulation. PMID:16673412

Chang, Jyh-Lurn; Chen, Gang; Lampe, Johanna W; Ulrich, Cornelia M



Malignant transformation of mammalian cells in culture, including human cells  

SciTech Connect

This overview of the malignant transformation of mammalian cells in culture, including human cells, describes the earliest evidence of spontaneous, virus-induced, and carcinogen-induced transformation. It discusses several systems developed to assay the carcinogen-induced transformation of highly selected infinite life span (established) cell lines as well as finite life span diploid cells. Evidence is presented to support the multistep hypothesis of the process of malignant transformation, and the theoretical requirement for acquisition of an infinite, or greatly extended, life span in culture if a cell is to become malignant is explained in light of the multistep nature of the process. The use of oncogene transfection studies to analyze the number and kinds of changes involved is discussed, with emphasis on studies using human cells. Finally, the results of earlier studies on viral- and carcinogen-induced transformation of mammalian cells (or chicken cells) are reinterpreted in the light of more recent insights into the process of carcinogenesis.

McCormick, J.J.; Maher, V.M. (Michigan State Univ., East Lansing (USA))



Optimization and characterization of an in vitro bovine mammary cell culture system to study regulation of milk protein synthesis and mammary differentiation  

SciTech Connect

A long term bovine mammary cell culture system that maintains normal mammary cell function was established and optimized to study milk protein synthesis and secretion and mammary differentiation. This culture system used bovine mammary acini isolated from developing or lactating mammary gland by enzymatic dissociation, and cryopreserved until thawed and plated for growth in vitro for these studies. Cells in M199 with lactogenic hormones {plus minus} fetal calf serum (FCS) were cultured on plastic, 100ul and 500ul type I collagen, and Matrigel, or embedded within type I collagen. Cell morphology, cell number, and total TCA-precipitable {sup 35}S-labelled proteins were monitored. Milk protein ({alpha}{sub s,1}-casein, lactoferrin (LF), {alpha}-lactalbumin, and {beta}-lactoglobulin) secretion and intracellular levels were determined by an ELISA assay.

Talhouk, R.S.



Isolation, culture and characterization of human peritoneal mesothelial cells  

Microsoft Academic Search

Isolation, culture and characterization of human peritoneal mesothelial cells. This study establishes a reproducible technique for the culture of human peritoneal mesothelial cells. Direct explants, as well as enzymatically degraded specimens, of human omentum have been used as the source of cells. Cells were grown on collagen and gelatin coated matrices and were maintained in supplemented Ham's F-12 medium containing

Eleni Stylianou; Lucy A Jenner; Malcolm Davies; Gerald A Coles; John D Williams



Primary cultures of corticostriatal cells from newborn rats: A model to study muscarinic receptor subtypes regulation and function  

Microsoft Academic Search

In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary\\u000a culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of\\u000a choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes\\u000a increasing amounts of acetylcholine (ACh) from the third day

Carola Eva; Patrizia Bovolin; Fiorella Balzac; Cristina Botta; Silvana Ricci Gamalero; Flora M. Vaccarino



'Pseudomonas aeruginosa' Exotoxin: Effect on Cell Cultures.  

National Technical Information Service (NTIS)

An exotoxin, toxic to both mice and cultured cells, was isolated from cultures of Pseudomonas aeruginosa. Relatively small amounts of the exotoxin inhibited the uptake of uridine and amino acids by Vero cells. Within limits, this toxic action was reversib...

O. R. Pavlovskis F. B. Gordon



Beta vulgaris L. suspension cultures permeabilized with triton X-100 retain cell viability and betacyanines production ability: a digital image analysis study.  


The influence of Triton X-100 on Beta vulgaris L. permeabilized cell culture viability, regrowth, and ability to produce betacyanines was evaluated in this study. A non-destructive method based on the analysis of images in the RGB (red, green, blue) system was developed to estimate betacyanines content. A treatment for 15 min with 0.7 mM Triton X-100 induced the release of 30% of betacyanines without loss of cell viability (>or=70%). After this permeabilization treatment, B. vulgaris cultures regrew normally, reaching a maximum biomass concentration of 48% higher than non-permeabilized cultures after 14 days of culture. Also, maximum betacyanines concentration was only 25% lower than that of non-permeabilized cultures. PMID:17253724

Trejo-Tapia, Gabriela; Cuevas-Celis, Jonathan; Salcedo-Morales, Guadalupe; Trejo-Espino, José Luis; Arenas-Ocampo, Martha L; Jiménez-Aparicio, Antonio



Studies of Biologically Active Agents in Cell and Tissue Cultures. Part I.  

National Technical Information Service (NTIS)

Toxins Produced by Microrganisms - The cytotoxicity of Staphylococcal enterotoxin B to cells of human embryonic intestine is markedly reduced by trypsin. The temporary resistance increases proportionally with increased time of exposure to trypsin and last...

J. Gabliks



Chemo-radionuclide therapy for thyroid cancer: Initial experimental study with cultured cells  

Microsoft Academic Search

Radioiodine therapy has long been used for distant metastases of thyroid cancer. Although partially effective in most cases,\\u000a it can render a complete cure only in a limited number of patients. One way to enhance its efficacy would be to combine it\\u000a with antineoplastic agents. Here we describe an initialin vitro evaluation with 4 thyroid cancer cell lines.Methods: Cells were

Takashi Misaki; Masahiro Iwata; Yasuhiro Iida; Kanji Kasagi; Junji Konishi



Comparative methodologic study of NFkappaB activation in cultured endothelial cells.  


The transcriptional regulatory protein nuclear factor kappaB (NFkappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NFkappaB release and nuclear translocation after phosphorylation and proteolytic degradation of IkappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting to detect NFkappaB activation in endothelial cells (ECs) and to investigate the role of homocysteine (Hcy) in NFkappaB activation. ECs were treated with interleukin-1B (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NFkappaB activators. Hcy, a thiol-containing amino acid, has been shown to directly damage ECs in vitro. Experimental evidence suggests that the atherogenic propensity associated with hyperhomocysteinemia results from EC dysfunction. When ECs were pretreated with an inhibitor (pyrrolidine dithiocarbamate, 100 micromol/L) or with staurosporine (5 microL/mL), no NFkappaB activation was observed. NFkappaB activation in ECs could be detected with all five techniques, clearly showing NFkappaB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference method, has the disadvantages of being radioactive, expensive, and time consuming. Western blot analysis detected the NFkappaB p50 subunit implicated in NFkappaB activation. The techniques usually used to detect NFkappaB activation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use. PMID:11079468

Mercié, P; Belloc, F; Bihlou-Nabera, C; Barthe, C; Pruvost, A; Renard, M; Seigneur, M; Bernard, P; Marit, G; Boisseau, M R



Establishing a liquid-covered culture of polarized human airway epithelial Calu-3 cells to study host cell response to respiratory pathogens in vitro.  


The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture. Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult, pharmacological compounds, and bacterial and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome-associated coronavirus. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE. Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells. To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments . Once TEER plateaus at or above 1,000 ?×cm(2), Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens. PMID:23426201

Harcourt, Jennifer L; Haynes, Lia M



Wound Coverage by Cultured Skin Cells.  

National Technical Information Service (NTIS)

At the conclusion of a three year study on ways to improve wound healing by cultured epidermal grafts, we have found that: We were able to grow epidermal cells on collapsed collagen sponges. As a result, we can create a skin transplant with a quarter of t...

M. Eisinger



Growth of Cell-strains and Primary Cells on Micro-carriers in Homogeneous Culture  

Microsoft Academic Search

THERE are obvious advantages in culturing tissue cells in suspension instead of in monolayers and in using the suspension as a substrate for virus multiplication. Culture conditions for cell growth and virus multiplication can be studied and large scale production can be achieved more easily. The growth of cells from cell-lines, in suspension culture, as separate units or small clumps,

A. L. van Wezel



Effect of plasma needle on cultured cells  

NASA Astrophysics Data System (ADS)

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 "plasma needle". Plasma needle is a small (below 1mm) non-thermal radio-frequency glow, operating in helium mixtures with air at ambient pressure. Plasma treatment of cultured cells resulted in detachment of the cells. Viability tests using propidium iodide staining in combination with confocal laser scanning microscopy confirmed that detached cells as well as surrounding cells remained alive. When the cells received a low dose of plasma treatment, they reattached within a few hours to the surface of the culture flask and to each other. Removal of cells with high precision, without damage to adjacent cells, promises to become a new surgical technique. For investigation of the mechanism causing this detachment we investigated the gas mixture of the plasma with Raman scattering measurements. Radicals diffusing from the plasma into a liquid were detected by means of fluorescent probe in combination with laser-induced fluorescence spectroscopy.

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



In vitro culturing of ciliary respiratory cells—a model for studies of genetic diseases  

Microsoft Academic Search

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by the impaired functioning of ciliated cells. Its diagnosis is based on the analysis of the structure and functioning of cilia present in the respiratory epithelium (RE) of the patient. Abnormalities of cilia caused by hereditary mutations closely resemble and often overlap with defects induced by the environmental factors. As

Zuzanna Bukowy; Ewa Zi?tkiewicz; Micha? Witt



Cytotoxicity and oxidative stress study in cultured rat Sertoli cells with methyl tert-butyl ether (MTBE) exposure.  


Cultured Sertoli cells were tested for their cytotoxicity and oxidative stress induced by methyl tert-butyl ether (MTBE) which has been extensively used as a gasoline additive. In cytotoxic experiments, Sertoli cells were cultured with medium alone (control), 5, 500, or 50,000 microM MTBE. Lactate dehydrogcnase (LDH) leakage assay, staining with fluorescein diacetate (FDA) and propidium iodide (PI), and flow cytometric analyses were used. In oxidative stress experiments, Sertoli cells were cultured with medium alone (control), 0.5, 50, or 5000 microM MTBE. The production of reactive oxygen species (ROS), maleic dialdehyde (MDA) content and the level of superoxide dismutase (SOD) activity in cell supernatants were measured. Meanwhile, the expression level of 8-oxoguanine DNA glycosidase (OGG1) and extracellular form of superoxide dismutase (SOD(EX)) in Sertoli cells were determined by RT-PCR. We also compared the current findings with the previous findings in rat spermatogenic cells exposed to MTBE. The present data indicate that high dose MTBE may exert a direct toxic effect on Sertoli cells. Oxidative stress induced by MTBE is a possible mechanism of cytotoxicity. PMID:19150650

Li, Dongmei; Liu, Qin; Gong, Yi; Huang, Yufeng; Han, Xiaodong



Impact of Culture Conditions, Culture Media Volumes, and Glucose Content on Metabolic Properties of Renal Epithelial Cell Cultures  

Microsoft Academic Search

When renal proximal tubular cells are brought into tissue culture, they revert from oxidative metabolism and gluconeogenesis to high rates of glycolysis. Among the factors possibly responsible for this metabolic conversion, limited oxygen availability and\\/or substrate supply are discussed. In order to study the role of these factors on long-term cultures, the impact of growth conditions, culture media volume, and

Gerhard Gstraunthaler; Thomas Seppi; Walter Pfaller



Fetal rat intestinal cells in monolayer culture: a new in vitro system to study the glucagon-like immunoreactive peptides.  


To establish an in vitro model to investigate the glucagon-related peptides, fetal rat intestinal cells were enzymatically dispersed and placed into culture for up to 7 days. After 1 day in culture, the presence of epithelial-like cells containing glucagon-like immunoreactivity (GLI) was demonstrated using immunocytochemical techniques. The cell peptides were extracted by passage through a cartridge of octadecylsilyl silica and characterized by gel filtration and RIA. Two GLI moieties were detected with apparent mol wts of 11,000-12,000 and 5,000-6,000. The immunoreactive profile obtained for the cells in culture was identical to that of both whole fetal rat intestine and adult rat ileum. The presence of glucagon could not be demonstrated in any of the extracts. The basal levels of GLI and apparent immunoreactive glucagon (IRGa) were 1,457 +/- 381 and 198 +/- 57 pg/dish, respectively, on day 1 of culture. The GLI content of the cells, but not the IRGa, declined with time in culture for up to 5-7 days (P less than 0.03). Addition of insulin to the culture medium (10 or 100 mU/ml) did not influence the decrease in GLI content of the cells, but did inhibit the production of IRGa (P less than 0.05). Addition of 500 mg/dl glucose to the cells in the presence of 20 microU/ml insulin increased the secretion of GLI by 42 +/- 7% over 2 h (P less than 0.05). The stimulation by glucose was not seen in the absence of insulin or with higher insulin concentrations (100 microU/ml), nor did insulin alone (100 microU/ml) have any effect on the release of GLI. Thus, fetal rat intestinal cells in culture produce the GLI peptides, and secrete them in response to glucose. This system may provide a means by which the synthesis and control of secretion of the glucagon-related peptides can be investigated. PMID:3552626

Brubaker, P L; Vranic, M



Paclitaxel loaded nanosponges: in-vitro characterization and cytotoxicity study on MCF-7 cell line culture.  


Beta cyclodextrin (?-CD) based nanosponges were synthesized and paclitaxel inclusion complex with nanosponges were prepared using techniques of inclusion complex formation. The paclitaxel nanosponge's complexes were evaluated for their release. The nanosponges complexes were also evaluated using DSC, FTIR, and NMR techniques for confirmation of inclusion complex formation between paclitaxel and nanosponges. Particle size and morphology of paclitaxel nanosponge's complex were estimated using SEM, TEM and dynamic light scattering techniques. The particle sizes were found out to be in range of 400 to 600 nm. Cytotoxic efficacy of paclitaxel nanosponge complex was determined against MCF-7 cells and paclitaxel nanosponge's complex was found to be cytotoxic and more effective against this cell line. PMID:21235471

Ansari, Khalid A; Torne, Satyen J; Vavia, Pradeep R; Trotta, Francesco; Cavalli, Roberta



Shh Pathway Activity Is Down-Regulated in Cultured Medulloblastoma Cells: Implications for Preclinical Studies  

Microsoft Academic Search

Gene expression profiling indicates that the Sonic Hedgehog (Shh) pathway is active in f30% of human medulloblastomas, suggesting that it could provide a useful therapeutic target. Previously, we showed that spontaneous medulloblastomas in Ptc1+\\/p53\\/ mice could be eradicated by treatment with a small-molecule inhibitor (HhAntag) of Smoothened (Smo). Here, we compared the responses of mouse medulloblastoma cells propagated in flank

Ken Sasai; Justyna T. Romer; Youngsoo Lee; David Finkelstein; Christine Fuller; Peter J. McKinnon; Tom Curran



Ultrastructure of cultured cells from Schistosoma japonicum  

Microsoft Academic Search

Ultrastructures and their dynamic changes of the cultured cells from Schistosoma japonicum were observed in the present experiments. Several types—including polygonal, round granular, deltaic fan-shaped and flagellated cells—were found in the cultures. The polygonal cells took a major ratio in the cultures from adult S. japonicum, while the majority from schistosomula was round granular cells. The ultrastuctures on the cell

Hui-Fen Dong; Xiao-Bei Chen; Zhen-Ping Ming; Qin-Ping Zhong; Ming-Sen Jiang



Establishment of primary cell cultures: Experiences with 155 cell strains  

Microsoft Academic Search

Summary Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: (1) Disintegration, (2) culture media supplemented with basal additions, (3) special supplements

M. Dietel; H. Arps; D. Gerding; M. Trapp; A. Niendorf



Isolated identified Aplysia neurons in cell culture.  


Methods have been developed for primary culture of large identified Aplysia neurons. Aplysia ganglia were treated with neutral protease to soften the connective tissue sheath. Individual neurons were isolated either by manipulation with tungsten needles or by tying off their axons with fine nylon filament and were immobilized in a chick plasma clot or a solution of methylcellulose. Somata up to approximately 300 micrometers in diameter extended long processes within several hours in culture. A single neuron produced as many as 10 processes which could grow at different rates. Intracellular recordings showed spontaneous and evoked action potentials in neurons cultured for up to 6 weeks. Electrical synapses formed between pairs of neurons in culture. In several culture dishes containing neurons from buccal ganglia, electrical coupling was observed between 90% of the cell pairs tested. This primary culture system currently is being used to compare the electrical and biochemical properties of neuronal processes with those of cell bodies and to study the conditions necessary for process regeneration and synapse formation between isolated identified neurons. PMID:7346582

Dagan, D; Levitan, I B



Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of the regulation of enzyme synthesis  

Microsoft Academic Search

Summary  Parenchymal cells from adult rat liver which had been fully regenerated were isolated and cultured in nonproliferating monolayers\\u000a in vitro. The optimum conditions for attachment of these cells to Falcon plastic dishes were determined. When approximately\\u000a 1.0105 nuclei per cm2 suspended in Ham's F-12 medium with 0.5 ?g of insulin per ml and 25% fetal calf serum were incubated at

Robert J. Bonney; Joyce E. Becker; P. Roy Walker; R. Van Potter



Guineapig primary cell cultures provide a model to study expression and amyloidogenic processing of endogenous amyloid precursor protein  

Microsoft Academic Search

Until now guinea-pigs have been rarely used to investigate formation and deposition of Alzheimer's disease-associated amyloid ? peptides despite the sequence identity of human and guinea-pig amyloid ? peptides being known, and the overall similarity of human and guinea-pig amyloid precursor protein. We now describe a primary cell culture system of mixed fetal guinea-pig brain cells, which we have applied

M. Beck; M. K. Brückner; M. Holzer; S. Kaap; T. Pannicke; T. Arendt; V. Bigl



Evaluation of osteogenic cell culture and osteogenic/peripheral blood mononuclear human cell co-culture on modified titanium surfaces.  


This study aimed to determine the effect of a bioactive ceramic coating on titanium in the nanothickness range on human osteogenic cells, peripheral blood mononuclear cells (PBMC) and on osteogenic cells co-cultured with PBMC without exogenous stimuli. Cell viability, proliferation, adhesion, cytokine release (IL1?, TGF?1, IL10 and IL17) and intracellular stain for osteopontin and alkaline phosphatase were assessed. Morphologic evaluation showed smaller and less spread cell aspects in co-culture relative to osteogenic cell culture. Cell viability, proliferation and adhesion kinetics were differently influenced by surface texture/chemistry in culture versus co-culture. Cytokine release was also influenced by the interaction between mononuclear and osteogenic cells (mediators released by mononuclear cells acted on osteogenic cells and vice versa). In general, 'multi-cell type' interactions played a more remarkable role than the surface roughness or chemistry utilized on the in vitro cellular events related to initial stages of bone formation. PMID:23531996

Moura, Camilla G; Souza, Maria A; Kohal, Ralf J; Dechichi, Paula; Zanetta-Barbosa, Darceny; Jimbo, Ryo; Teixeira, Cristina C; Teixeira, Hellen S; Tovar, Nick; Coelho, Paulo G



A modified culture system for epidermal cells for grafting purposes: an in vitro and in vivo study.  


A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitro by culturing human or porcine epidermal keratinocytes on polyester filter substrate at the air-liquid interface. In addition, after 2 weeks of culture, hemidesmosome-like structures were formed along the basal area of the plasma membrane of the basal cells at the cell-filter interface. When grafted onto full-thickness skin wounds in pigs, the take of cell sheets detached from the filter with dispase was significantly higher (about 70%) in comparison to mechanically detached keratinocytes (about 15%). With dispase-treated keratinocytes alone, basement membrane formation took place within 7 days postgrafting as judged from the presence of a lamina lucida and positive staining for type IV collagen. Also, numerous hemidesmosomes and anchoring fibrils were observed at the basal cell-"neodermis" interface. The fully differentiated epidermis, generated by culturing keratinocytes at the air-liquid interface and detached from the substrate by dispase-treatment, is less fragile and easier to handle than epidermal autografts obtained by conventional culturing methods. Detachment by a short dispase-treatment appeared in our hands the only method for successful and complete epithelial regeneration in full-thickness wounds. PMID:10781213

van Dorp, A G; Verhoeven, M C; Nat-Van Der Meij, T H; Koerten, H K; Ponec, M


An in vitro cell culture system to study the influence of external pneumatic compression on endothelial function  

Microsoft Academic Search

Purpose: External pneumatic compression (EPC) is an effective means of prophylaxis against deep venous thrombosis. However, its mechanism remains poorly understood. Understanding of the biological consequences of EPC is an important goal for optimizing performance of the EPC-generating device and providing guidance for clinical use. We present a new in vitro cell culture system (Venous Flow Simulator) that simulates blood

Guohao Dai; Olga Tsukurov; Roslyn W Orkin; William M Abbott; Roger D Kamm; Jonathan P Gertler



Somaclonal Variation Is Induced De Novo via the Tissue Culture Process: A Study Quantifying Mutated Cells in Saintpaulia  

PubMed Central

Background The origin of somaclonal variation has not been questioned previously, i.e., “pre-existing mutations” in explants and “newly induced mutations” arising from the tissue culture process have not been distinguished. This is primarily because there has been no reliable molecular method for estimating or quantifying variation. Methodology/Principal Findings We adopted a petal-variegated cultivar of Saintpaulia ‘Thamires’ (Saintpaulia sp.) as the model plant. Based on the difference between the pre- and post-transposon excision sequence of the promoter region of flavonoid 3?, 5?-hydoroxylase (F3?5?H), we estimated mutated (transposon-excised) cell percentages using a quantitative real-time PCR. Mutated cell percentages in leaf laminae used as explants was 4.6 and 2.4% in highly or low variegation flower plants, respectively, although the occurrences of blue color mutants in their regenerants were more than 40%. Preexisting mutated cell percentages in cultured explants were considerably lower than the mutated plant percentage among total regenerants via tissue culture. Conclusions/Significance The estimation of mutated cell percentages became possible using the quantitative real-time PCR. The origins of mutations were successfully distinguished; it was confirmed that somaclonal variations are mainly caused by newly generated mutations arising from tissue culture process.

Sato, Mitsuru; Hosokawa, Munetaka; Doi, Motoaki



A cytophotometric and karyometric study of the action of ThioTEPA and protamine on tumor cells in tissue culture  

Microsoft Academic Search

The results of previous investigations conducted in the Laboratory of Medical Cytology at the Central Postgraduate Medical Institute showed that a more marked depression of mitotic activity is produced in tissue cultures by the combined action of TMoTEPA andprotamine thenby ThioTEPA alone. With the combined use of the two preparations, degeneration of many more of the cells was also observed.

I. I. Smertenko; V. Ya. Brodskii; A. I. Braude



Enhanced lymphatic transport of bioactive lipids: cell culture study of polymethoxyflavone incorporation into chylomicrons.  


Polymethoxyflavones (PMFs) are bioactive flavonoids found in citrus fruits that have been shown to have potential health promoting properties. However, their application as nutraceuticals in functional foods and beverages is currently limited due to their low water solubility and high melting point. The oral bioavailability of lipophilic compounds can be enhanced by promoting their intestinal lymphatic transport through co-administration with digestible lipids. We investigated the effects of chylomicron-mediated intestinal lymphatic transport on the bioavailability of 5-hydroxy-6,7,8,3',4'-pentamethoxylflavone (5-HPMF), one of representative PMFs in Caco-2 cells. Our results demonstrated that oleic acid and bile acid promoted secretion of chylomicrons in Caco-2 cells, with mean diameter ranged from 70 to 150 nm. The intracellular level of 5-HPMF increased 3-fold by co-incubation with the mixed micelle solution. Moreover, the basolateral level of 5-HPMF increased 3-fold due to enhanced chylomicron-mediated transport. Overall, our results demonstrated for the first time that the bioavailability of polymethoxyflavones can be enhanced by promoting their incorporation into chylomicrons. PMID:24084938

Yao, Mingfei; Chen, Jingjing; Zheng, Jinkai; Song, Mingyue; McClements, David Julian; Xiao, Hang



Alternative methods for mechanistic studies in toxicology. Screening of hepatotoxicity of pesticides using freshly isolated and primary cultured hepatocytes and non-liver-derived cells, SIRC cells.  


We constructed a screening battery for the evaluation of hepatotoxicity using freshly isolated and primary cultured rat hepatocytes (abbreviated to FIH and PCH, respectively) and rabbit eye derived cell line, SIRC cells. Effects on cell viability and drug metabolizing enzyme activities were examined by several pesticides and compared to those of in vivo. Among the pesticides studied, prometryn and ametryn showed cytotoxicity on PCH at lower concentration than on SIRC cells. Cytotoxicities of these chemicals on FIH were inhibited by metyrapone. They also increased rat serum AST in vivo. On the other hand, cytotoxicities of IBP, erusan, alanicarb, benfuracarb, and swep in PCH were observed at similar concentration to those in SIRC cells. Linuron, nitrofen, and chlomethoxifen increased ethoxycoumarin O-deethylation activities by almost similar concentration to those of benzo[a]pyrene. Linuron also induced ethoxycoumarin O-deethylation activity in vivo. These findings indicated that a battery of in vitro tests consisting of FIH, PCH and SIRC cells was useful to screen the hepatotoxicity of pesticides. PMID:10022315

Ohno, Y; Miyajima, A; Sunouchi, M



Effect of Light on Cell Division in Plant Tissue Cultures  

Microsoft Academic Search

LIGHT strongly influences many aspects of growth in plants. There have, however, been few studies on the effects of light on cell division in non-green plant tissue cultures. In the course of investigating the physiology of cell division in developing callus cultures of Helianthus tuberosus it has been observed that light can have an inhibitory effect on cell division.

R. S. S. Fraser; U. E. Loening; M. M. Yeoman



Development and mechanism of barbiturate tolerance in glial cell cultures  

Microsoft Academic Search

The effects of exposure of glial cells in primary culture and in continuous line (clone NN) to pentobarbital over various periods of time on cellular respiration and activities of enzymes involved in carbohydrate metabolism were studied. The results obtained in glial cells in primary culture were qualitatively identical to those obtained in glial cells in clonal line (NN). Both types

B. F. Roth-Schechter; G. Tholey; P. Mandel



Culture and differentiation of embryonic stem cells  

Microsoft Academic Search

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

Austin G. Smith



Use of TH-EGFP transgenic mice as a source of identified dopaminergic neurons for physiological studies in postnatal cell culture  

Microsoft Academic Search

The physiological and pharmacological properties of dopaminergic neurons in the brain are of major interest. Although much has been learned from cell culture studies, the physiological properties of these neurons remain difficult to study in such models because they are usually in minority and are difficult to distinguish from other non-dopaminergic neurons. Here we have taken advantage of a recently

C. Jomphe; M.-J. Bourque; G. D. Fortin; H. Okano; K. Kobayashi; L.-E. Trudeau



Plant Tissue Culture Studies.  

ERIC Educational Resources Information Center

|Plant tissue culture has developed into a valid botanical discipline and is considered a key area of biotechnology, but it has not been a key component of the science curriculum because of the expensive and technical nature of research in this area. This manual presents a number of activities that are relatively easy to prepare and perform. The…

Smith, Robert Alan


In vitro culture studies of Sutherlandia frutescens on human tumor cell lines  

Microsoft Academic Search

Sutherlandia frutescens is a South African herb used traditionally by the natives to treat cancer, and more recently to improve the overall health in HIV\\/AIDS patients. Gas chromatography\\/mass spectrometer profiling and liquid chromatographic\\/mass spectral investigation confirmed and quantified the presence of canavanine, GABA and arginine in the herbal preparation used in this study. In vitro study demonstrated a concentration dependent

Joseph Tai; Susan Cheung; Edwin Chan; David Hasman



ESR and cell culture studies on free radical-scavenging and antioxidant activities of isoflavonoids  

Microsoft Academic Search

Isoflavonoids are thought to be the biologically active components in soy that play a role in the prevention of coronary heart disease and breast and prostate cancer. Mechanisms to explain how isoflavonoids mediate beneficial effects have not yet been clearly established. This study was undertaken to investigate the free radical-scavenging and antioxidant activities of various structure-related isoflavonoids including genistein, daidzein,

Qiong Guo; Gerald Rimbach; Hadi Moini; Stefan Weber; Lester Packer



Critical illness polyneuropathy: clinical findings and cell culture assay of neurotoxicity assessed by a prospective study  

Microsoft Academic Search

Objective: First, to evaluate the role of typical intensive care-related conditions like sepsis, prolonged ventilation, drug effects and metabolic disorders in the pathogenesis of critical illness polyneuropathy (CIP); second, to investigate the possible significance of patient serum neurotoxicity assessed by an in vitro cytotoxicity assay with respect to CIP development. Design: Prospective study. Setting: Neurological intensive care unit. Patients and

A. Druschky; M. Herkert; M. Radespiel-Tröger; K. Druschky; E. Hund; C.-M. Becker; M. J. Hilz; F. Erbguth; B. Neundörfer



An endothelial-cell-enriched primary culture system to study vascular endothelial growth factor (VEGF A) expression in a teleost, the Japanese eel (Anguilla japonica).  


A partial gene for eel (Anguilla japonica) vascular endothelial growth factor (VEGF) has been cloned and an endothelial-cell-enriched primary culture derived from rete mirabile established to study regulation of the expression of the eel VEGF gene. Cells were cultured in M199 medium containing 0.1% fetal calf serum (FCS) and serum-free M199 medium for long-and short-term experiments, respectively. Cells were separately treated with cobalt ions (Co2+), basic fibroblast growth factor (bFGF), and estradiol (E2), which have been demonstrated to stimulate mammalian VEGF A expression, followed by quantification of the VEGF mRNA levels by real-time reverse transcription polymerase chain reaction. Our results show that: (1) the deduced eel VEGF protein encoded by the cloned gene is about 130 amino acids in length, and is closely related to a zebrafish (Danio rerio) VEGF A; (2) the endothelial-cell-enriched rete mirabile primary culture containing mainly (over 70%) the capillary endothelial cells; (3) the expression levels of the eel VEGF transcript were increased by Co2+, bFGF, and E2 treatments in a dose-and time-dependent manner. Our data demonstrate that an eel partial VEGF gene has been cloned and its regulation of expression in endothelial-cell-enriched rete mirabile cell culture is similar to that in higher vertebrates. PMID:16807025

Huang, Yung-Sen; Huang, Wen-Lian; Lin, Wei-Fan; Chen, Ming-Chyuan; Jeng, Shan-Ru



Gap junction currents in cultured muscle cells from human myometrium  

Microsoft Academic Search

Objective: The electrophysiologic properties of gap junctions between human myometrial smooth muscle cells were studied. Study Design: Double whole-cell patch clamp recordings were made on pairs of cells from primary cultures of myometrial cells from women undergoing cesarean section. Macroscopic gap junction currents were measured as the change in current in a cell held at a constant voltage while the

Hiroshi Miyoshi; Mary B. Boyle; Lynette B. MacKay; Robert E. Garfield



Mucin secretion is stimulated by purinergic drugs: Studies with hamster tracheal surface epithelial (HTSE) cells in primary culture  

SciTech Connect

Mucous glycoproteins or mucins are a major component of the mucus and are responsible for the viscoelastic property of the mucus. Confluent primary cultures of HTSE cells are highly enriched with secretory cells and secrete mucins, resembling airway goblet cells (Kim et al, JBC 260:4021, 1985). Using this cell culture system, the authors examined effects of purinergic drugs on mucin secretion. Confluent HTSE cells were metabolically labeled with 3H-glucosamine for 24 h and chased under various drug treatments. 3H-Mucins were quantified by Sepharose CL-4B gel-filtration column chromatography eluting with 50 mM sodium acetate/0.1% SDS. They found that 20 uM ATP stimulates mucin secretion by two fold while the same concentration of adenosine shows no effect. The order of potency was ATP>GTP=GDP{much gt}adenosine. Both ATP{sub {alpha}}S and GTP{sub {alpha}}S, non-hydrolyzable forms of ATP and GTP respectively, had almost identical potencies with their hydrolyzable counterparts indicating that the stimulatory effect of these nucleotides does not require the hydrolysis of high energy phosphate groups. They conclude that HTSE cells contain P2 receptors stimulation of which results in secretion of mucins. This may be an important mechanism by which airway goblet cell mucin secretion is regulated in vivo.

Kim, K.C.; Wilson, A.K. (Boston Univ. School of Medicine, MA (United States))



Cultural studies as a construct  

Microsoft Academic Search

This article discusses cultural studies as a social construct, and especially how it emerged in Finland. The 'discovery' of cultural studies in Finland in the early 1980s was made in a situation within Finnish sociology where some scholars began to adopt an orientation which resembled the Birmmgham orientation, but the influences to it stemmed from several sources. It was only

Pertti Alasuutari



The Ethics of Cultural Studies.  

ERIC Educational Resources Information Center

|To achieve its expressed political goals of social empowerment and transformation, cultural studies must maintain some significant ethical and political commitments. The growing field of cultural studies is analyzed in terms of its definition within a specific ethical and social justice agenda and its links to the critical pedagogy tradition in…

Hytten, Kathy



Comparative methodologic study of NF?B activation in cultured endothelial cells  

Microsoft Academic Search

The transcriptional regulatory protein nuclear factor ?B (NF?B) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NF?B release and nuclear translocation after phosphorylation and proteolytic degradation of I?B. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting

Patrick Mercié; Francis Belloc; Chrystèle Bihlou-Nabera; Christophe Barthe; Annie Pruvost; Martine Renard; Martine Seigneur; Philippe Bernard; Gérald Marit; M. R. Boisseau



Microcoil-based MRI: feasibility study and cell culture applications using a conventional animal system  

Microsoft Academic Search

Object  The aim of this study was to demonstrate the feasibility of MR microimaging on a conventional 9.4 T horizontal animal MRI\\u000a system using commercial available microcoils in combination with only minor modifications to the system, thereby opening this\\u000a field to a larger community.\\u000a \\u000a \\u000a \\u000a \\u000a Materials and methods  Commercially available RF microcoils designed for high-resolution NMR spectrometers were used in combination with a

Hans Weber; Nicoleta Baxan; Dominik Paul; Julian Maclaren; Daniel Schmidig; Mohammad Mohammadzadeh; Jürgen Hennig; Dominik von Elverfeldt



Study on characteristics of in vitro culture and intracellular transduction of exogenous proteins in fibroblast cell line of Liaoning cashmere goat.  


Establishment of fibroblast cell lines of endangered goat breeds and research on the gene or protein functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. In this study, a fibroblast cell line of Liaoning cashmere goat, frozen in 174 cryovials with 5 × 10(6) cells each, was successfully established from 60 goats ear marginal tissues using explant culture and cryopreservation techniques. Biological analysis of in vitro cultured cell line showed that, the cells were morphologically consistent with fibroblasts; the average viability of the cells was 94.9 % before freezing and 90.1 % after thawing; the growth process of cells was consisted of a lag phase, a logarithmic phase and a plateau phase; cell population doubling time was 65.5 h; more than 90 % of cells were diploid prior to the 6th generation; Neither microbial contamination nor cross-contamination was detected. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, a TAT protein transduction domain was fused to the C-terminus of enhanced green fluorescent protein, the established fibroblast cell line was treated with the purified exogenous proteins at various concentrations by adding them to the cell culture media for 1-24 h and assayed cell morphology and protein presence, it was found that the purified exogenous proteins readily entered cells at a concentration of 0.1 mg/ml within 1.5 h and some of them could translocate into nucleus, moreover, the exogenous proteins appeared to be stable inside cells for up to 24 h. PMID:23065271

Hu, P F; Guan, W J; Li, X C; Zhang, W X; Li, C L; Ma, Y H



Organotypic 3D cell culture models: using the rotating wall vessel to study host–pathogen interactions  

Microsoft Academic Search

Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host–pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture

Jennifer Barrila; Andrea L. Radtke; Aurélie Crabbé; Shameema F. Sarker; Melissa M. Herbst-Kralovetz; C. Mark Ott; Cheryl A. Nickerson



Study of some enzyme activities in cultured chick embryo brain nerve cells treated by chick embryo brain extracts  

Microsoft Academic Search

Brain extracts from 8-day-old chick embryos have been shown to influence morphological development of dissociated brain cells from 7-day-old chick embryos in culture. Stimulatory, effects on size of the neuronal somas and on growth of long processes were observed by adding the cytosol of the brain extract or the dialysate of the cytosol. These morphological changes parallel modifications of various

Y. Cam; M. Ledig; A. Ebel; M. Sensenbrenner; P. Mandel



Cell Culture as an Alternative in Education.  

ERIC Educational Resources Information Center

|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)|

Nardone, Roland M.



Studies on vacuolar membrane microdomains isolated from Arabidopsis suspension-cultured cells: local distribution of vacuolar membrane proteins.  


The local distribution of both the vacuolar-type proton ATPase (V-ATPase) and the vacuolar-type proton pyrophosphatase (V-PPase), the main vacuolar proton pumps, was investigated in intact vacuoles isolated from Arabidopsis suspension-cultured cells. Fluorescent immunostaining showed that V-PPase was distributed evenly on the vacuolar membrane (VM), but V-ATPase localized to specific regions of the VM. We hypothesize that there may be membrane microdomains on the VM. To confirm this hypothesis, we prepared detergent-resistant membranes (DRMs) from the VM in accordance with well established conventional methods. Analyses of fatty acid composition suggested that DRMs had more saturated fatty acids compared with the whole VM in phosphatidylcholine and phosphatidylethanolamine. In the proteomic analyses of both DRMs and detergent-soluble mebranes (DSMs), we confirmed the different local distributions of V-ATPase and V-PPase. The observations of DRMs with an electron microscope supported the existence of different areas on the VM. Moreover, it was observed using total internal reflection fluorescent microscopy (TIRFM) that proton pumps were frequently immobilized at specific sites on the VM. In the proteomic analyses, we also found that many other vacuolar membrane proteins are distributed differently in DRMs and DSMs. Based on the results of this study, we discuss the possibility that VM microdomains might contribute to vacuolar dynamics. PMID:23903016

Yoshida, Katsuhisa; Ohnishi, Miwa; Fukao, Yoichiro; Okazaki, Yozo; Fujiwara, Masayuki; Song, Chihong; Nakanishi, Yoichi; Saito, Kazuki; Shimmen, Teruo; Suzaki, Toshinobu; Hayashi, Fumio; Fukaki, Hidehiro; Maeshima, Masayoshi; Mimura, Tetsuro



Immunocytochemical characterisation of cultures of human bladder mucosal cells  

PubMed Central

Background The functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts. The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical characterisation. Methods Primary cell cultures were established from human bladder biopsies (n = 10). Individual populations of urothelial and myofibroblast-like cells were isolated using magnetic activated cell separation (MACS). Cells were slow growing, needing 3 to 5 weeks to attain confluence. Results Cytokeratin 20 positive cells (umbrella cells) were isolated at primary culture and also from patients' bladder washings but these did not proliferate. In primary culture, proliferating cells demonstrated positive immunocytochemical staining to cytokeratin markers (AE1/AE3 and A0575) as well fibroblasts (5B5) and smooth muscle (?SMA) markers. An unexpected finding was that populations of presumptive urothelial and myofibroblast-like cells, isolated using the MACS beads, stained for similar markers. In contrast, staining for cytokeratins and fibroblast or smooth muscle markers was not co-localised in full thickness bladder sections. Conclusions Our results suggest that, in culture, bladder mucosal cells may undergo differentiation into a myoepithelial cell phenotype indicating that urothelial cells have the capacity to respond to environmental changes. This may be important pathologically but also suggests that studies of the physiological function of these cells in culture may not give a reliable indicator of human physiology.



Phosphoribosylpyrophosphate Synthesis in Cultured Human Cells  

Microsoft Academic Search

Phosphoribosylpyrophosphate (PRPP) concentrations and PRPP synthetase activity were studied in cultured human fibroblasts with control and mutant hypoxanthine-guanine phosphoribosylpyrophosphate (HPRT) activity. The PRPP concentrations increased 20- to 50-fold and PRPP synthetase activity 3-fold in cells from patients with the Lesch-Nyhan syndrome when aminopterin, an inhibitor of de novo purine synthesis, was added to the medium. Concentrations of PRPP and PRPP

Paul J. Benke; David Dittmar



Genomics in mammalian cell culture bioprocessing.  


Explicitly identifying the genome of a host organism including sequencing, mapping, and annotating its genetic code has become a priority in the field of biotechnology with aims at improving the efficiency and understanding of cell culture bioprocessing. Recombinant protein therapeutics, primarily produced in mammalian cells, constitute a $108 billion global market. The most common mammalian cell line used in biologic production processes is the Chinese hamster ovary (CHO) cell line, and although great improvements have been made in titer production over the past 25 years, the underlying molecular and physiological factors are not well understood. Confident understanding of CHO bioprocessing elements (e.g. cell line selection, protein production, and reproducibility of process performance and product specifications) would significantly improve with a well understood genome. This review describes mammalian cell culture use in bioprocessing, the importance of obtaining CHO cell line genetic sequences, and the current status of sequencing efforts. Furthermore, transcriptomic techniques and gene expression tools are presented, and case studies exploring genomic techniques and applications aimed to improve mammalian bioprocess performance are reviewed. Finally, future implications of genomic advances are surmised. PMID:22079893

Wuest, Diane M; Harcum, Sarah W; Lee, Kelvin H



Cell culture of Peyronie's disease plaque and normal penile tissue.  


Cell cultures derived from Peyronie's disease plaque and normal penile tissue were characterized morphologically and examined by immunofluorescence for actin cable formation, and their growth properties were compared. Relative to normal penile cell cultures which grew as contact inhibited, poorly refractile fibroblast-like cells, plaque derived cell cultures consisted of round and spindle shaped cells that were more refractile and exhibited random crisscross growth patterns. Scanning electron microscopy of plaque derived cell cultures revealed changes in cell surface topography characterized by the appearance of surface membrane blebs amd microvilli. Transmission electron microscopy demonstrated cells containing organized cytoplasmic microfilament bundles and nuclear indentations which resembled myofibroblasts. Such alterations were less extensive or absent in normal penile cell cultures. The amount and extent of actin cable formation was increased in plaque derived compared to normal penile cell cultures. Plaque derived cells also exhibited differences in growth properties and grew to higher saturation densities than their normal counterparts. These results demonstrate that cells derived from Peyronie's disease plaque can be grown in vitro and that these cells are morphologically altered and have an enhanced proliferative capacity. The availability of these cell cultures will permit studies directed at understanding the etiology and pathogenesis of Peyronie's disease. PMID:7038152

Somers, K D; Dawson, D M; Wright, G L; Leffell, M S; Rowe, M J; Bluemink, G G; Vande Berg, J S; Gleischman, S H; Devine, C J; Horton, C E



Adherence of Staphylococcus aureus to cultured epidermal cells during differentiation  

Microsoft Academic Search

Summary. The adherence of clinical isolates of Staphylococcus aureus to cultured mouse epidermal cells was studied. Adherence of the isolates to the cells varied from strain to strain. When epidermal cell differentiation was induced by raising the calcium concentration in the medium, three out of 10 strains tested adhered better to calcium-induced differentiated cells than to undifferentiated cells, and one




Establishment of callus and cell suspension cultures from Gypsophila paniculata leaf segments and study of the attachment of host cells by Erwinia herbicola pv. gypsophilae  

Microsoft Academic Search

Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l-1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of

Mazen Nayef Salman



Three-dimensional tissue culture based on magnetic cell levitation.  


Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies. PMID:20228788

Souza, Glauco R; Molina, Jennifer R; Raphael, Robert M; Ozawa, Michael G; Stark, Daniel J; Levin, Carly S; Bronk, Lawrence F; Ananta, Jeyarama S; Mandelin, Jami; Georgescu, Maria-Magdalena; Bankson, James A; Gelovani, Juri G; Killian, T C; Arap, Wadih; Pasqualini, Renata



Three-dimensional tissue culture based on magnetic cell levitation  

NASA Astrophysics Data System (ADS)

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

Souza, Glauco R.; Molina, Jennifer R.; Raphael, Robert M.; Ozawa, Michael G.; Stark, Daniel J.; Levin, Carly S.; Bronk, Lawrence F.; Ananta, Jeyarama S.; Mandelin, Jami; Georgescu, Maria-Magdalena; Bankson, James A.; Gelovani, Juri G.; Killian, T. C.; Arap, Wadih; Pasqualini, Renata



Esophageal stem cells and 3D-cell culture models.  


The following on esophageal stem cells and 3D-cell culture models contains commentaries on metaplasia through transdifferentiation and through stem cells; transcription factors that may determine an intestinal-like phenotype; the?in vitro, organotypic cell culture models; and the role of stem cells in Barrett's esophagus and its dysplastic progression. PMID:21950821

Souza, Rhonda F; Schwartz, Robert E; Mashimo, Hiroshi



Cell cultures from zebrafish embryos and adult tissues  

Microsoft Academic Search

Summary The zebrafish is increasingly popular as a nonmammalian model for studies of vertebrate developmental biology, genetics, and toxicology. The availability of cell culture systems makes it possible to address many basic questions using in vitro approaches. Here we describe materials and procedures for initiating cell cultures from zebrafish early (blastula- and gastrula-stage) diploid and haploid embryos and adult tissues

C. Samuel Bradford; Le Sun; Paul Collodi; David W. Barnes



Use of an insect cell culture growth medium to isolate bacteria from horses with effusive, fibrinous pericarditis: a preliminary study.  


Effusive, fibrinous pericarditis is an uncommon disease entity in horses. In 2001, pericarditis occurred in conjunction with an epizootic in central Kentucky that was associated with exposure to eastern tent caterpillars (ETCs). Bacterial isolation from equine pericardial fluid samples was attempted using an insect cell culture growth medium (ICCGM). Using previously cultured, stored frozen samples from four horses with fibrinous pericarditis, inoculation of 10% blood agar plates yielded no growth, whereas simultaneous inoculation of ICCGM resulted in the isolation of Proprionibacterium acnes, Staphylococcus equorum, a Streptococcus sp. and Pseudomonas rhodesiae from pericardial fluid samples. A similar or novel caterpillar-associated bacteria was not identified; however, use of an ICCGM might enhance isolation of bacteria from equine pericardial fluid. PMID:17204376

Jones, Samuel L; Valenzisi, Amy; Sontakke, Sushama; Sprayberry, Kimberly A; Maggi, Ricardo; Hegarty, Barbara; Breitschwerdt, Edward



On the origin of lipofuscin; the iron content of residual bodies, and the relation of these organelles to the lysosomal vacuome. A study on cultured human glial cells  

SciTech Connect

Cultured human glial cells constitute a suitable model system for the study of lipofuscinogenesis in vitro. These cells, although not post-mitotic, can be kept for several months in stable monolayers due to their display of very pronounced density-dependent inhibition of cell growth. Residual bodies, or lipofuscin pigment granules, accumulate over time in this pseudo post-mitotic cell system. I. In early dense cultures, exposed to purified rat liver mitochondriae, it was possible to follow the uptake of mitochondriae and their degradation, which was found to be incomplete and result in the formation of numerous residual bodies containing lipofuscin-type material. It was concluded that incomplete degradation of mitochondriae may be an important origin of lipofuscin. II. Dense, older cultures exposed to electron dense marker particles (colloidal thorium dioxide) accumulated these markers within endosomes, and later in secondary lysosomes of various types, including residual bodies. It was concluded that residual bodies constitute an integral part of the lysosomal vacuome system. III. Phase III glial cells were cultured on formvar-coated gold EM-grids and studied by whole cell transmission electron microscopy using TEM and STEM techniques in combination with energy dispersive X-ray microanalysis. It was found that residual bodies contained iron. This fact was taken as a further indication that lipofuscin has its origin in autophagocytosed mitochondriae and ER-material rich in metallo-enzymes. Due to their high concentration of iron, residual bodies may constitute unstable structures within the cells. Since iron is a well known catalyst of various peroxidative processes, the surrounding lysosomal membrane might be damaged, e.g. by oxidative stress, with risk for leakage of degradative lysosomal enzymes into the cell sap.

Brunk, U.T. (Linkoeping Univ. (Sweden))



Acetaldehyde and hexanaldehyde from cultured white cells  

PubMed Central

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.

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



Oscillatory behavior of cells in tissue culture  

Microsoft Academic Search

Fibroblasts and epithelial cells organize themselves in distinct patterns in tissue culture which indicates that neighboring cells communicate. A striking example of such communication is the oscillatory behavior of Madin-Darby canine kidney (MDCK) cells reported here. These oscillations were discovered using a biosensor referred to as ECIS (Electric Cell-substrate Impedance Sensing). In this measurement cells are seeded out on a

Ivar Giaever; Michael F. A. Linton; Charles R. Keese



Culture of Cells from Amphibian Embryos.  

ERIC Educational Resources Information Center

|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)|

Stanisstreet, Martin



Liver cells culture on three-dimensional micropatterned polydimethylsiloxane surfaces  

Microsoft Academic Search

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

Fanny Evenou; Teruo Fujii; Yasuyuki Sakai


Serial culturing of human bronchial epithelial cells derived from biopsies  

Microsoft Academic Search

Summary  In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies\\u000a obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies)\\u000a using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation\\u000a and the number of passages

Petra M. de Jong; Marianne A. J. A. van Sterkenburg; Johanna A. Kempenaar; Joop H. Dijkman; Maria Ponec



Suspension culture of drosophila cells employing a gyratory shaker  

Microsoft Academic Search

Summary  To develop a method for culturing a large number of small-scale suspension cultures ofDrosophila melanogaster cells simultaneously, basic conditions were studied using a cell line GM2 and a gyratory shaker. Under gyration at more than 180 rpm, a majority (>80%) of the cells still remained as suspension and\\u000a grew normally. Lower speed of gyration caused adhesion of the cells to

T. Miyake; K. Saigo; T. Marunouchi; T. Shiba



Dichlorodiphenyltrichloroethane Specifically Depletes Dopaminergic Neurons in Primary Cell Culture  

Microsoft Academic Search

Toxicity of dichlorodiphenyltrichloroethane (DDT) to dopaminergic neurons in primary cell culture was investigated in the present study. Developing neurons from the substantia nigra of neonatal rats were cultured. After treatments with different concentrations of DDT (5– 12.5 ?M), specific cell death of tyrosine-hydroxylase-immunoreactive dopaminergic neurons was observed in the culture by flow cytometric analysis. More than 60% of dopaminergic neurons

K. W. Leung; Y. S. Chan; K. K. L. Yung



Initiation of primary cell culture from amphioxus Branchiostoma belcheri tsingtauense  

Microsoft Academic Search

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell\\u000a culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells,\\u000a primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The

Changliu Wang; Shicui Zhang; Feng Su; Lei Wang; Hongyan Li



Regenerating holothurian tissues as a source of cells for long-term cell cultures  

Microsoft Academic Search

Cell cultures of marine invertebrates can provide simple and adequate model systems for studying different aspects of complex biological processes, with the additional advantage of allowing study under controlled experimental conditions. In the present study an attempt was made to establish long-term cell cultures from regenerating intestine of the sea cucumber Apostichopus japonicus and showed that cells obtained at different

N. A. Odintsova; I. Yu. Dolmatov; V. S. Mashanov



Lactate Transport in Cultured Glial Cells  

Microsoft Academic Search

Uptake of L-lactate was investigated with a radioactive tracer method in cultured rat glioma cells and in astroglia-rich primary cultures derived from rat brain. In the glioma cells, a saturable component of uptake was identified with half-maximal uptake occurring at 1.0±0.4 mM lactate. In addition, a non-saturable component dominated the uptake at high concentrations of lactate. In astroglia-rich primary cultures,

Ralf Dringen; Hajo Peters; Heinrich Wiesinger; Bernd Hamprecht



Glucosylation of taxifolin with cultured plant cells.  


Cultured plant cells of Eucalyptus perriniana glucosylated taxifolin to its 3'- and 7-O-beta-D-glucosides and 3',7-O-beta-D-diglucoside. On the other hand, taxifolin was converted into 3'- and 7-O-beta-D-glucosides by cultured cells of Nicotiana tabacum and Catharanthus roseus. PMID:23980419

Shimoda, Kei; Kubota, Naoji; Hamada, Manabu; Sugamoto, Masahiro; Ishihara, Kohji; Hamada, Hatsuyuki; Hamada, Hiroki



Crustacean primary cell culture: A technical approach  

Microsoft Academic Search

Crustacean cell culture has gained attention as a potent model to assist in the development of diagnostic reagents and probes for use in the shrimp, crayfish and lobster industries. The availability of such cellular tools is especially important to industries which use intensive aquaculture methods and thus have increased risk of disease problems. Indeed, crustacean cell cultures offer potential for

Jean-Yves Toullec



A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.  


The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72?h, INS-1 cells co-cultured with HUVEC separated by a distance of 100?µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200?µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered. PMID:22949028

Sabra, Georges; Vermette, Patrick



21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2010 CFR

...2009-04-01 false Cultured animal and human cells. 864.2280 Section 864...864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell...



21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 false Cultured animal and human cells. 864.2280 Section 864...864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell...



p53 Localizes to the Centrosomes and Spindles of Mitotic Cells in the Embryonic Chick Epiblast, Human Cell Lines, and a Human Primary Culture: An Immunofluorescence Study  

Microsoft Academic Search

Immunofluorescent staining of mitotic centrosomes and spindles by anti-p53 antibodies was observed in the embryonic chick epiblast by epifluorescence microscopy and in three human cancer cell lines, an SV40-immortalized cell line, and a normal human fibroblast culture by confocal microscopy. In the chick epiblast, the centrosomes stained from early prophase through to the formation of the G1 nuclei and the

Valerie B. Morris; Jennifer Brammall; Jane Noble; Roger Reddel



Skeletal muscle satellite cells cultured in simulated microgravity  

Microsoft Academic Search

Summary  Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells.\\u000a 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\\u000a muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation

Greg Molnar; Nancy A. Schroedl; Steve R. Gonda; Charles R. Hartzell



Cultural Studies, Multiculturalism, and Media Culture  

Microsoft Academic Search

Radio, television, film, and the other products of media culture provide materials out of which we forge our very identities; our sense of selfhood; our notion of what it means to be male or female; our sense of class, of ethnicity and race, of nationality, of sexuality; and of \\

Douglas Kellner


Primary cell-culture of the digestive gland of the marine mussel Mytilus edulis: a time-course study of antioxidant- and biotransformation-enzyme activity and ultrastructural changes  

Microsoft Academic Search

A primary culture of a mixed-cell population from the digestive gland of the marine mussel Mytilus edulis L. was developed for potential use in toxicological studies. Cells were maintained for up to 2 wk in a suspension culture,\\u000a and their survival rate was monitored with regard to different cell types. No cell proliferation was observed. Smaller cell\\u000a types survived with

C. Birmelin; R. K. Pipe; P. S. Goldfarb; D. R. Livingstone



Snythesis and differentiation of plasma proteins in cultured embryonic chicken liver cells: a system for study of regulation of protein synthesis.  

PubMed Central

A new system is described for studying the control of protein synthesis. In a monolayer culture of chick embryo liver cells, plasma proteins are synthesized for three days at in vivo rates. The plasma proteins are secreted into the culture medium and without concentration are detected there simply and sensitively by a modified Laurell electronimmunoassay. Secretion of the newly synthesized plasma proteins occurs within 30 min of their synthesis. Thus, rates of synthesis of the plasma proteins can be followed readily from rates of their accumulation in the culture medium. This system has the following advantages for the study of protein synthesis: cells do not have to be disrupted for the assay; the cell population can be followed over several days; it is not necessary to label the proteins radioactively; and turnover of plasma proteins is negligible and need not be taken into account. The usefulness of the system is illustrated by a number of findings. The spectrum of plasma proteins synthesized in culture changed qualitatively and quantitatively. Albumin synthesis steadily decreased with culture time and stopped at the third day, whereas the synthesis of some new plasma proteins ("adult") was induced. These qualitative changes suggest differential gene expression in culture and a special control of albumin synthesis in vivo, different from the synthesis of the other plasma proteins. Quantitative changes in the rates of synthesis of specific plasma proteins suggest a competition among their messenger RNAs for components of the translational machinery. Insulin has a differential effect on the synthesis of specific plasma proteins at concentrations within the physiological range of the hormone. Images

Grieninger, G; Granick, S



Replication of cultured lung epithelial cells  

SciTech Connect

The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to (/sup 3/H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems.

Guzowski, D.; Bienkowski, R.



Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of a Thyroid Hormone-regulated Secretome in Human Hepatoma Cells*  

PubMed Central

The thyroid hormone, 3, 3?,5-triiodo-l-thyronine (T3), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T3 are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T3-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TR?1 (HepG2-TR?1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T3 target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions –327/–312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T3 induced PAI-1 expression in J7-TR?1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T3/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra- and intracellular space of T3-treated HepG2-TR?1 cells. The T3-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T3-associated tumor progression and prognosis.

Chen, Cheng-Yi; Chi, Lang-Ming; Chi, Hsiang-Cheng; Tsai, Ming-Ming; Tsai, Chung-Ying; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Wei-Jan; Huang, Ya-Hui; Lin, Kwang-Huei



Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation  

Microsoft Academic Search

BACKGROUND: The relationship between cell shape, proliferation, and extracellular matrix (ECM) production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D) culture. RESULTS: Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1)

Helen E Gruber; Edward N Hanley Jr



Medium and Culture of Embryonic Stem Cells.  

National Technical Information Service (NTIS)

Previous methods for culturing human embryonic stem cells have required either fibroblast feeder cells or a medium which has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state. It has now been found th...

J. A. Thomson T. Ludwig



Longterm cultures of sheep thyroid cells.  


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

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



Cell culturing of human and murine microglia cell lines.  


Despite the fact that microglia cells were first described almost a century ago, microglia-derived immortalized cell lines have only been established in the last two decades. One should be aware of their limitations but also of their advantages. Cell lines offer a potentially powerful tool to investigate some functional aspects of microglia. Cell culturing of human and murine microglia cell lines will be described in this chapter. It includes a presentation of equipment needed, cell culture medium and supplements, cell culture monitoring, and a protocol describing the steps for subculturing of microglia cell lines. PMID:23813364

Rodhe, Johanna



A digital microfluidic platform for primary cell culture and analysis.  


Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-? than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients. PMID:22094822

Srigunapalan, Suthan; Eydelnant, Irwin A; Simmons, Craig A; Wheeler, Aaron R



Feeder cell cultures for zebrafish embryonal cells in vitro.  


Use of fibroblast cells derived from mouse embryos as feeder layers was one of the major steps leading to the establishment of pluripotential mouse embryonal stem (ES) cells in culture. In attempts to obtain a culture of pluripotential ES cells from zebrafish, a culture of fibroblastoid cells, designated zebrafish embryo fibroblast (ZEF), was established from early gastrula stage zebrafish embryos for use as feeder layer. In primary cultures initiated from early embryos of zebrafish without feeder layers, melanocytes appeared on the second day of culture. In contrast, melanogenesis was markedly suppressed in cocultures containing confluent monolayers of ZEF or Buffalo rat liver (BRL) cells. BRL cells are commonly used feeder layer cells for mouse ES cells. Suppression of melanogenesis was not observed in primary cultures initiated in medium containing human recombinant differentiation-inhibiting activity (DIA) or in medium conditioned by cultures of BRL feeder cells. Proliferation of zebrafish embryonal cells was enhanced significantly in cocultures with either feeder layer. Zebrafish embryonal cells cocultured short-term on ZEF and BRL feeder layers gave rise to melanocytes and formed embryoid body-like structures when removed from feeder layers and cultured in suspension, suggesting that the cells remained pluripotent in culture. PMID:7749465

Sun, L; Bradford, C S; Barnes, D W



Method to Obtain Endoscopic Esophageal Samples for Primary Cell Culture  

Microsoft Academic Search

Cell culture techniques hold great importance for the development of molecular biology. However, when used to study oncology, most of the samples come from surgical specimens. Endoscopy is a interesting alternative to get samples for culture. We studied a protocol to allow the control of infectious contamination potentially related to endoscopy, which could preclude it as a method to obtain

M. Constant-Neto; M. Falavigna; A. C. S. Castro; D. C. Machado



Epithelial cell characteristics of cultured human limbal explants  

PubMed Central

Aim: To determine the immunohistochemical characteristics of putative corneal epithelial stem cells remaining on limbal explants maintained in culture. Methods: Human limbal explant cultures were generated from 25 residual corneoscleral donor rims following penetrating keratoplasty. Serial sections of these explants were studied using immunohistochemical techniques with a panel of antibodies, on day 0 and 1, 2, and 3 weeks. Results: The number of epithelial cells expressing cytokeratin 19 and vimentin increased with duration in culture, while the number of cells expressing cytokeratin 3 decreased. Connexin 43 expression was lost by 1 week in culture. p63 was expressed by cells that had migrated around the explant and the number of p63 positive cells decreased with longer duration in culture. The explants were initially negative for Ki67, but the epithelial cells were positive at 1 week, and expression of Ki67 was progressively lost with increasing duration in culture. The initial uniform staining of the epithelium for epidermal growth factor receptor and ? enolase remained unchanged at 3 weeks. Conclusions: There is an expansion of less differentiated (cytokeratin 3 negative and CK19/vimentin positive) epithelial cells on corneoscleral explants maintained in culture for 3 weeks. The pattern of expression of p63 noted in this study does not support the suggestion that it is a marker of limbal stem cells. The decline in p63 and Ki67 expression among the epithelial cells of the cultured explant button implies that as the epithelial sheet outgrowing from the explant button reaches confluence, the proliferative status of the cells remaining on the explant button declines. These findings are of clinical relevance as explants of limbal tissue are used in limbal stem cell transplantation. There is no information available to date on the fate of epithelial cells on such explants. This study provides some insight into this and suggests that an expansion of the stem cell pool or its progeny may occur in limbal explants.

Joseph, A; Powell-Richards, A O R; Shanmuganathan, V A; Dua, H S



Electrochemical study of Type 304 and 316L stainless steels in simulated body fluids and cell cultures  

Microsoft Academic Search

The electrochemical corrosion behaviour of Type 304 and 316L stainless steels was studied in Hanks’ solution, Eagle’s minimum essential medium (MEM), serum containing medium (MEM with 10% of fetal bovine serum) without cells, and serum containing medium with cells over a 1-week period. Polarization resistance measurements indicated that the stainless steels were resistant to Hanks’ and MEM solutions. Type 304

Yee-Chin Tang; Shoji Katsuma; Shinji Fujimoto; Sachiko Hiromoto



Primary cell cultures from fetal bovine hypothalamus and cerebral cortex: A reliable model to study P450 Arom and ? and ? estrogen receptors in vitro  

Microsoft Academic Search

Estrogens synthesized by neural P450 aromatase (P450Arom) are implicated in many aspects of mammalian brain development and particularly in sexual differentiation of the central nervous system (CNS). This study analyzes the usefulness of an in vitro model based on bovine primary cell cultures from the hypothalamus and frontal cortex to investigate the role of P450Arom and estrogen receptors (ERs) in

Antonella Peruffo; Genny Buson; Bruno Cozzi; Cristina Ballarin



Insect cell culture in research: Indian scenario.  


Insect cell cultures are widely used in viral diagnosis and biotechnology, for the production of recombinant proteins, viral pesticides and vaccines as well as in basic research in genetics, molecular biology, biochemistry, endocrinology and virology. Following KRP Singh's pioneering research in 1967, a large number of cell lines from diptera, hemiptera, and lepidopteran insects were established and characterized in India. With the availability of the modern tools in molecular biology and the advancements made in biotechnology, the indigenous cell lines may prove useful in creating a future without biohazardous chemical pesticides as well as producing life saving pharmaceuticals and vaccines for many diseases. This review summarizes information gathered regarding the insect cell lines established so far in India. It also covers the familiarization of the well characterized continuous cell lines and their potential applications. Special attention is given to virus susceptibility of the cell lines, the yield of virus with a comparative analysis with other conventional systems. The potential applications of dipteran and lepidopteran cell lines in agriculture and biotechnology are also briefly discussed for prospective studies. PMID:16037617

Sudeep, A B; Mourya, D T; Mishra, A C



Studies on the synthesis of sterol carrier protein-2 in rat adrenocortical cells in monolayer culture. Regulation by ACTH and dibutyryl cyclic 3',5'-AMP  

SciTech Connect

The effects of ACTH or dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of sterol carrier protein-2 (SCP2) have been studied in rat adrenocortical cells in monolayer culture. Radiolabeling of total cellular proteins with (/sup 35/S)methionine and immunoprecipitation with antibodies directed against rat liver SCP2, followed by polyacrylamide gel electrophoresis and fluorography, showed a 3-4-fold increase in the rate of synthesis of SCP2 in cells treated for 48 h with ACTH (1 microM) or Bt2cAMP (0.1 mM). The induction of SCP2 synthesis depended upon the concentrations of ACTH or Bt2cAMP with an ED50 of 8 and 100 nM, respectively, and increased linearly with time between 12 and 48 h of treatment. Immunoprecipitation of SCP2 synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from cells treated with ACTH or Bt2cAMP revealed increased synthesis of SCP2 compared to RNA from control cells. The immunoprecipitable rat adrenal SCP2, synthesized in a cell-free translation system, showed mobility corresponding to Mr of 14,400 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was clearly larger than immunodetectable SCP2 synthesized in cultured adrenal cells (Mr = 11,300). The electrophoretic mobilities of rat liver SCP2 synthesized in cultured cells and in a cell-free translation system were the same as the respective forms from rat adrenal.

Trzeciak, W.H.; Simpson, E.R.; Scallen, T.J.; Vahouny, G.V.; Waterman, M.R.



Novel insights into the distribution and functional aspects of the calcium binding protein secretagogin from studies on rat brain and primary neuronal cell culture.  


Secretagogin is a calcium binding protein (CBP) highly expressed in neuroendocrine cells. It has been shown to be involved in insulin secretion from pancreatic beta cells and is a strong candidate as a biomarker for endocrine tumors, stroke, and eventually psychiatric conditions. Secretagogin has been hypothesized to exert a neuroprotective role in neurodegenerative diseases like Alzheimer's disease. The expression pattern of Secretagogin is not conserved from rodents to humans. We used brain tissue and primary neuronal cell cultures from rat to further characterize this CBP in rodents and to perform a few functional assays in vitro. Immunohistochemistry on rat brain slices revealed a high density of Secretagogin-positive cells in distinct brain regions. Secretagogin was found in the cytosol or associated with subcellular compartments. We tested primary neuronal cultures for their suitability as model systems to further investigate functional properties of Secretagogin. These cultures can easily be manipulated by treatment with drugs or by transfection with test constructs interfering with signaling cascades that might be linked to the cellular function of Secretagogin. We show that, like in pancreatic beta cells and insulinoma cell lines, also in neurons the expression level of Secretagogin is dependent on extracellular insulin and glucose. Further, we show also for rat brain neuronal tissue that Secretagogin interacts with the microtubule-associated protein Tau and that this interaction is dependent on Ca(2+). Future studies should aim to study in further detail the molecular properties and function of Secretagogin in individual neuronal cell types, in particular the subcellular localization and trafficking of this protein and a possible active secretion by neurons. PMID:22888312

Maj, Magdalena; Milenkovic, Ivan; Bauer, Jan; Berggård, Tord; Veit, Martina; Ilhan-Mutlu, Aysegül; Wagner, Ludwig; Tretter, Verena



[The study of quantitative karyotypic variability by induction of chromosomal instability in cultured cells of the Indian muntjac skin fibroblasts].  


The influence of mycoplasmal contamination and somatic cell hybridization on the character of karyotypic variability in cell cultures of Indian muntjac skin fibroblasts has been investigated. Mycoplasma arginini and Acholeplasma laidlawii, used as factors inducing chromosomal instability, do not break the main regulations peculiar to intact control. They regulations are: 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations of each chromosome from MSVK; 3) presence of significant connections between separate chromosomes by simultaneous mainly single directed numeral deviations. However, mycoplasmal contamination promotes the increase in the number of deviations in the direction of a decreasing chromosomes number. There is a breach of some connections between chromosomes by simultaneous deviations. They are chromosomes with broken connections according to the number of deviations which form telomeric associations (dicentrics). The number of these associations excel essentially intact control. The formation of new MSVK in subline M2 cells of the Indian muntjac in the process of chromosomal segregation in cell hybrid (M2 x clone of JF1 rat Jensen sarcoma) depends on the presence of significant connections between chromosomes by simultaneous numerical deviations in direction of MSVK formation. They are chromosomes that take part in the formation of new MSVK which form telomeric associations. These associations can be observed till stabilization of new MSVK. Probably, the support of the balance of karyotypic structure by factors inducing chromosomal instability is connected with change of some connections between chromosomes according to the number by simultaneous deviations as well as with the formation of dicentrics. PMID:10591121

Polianskaia, G G; Samokish, V A



Antibacterial effect of theaflavin, polyphenon 60 (Camellia sinensis) and Euphorbia hirta on Shigella spp.--a cell culture study.  


Antibacterial effect of compounds extracted from Camellia sinensis L. and the methanol extract of Euphorbia hirta L. were studied against dysentery causing Shigella spp. using the Vero cell line. Cytotoxicity studies of the extracts were performed using the cell line and the non-cytotoxic concentration of the extract was tested for antibacterial activity against the cytopathic dose of the pathogen. These extracts were found to be non-cytotoxic and effective antibacterial agents. PMID:8847884

Vijaya, K; Ananthan, S; Nalini, R



Bioprocessing technology for plant cell suspension cultures  

Microsoft Academic Search

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

Wei wen Su



Galactose-1-phosphate uridyltransferase in cultured cells.  


A method to measure the enzyme galactose-1-phosphate uridyltransferase in cultured cells is described. The optimun pH was 8.7 and no enzyme activity was found without preincubation with dithiothreitol. The KM values for Gal-1-P and UDPG for the wild type enzyme were found to be 0.2 mM and 0.08 mM, respectively. Values for the Duarte variant were found to be identical. No significant change in enzyme activity with time after subculture was found in either cultured skin fibroblasts or cultured amniotic fluid cells. Different transferase genotypes were clearly distinguished and reference range established. Transferase levels found in normal cultured amniotic fluid cells were the same as in normal cultured skin fibroblasts. The results of a prenatal diagnosis was obtained within 3 weeks of amniocentesis. PMID:11916

Monk, A M; Holton, J B



21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2010 CFR

...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....



p27 small interfering RNA induces cell death through elevating cell cycle activity in cultured cortical neurons: a proof-of-concept study.  


Recent research has demonstrated that cell cycle-associated molecules are activated in multiple forms of cell death in mature neurons, and raised a hypothesis that unscheduled cell cycle activity leads to neuronal cell death. But there is little evidence that changes in endogenous level of these molecules are causally associated with neuronal cell death. Here we transfected small interfering RNA (siRNA) targeting cyclin-dependent kinase (CDK) inhibitor p27, which plays an important role in cell cycle arrest at G1-S phase, into cultured cortical neurons. Transfection of p27 siRNA reduced neuronal viability in a time-dependent manner. p27 siRNA induced phosphorylation of retinoblastoma protein (Rb), a marker of cell cycle progression at late G1 phase. Moreover, phosphorylation of Rb and neuronal cell death provoked by p27 siRNA were abrogated by pharmacological CDK inhibitors, olomoucine and purvalanol A. Our data demonstrate that a decrease in endogenous p27 induces neuronal cell death through elevating cell cycle activity. PMID:17006629

Akashiba, H; Matsuki, N; Nishiyama, N



Verbascoside production by plant cell cultures  

Microsoft Academic Search

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g\\/l. Of the three species, suspension cultures of L.

Nobuyuki Inagaki; Hiroaki Nishimura; Minoru Okada; Hiroshi Mitsuhashi



Interactions of Cultured Human Endothelial Cells with Polymeric Surfaces.  

National Technical Information Service (NTIS)

The aim of the study was to elucidate the mechanisms involved in the 'in vitro' interactions between seeded cultured human endothelial cells (EC) and polymers with different surface properties. Adhesion, spreading, proliferation and several other function...

P. B. van Wachem



Human nasal and tracheo-bronchial respiratory epithelial cell culture.  


Human airway epithelial (hAE) cell cultures are instrumental for studying basic and applied aspects of respiratory tract biology, disease, and therapy. When primary epithelial cells from the human nasal passages or tracheo-bronchial airways are grown on porous supports at an air-liquid interface (ALI) they undergo mucociliary differentiation, reproducing both the in vivo morphology and key physiologic processes. These cultures are useful for studying basic biology, disease pathogenesis, gene therapy and aerosol administration of drugs. This chapter gives detailed protocols for tissue procurement, cell isolation, production of complex media, and cell culture initiation and maintenance needed for hAE cell ALI cultures with non-proprietary reagents. PMID:23097104

Fulcher, M Leslie; Randell, Scott H



Cultural Language Study: Grade 7.  

ERIC Educational Resources Information Center

|This course guide, the first in a two-year sequence, is designed to give students an overview of Greek and Roman culture and language from the era of the early Aegean civilizations in Greece and Asia Minor to the Augustan Age in Rome. Six units of study are concerned with the growth and development of Greece and with the metamorphosis of Rome…

Schwartz, Betty L.; Tappenden, Jacqueline W.


Plasminogen carbohydrate side chains in receptor binding and enzyme activation: A study of C6 glioma cells and primary cultures of rat hepatocytes  

SciTech Connect

The human (Glu1)-plasminogen carbohydrate isozymes, plasminogen type I (Pg 1) and plasminogen type II (Pg 2), were separated by chromatography and studied in cell binding experiments at 4{degrees}C with primary cultures of rat hepatocytes and rat C6 glioma cells. In both cell systems, Pg 1 and Pg 2 bound to an equivalent number of receptors, apparently representing the same population of surface molecules. The affinity for Pg 2 was slightly higher. With hepatocytes, the KD for Pg 1 was 3.2 +/- 0.2 microM, and the KD for Pg 2 was 1.9 +/- 0.1 microM, as determined from Scatchard transformations of the binding isotherms. The Bmax was approximately the same for both isozymes. With C6 cells, the KD for Pg 1 was 2.2 +/- 0.1 microM vs. 1.5 +/- 0.2 microM for Pg 2. Again, the Bmax was similar with both isozymes. 125I-Pg 1 and 125I-Pg 2 were displaced from specific binding sites by either nonradiolabeled isozyme. The KI for Pg 2 was slightly lower than the KI for Pg 1 with hepatocytes (0.9 vs. 1.3 microM) and with C6 cells (0.6 vs. 1.1 microM). No displacement was detected with miniplasminogen at concentrations up to 5.0 microM. Activation of Pg 1 and Pg 2 by recombinant two-chain tissue-plasminogen activator (rt-PA) was enhanced by hepatocyte cultures. The enhancing effect was greater with Pg 2. Hepatocyte cultures did not affect the activation of miniplasminogen by rt-PA or the activation of plasminogen by streptokinase. Unlike the hepatocytes, C6 cells did not enhance the activation of plasminogen by rt-PA or streptokinase; however, plasmin generated in the presence of C6 cells reacted less readily with alpha 2-antiplasmin.

Hall, S.W.; VandenBerg, S.R.; Gonias, S.L. (Univ. of Virginia Health Sciences Center, Charlottesville (USA))



Quantification of Cells in Culture  

Microsoft Academic Search

\\u000a Cell enumeration using the hemocytometer is applicable when determining the number of cells in a suspension, and when the\\u000a number of samples to be analyzed is relatively small. Hemocytometry is also useful for determining the proportion of singly\\u000a dispersed cells in a suspension, and for estimating the frequency of viable cells.

Arleen Richardson; Sergey Fedoroff


Alpha-naphthylisothiocyanate cytotoxicity in hepatocytes and other cultured cells  

SciTech Connect

Alpha-naphthylisothiocyanate (ANIT) is a model hepatotoxicant that causes injury to liver parenchymal and bile ductular cells in vivo. In this study, toxicity to various cells in culture was evaluated. In short term cultures of rat hepatocytes (HCs), a 4hr exposure to ANIT caused a concentration dependent increase in cytotoxicity as measured by lactate dehydrogenase (LDH) release. HCs cultured for 24hr or longer demonstrated a delay in ANIT-induced LDH release when compared to 2.5hr cultures. In addition, the magnitude of the cytotoxic response was greater in longer term cultures. The threshold for ANIT-induced cytotoxicity in HCs was between 20 and 63uM. In porcine endothelial cell cultures, ANIT cytotoxicity was similar to that seen in HCs. In two transformed cells lines, the Swiss 3T3 fibroblast and WB cell, a 24hr exposure to ANTI caused a concentration dependent increase in LDH release. Like the HCs, the threshold concentration was between 20 and 63uM. These results indicate that ANIT is directly cytotoxic to various cells in culture. Since endothelium and fibroblasts are deficient in cytochrome P-450 mixed function oxidase activity, ANIT toxicity in culture may be largely independent of this xenobiotic metabolizing system.

Bailie, M.B.; Roth, R.A. (Michigan State Univ., East Lansing (United States))



Stimulation of menthol production in Mentha piperita cell culture  

Microsoft Academic Search

Plant cell culture provides an alternative means for producing secondary metabolites. In this study, experiments were carried\\u000a out to study the impact of several parameters, independently and in combination, on the stimulation of menthol production\\u000a in the cell suspension culture of Mentha piperita. Callus was obtained from leaf segments of in vitro grown plantlets on Murashige and Skoog (MS) medium

Amrita Chakraborty; Sharmila Chattopadhyay



Culture and Manipulation of Embryonic Cells  

PubMed Central

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.

Edgar, Lois G.; Goldstein, Bob



Integrated Bioprocessing for Plant Cell Cultures  

Microsoft Academic Search

Plant cell suspension culture has become the focus of much attention as a tool for the production of secondary metabolites\\u000a including paclitaxel, a well-known anticancer agent. Recently, it has also been regarded as one of the host systems for the\\u000a production of recombinant proteins. In order to produce phytochemicals using plant cell cultures, efficient processes must\\u000a be developed with adequate

Jeong-Woo Choi; Gyu Heon Cho; S ang Yo Byun; Dong-Il Kim


Oscillatory behavior of cells in tissue culture.  

NASA Astrophysics Data System (ADS)

Fibroblasts and epithelial cells organize themselves in distinct patterns in tissue culture which indicates that neighboring cells communicate. A striking example of such communication is the oscillatory behavior of Madin-Darby canine kidney (MDCK) cells reported here. These oscillations were discovered using a biosensor referred to as ECIS (Electric Cell-substrate Impedance Sensing). In this measurement cells are seeded out on a small electrode deposited at the bottom of a tissue culture well and immersed in ordinary culture medium. By measuring the changes in the impedance of the electrode as a function of time, many important properties of the cells on the electrode can be inferred, such as motion, morphology changes and membrane capacitance. The impedance oscillations of MDCK cells were observed with highly confluent cell layers, where the approximately 100 cells on the electrode acted in unison. The communication between cells can be demonstrated directly by a variation of the ECIS concept, where cells are cultured on two closely spaced electrodes. The impedance fluctuations are measured independently on each electrode and compared by using a cross-correlation function.

Giaever, Ivar; Linton, Michael F. A.; Keese, Charles R.



In vitro Assay for Dermatotoxicity Using Cultured Human Epidermal Cells.  

National Technical Information Service (NTIS)

Since cultures of human epidermal cells may serve as a model for the living part of the epidermis, the suitability of this culture system in assessing skin irritancy has been studied. Five chemicals with well known dermatotoxic actions have been examined ...

M. A. E. Mol O. L. Wolthuis



Effect of culture conditions on endothelial cell growth and responsiveness  

Microsoft Academic Search

The in vitro culture of endothelial cells (EC) is dependent on the presence of a coated surface and the availability of growth factors in the medium. The aim of the present research is to investigate whether in vitro EC culture conditions, such as serum source and surface coating, determine the growth characteristics of EC. The phenotype of EC was studied

Ingrid A. M. Relou; Cora A. Damen; Gerard Groenewegen; Arjan W. Griffioen



Cell culture systems for hepatitis C virus.  


Due to the obligatory intracellular lifestyle of viruses, cell culture systems for efficient viral propagation are crucial to obtain a detailed understanding of the virus-host cell interaction. For hepatitis C virus (HCV) the development of permissive and authentic culture models continues to be a challenging task. The first efforts to culture HCV had limited success and range back to before the virus was molecularly cloned in 1989. Since then several major breakthroughs have gradually overcome limitations in culturing the virus and sequentially permitted analysis of viral RNA replication, cell entry, and ultimately the complete replication cycle in cultured cells in 2005. Until today, basic and applied HCV research greatly benefit from these tremendous efforts which spurred multiple complementary cell-based model systems for distinct steps of the HCV replication cycle. When used in combination they now permit deep insights into the fascinating biology of HCV and its interplay with the host cell. In fact, drug development has been much facilitated and our understanding of the molecular determinants of HCV replication has grown in parallel to these advances. Building on this groundwork and further refining our cellular models to better mimic the architecture, polarization and differentiation of natural hepatocytes should reveal novel unique aspects of HCV replication. Ultimately, models to culture primary HCV isolates across all genotypes may teach us important new lessons about viral functional adaptations that have evolved in exchange with its human host and that may explain the variable natural course of hepatitis C. PMID:23463196

Steinmann, Eike; Pietschmann, Thomas



Endogenous amyloidogenesis in long-term rat hippocampal cell cultures  

PubMed Central

Background Long-term primary neuronal cultures are a useful tool for the investigation of biochemical processes associated with neuronal senescence. Improvements in available technology make it possible to observe maturation of neural cells isolated from different regions of the rodent brain over a prolonged period in vitro. Existing experimental evidence suggests that cellular aging occurs in mature, long-term, primary neuronal cell cultures. However, detailed studies of neuronal development in vitro are needed to demonstrate the validity of long-term cell culture-based models for investigation of the biochemical mechanisms of in vitro neuronal development and senescence. Results In the current study, neuron-enriched hippocampal cell cultures were used to analyze the differentiation and degeneration of hippocampal neurons over a two month time period. The expression of different neuronal and astroglial biomarkers was used to determine the cytochemical characteristics of hippocampal cells in long-term cultures of varying ages. It was observed that the expression of the intermediate filament nestin was absent from cultures older than 21 days in vitro (DIV), and the expression of neuronal or astrocytic markers appeared to replace nestin. Additionally, morphological evaluations of neuronal integrity and Hoescht staining were used to assess the cellular conditions in the process of hippocampal culture development and aging. It was found that there was an increase in endogenous production of A?1-42 and an increase in the accumulation of Congo Red-binding amyloidal aggregates associated with the aging of neurons in primary culture. In vitro changes in the morphology of co-existing astrocytes and cell culture age-dependent degeneration of neurodendritic network resemble features of in vivo brain aging at the cellular level. Conclusion In conclusion, this study suggests that long-term primary CNS culture is a viable model for the study of basic mechanisms and effective methods to decelerate the process of neuronal senescence.



Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: A preliminary study.  


This study examined the potential for osteogenesis via regenerative medicine using autologous tissues (umbilical cord (UC) and umbilical cord blood (UCB)) in nude mice. The study was designed to provide the three elements required for regenerative medicine (cell, scaffold, and growth factor) and autoserum for culture by means of autologous tissues. Mesenchymal stromal cells were obtained from UC (UC-MSCs). Fibrin, platelet-rich-plasma, and autoserum were obtained from UCB as scaffold, growth factor and serum for culture respectively. UC-MSCs were obtained from Wharton jelly and cultured with UCB-derived fibrin (UCB-fibrin) for 3-4 weeks to induce their differentiation into osteoblasts. They were implanted subcutaneously into the dorsum of male nude mice for 6 weeks prior to undergoing assessment. The assessments performed were haematoxylin and eosin, and alizarin red staining, immunohistochemical staining of human mitochondria, scanning electron microscopy, scanning electron microscopy with energy dispersive X-ray spectrometry and real-time reverse transcriptase-polymerase chain reaction to assess the expressions of osteoblast markers. Consequently, the differentiation of UC-MSCs into osteoblasts and the production of hydroxyapatite were verified. This study suggested the possible formation of bone tissue using biomedical materials obtained from UC and UCB. PMID:23465638

Baba, Kyoko; Yamazaki, Yasuharu; Ishiguro, Masashi; Kumazawa, Kenichi; Aoyagi, Kazuya; Ikemoto, Shigehiro; Takeda, Akira; Uchinuma, Eiju



Growth of melanocytes in human epidermal cell cultures  

SciTech Connect

Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

Staiano-Coico, L.; Hefton, J.M.; Amadeo, C.; Pagan-Charry, I.; Madden, M.R.; Cardon-Cardo, C. (Cornell Medical College, New York, NY (USA))



A quantum-dot nanocrystal study of GABAA receptor subunits in living cerebellar granule cells in culture.  


Quantum dots (QDs) are semiconductor nanocrystals emerging as a new class of fluorescent labels with large brightness, multi color fluorescence emission and resistance against photobleaching. Here we have used QDs as biological markers in an immunofluorescence approach. In this work GABA(A )receptors of rat cerebellar granule cells have been studied and in particular we have visualized the beta(2/3) and delta subunits in live cells. The results obtained were compared to those gathered with conventional probes. The images of the delta subunit in living cells appear to correspond to those expected for a subunit part of GABA(A )receptors mediating tonic inhibition in the granules cell bodies. PMID:17401675

Siano, Silvia; Cupello, Aroldo; Pellistri, Francesca; Robello, Mauro



Microtechnology for Stem Cell Culture  

Microsoft Academic Search

\\u000a Advances in stem cell research in recent decades have been aided by progress in the development of novel technologies aimed\\u000a at biological systems. At the same time mimicking stem cell niches in vitro has become crucial for both basic stem cell research\\u000a and the development of innovative therapies based on stem cells. Innovative microscale technologies can contribute to our\\u000a quantitative

Elena Serena; Elisa Cimetta; Camilla Luni; Nicola Elvassore


Immunocytochemical characterisation of cultures of human bladder mucosal cells  

Microsoft Academic Search

Background  The functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two\\u000a significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts.\\u000a The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical\\u000a characterisation.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Primary cell cultures were

Jacqueline R Woodman; Kylie J Mansfield; Vittoria A Lazzaro; William Lynch; Elizabeth Burcher; Kate H Moore



Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation  

Microsoft Academic Search

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation.

Ranjith Pathirana; Jocelyn R. Eason



Increased mechanosensitivity of cells cultured on nanotopographies  

PubMed Central

Enhancing cellular mechanosensitivity is recognized as a novel tool for successful musculoskeletal tissue engineering. We examined the hypothesis that mechanosensitivity of human mesenchymal stem cells (hMSCs) is enhanced on nanotopographic substrates relative to flat surfaces. hMSCs were cultured on polymer-demixed, randomly distributed nanoisland surfaces with varying island heights and changes in intracellular calcium concentration, [Ca2+]i, in response to fluid flow induced shear stress were quantifide. Stem cells cultured on specific scale nanotopographies displayed greater intracellular calcium responses to fluid flow. hMSCs cultured on 10-20 nm high nanoislands displayed a greater percentage of cells responding in calcium relative to cells cultured on flat control, and showed greater average [Ca2+]i increase relative to cells cultured on other nanoislands (45-80 nm high nanoislands). As [Ca2+]i is an important regulator of downstream signaling, as well as proliferation and differentiation of hMSCs, this observation suggests that specific scale nanotopographies provide an optimal milieu for promoting stem cell mechanotransduction activity. That mechanical signals and substrate nanotopography may synergistically regulate cell behavior is of significant interest in the development of regenerative medicine protocols.

Salvi, Joshua D.; Lim, Jung Yul; Donahue, Henry J.



Cell cultures from the symbiotic soft coral Sinularia flexibilis.  


The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of cultivation of S. flexibilis cells on different media and cell dissociation methodologies. Mechanical dissociation of coral tissue always yielded the highest number of cells and allowed subsequent cellular growth in all treatments. The best results from chemical dissociation reagents were found with trypsin-ethylene diamine tetraacetic acid. Coral cells obtained from spontaneous dissociation did not grow. Light intensity was found to be important for coral cell culture showing an enduring symbiosis between the cultured cells and their intracellular algae. The Grace's insect medium and Grace's modified insect medium were found to be superior substrates. To confirm the similarity of the cultured cells and those in the coral tissue, a molecular test with Internal Transcribed Spacer primers was performed. Thereby, the presence of similar cells of both the coral cells and zooxanthella in different culture media was confirmed. PMID:18661193

Khalesi, Mohammad K; Vera-Jiménez, N I; Aanen, D K; Beeftink, H H; Wijffels, R H



Cell Cultures and Retroviral Particles From a Tumor of a Moray Eel  

Microsoft Academic Search

Until recently, fish cell culture primarily has been useful only in the propagation and study of epidemic viruses significant to the fishing industry. Such fish cell lines derived were developed by appropriating classical techniques of mammalian cell culture, with serum as the major growth supplement. Using an approach in which culture medium is formulated in a cell-type-specific manner with minimal

Charles Buck; Charles Walsh; Raymond Davis; Araz Toumadje; Kenichi Kusamoto; Angela Helmrich; Christine Chapline; Patricia Mericko; David Barnes



Adult liver parenchymal cells in primary culture: Characteristics and cell recognition standards  

Microsoft Academic Search

Conclusions  Primary cultures provide one method for studying liver biochemistry in vitro. The short term incubations of isolated cells\\u000a employ homogenous cell populations, incubated in completely defined media. These cells are capable of exhibiting several of\\u000a the in vivo functions. The long term cultures derived from adult liver parenchymal cells offer different advantages. For example,\\u000a these cells can be cloned, isolated,

Robert J. Bonney



Control of fibronectin synthesis by rat granulosa cells in culture  

SciTech Connect

The secreted and cellular (/sup 35/S)methionine-radiolabeled proteins of cultured rat granulosa cells were separated by electrophoresis on sodium dodecylsulfate (SDS) polyacrylamide gradient slab gels. From 24 to 72 h of culture FSH increased the intensity of labeling of most of the secreted proteins. A 220,000-dalton protein, however, increased in intensity only in control cultures and became the major secreted protein after 72 h, comprising 20% of the total radiolabeled proteins. This protein was identified as fibronectin by immunoprecipitation. There was no increase in the secreted or cellular fibronectin in FSH- or testosterone- and insulin-treated cultures. These studies indicate that a component of extracellular matrix is a major secretory product of unstimulated immature granulosa cells. As hormones induce the differentiated functions of granulosa cells in culture, the secretion of fibronectin is inhibited.

Skinner, M.K.; Dorrington, J.H.



Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study.  


Fluorescein-labeled heavy meromyosin subfragment-1 (F-S-1) has been purified by ion exchange chromatography and characterized in terms of its ability to bind specifically to actin. F-S-1 activates the Mg++-adenosine triphosphatase activity of rabbit skeletal muscle actin and decorates actin as shown by negative stains and thin sections of rabbit actin and rat embryo cell microfilament bundles, respectively. Binding of F-S-1 to cellular structures is prevented by pyrophosphate and by competition with excess unlabeled S-1. The F-S-1 is used in light microscope studies to determine the distribution of actin-containing structures in wnterphase and mitotic rat embryo and rat kangaroo cells. Interphase cells display the familiar pattern of fluorescent stress fibers. Chromosome-to-pole fibers are fluorescent in mitotic cells. The glycerol extraction procedures employed provide an opportunity to examine cells prepared in an identical manner by light and electron microscopy. The latter technique reveals that actin-like microfilaments are identifiable in spindles of glycerinated cells before and after addition of S-1 or HMM. In some cases, microfilaments appear to be closely associated with spindle microtubles. Comparison of the light and electron microscope results aids in the evaluation of the fluorescent myosin fragment technique and provides further evidence for possible structural and functional roles of actin in the mitotic apparatus. PMID:71303

Schloss, J A; Milsted, A; Goldman, R D



What Is Video Game Culture? Cultural Studies and Game Studies  

Microsoft Academic Search

What is video game culture, however? What does it mean to have a culture defined by the consumption of a particular medium? Moreover, what are the implications of defining this culture in a particular way? While there has been a great deal of ink split on video game culture, the actual definition of the term is often treated as common

Adrienne Shaw



Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture  

Microsoft Academic Search

\\u000a Different types of bioreactors are used at Nestlé R&D Centre – Tours for mass propagation of selected plant varieties by somatic\\u000a embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have\\u000a been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative\\u000a propagation of

Jean-Paul Ducos; Bénédicte Terrier; Didier Courtois



Genome selection within cell cultures of potato and tobacco  

Microsoft Academic Search

The ploidy level of callus cultures, suspensions and cell cultures derived from single cells and protoplasts of Solanum tuberosum\\u000a L. and Nicotania tabascum L. was analysed with the aim of studying selection processes. Genome selection was tested using\\u000a chromosome number. Subculture of callus lines mostly resulted in an increase of cytogenetic destabilization, whereas subculture\\u000a of suspensions led to an increase

Günter Wersuhn; Ursula Dathe



Challenges in Using Cultured Primary Rodent Hepatocytes or Cell Lines to Study Hepatic HDL Receptor SR-BI Regulation by Its Cytoplasmic Adaptor PDZK1  

PubMed Central

Background PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Methodology/Principal Findings Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Conclusions/Significance Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

Tsukamoto, Kosuke; Buck, Lorenna; Inman, Walker; Griffith, Linda; Kocher, Olivier; Krieger, Monty



Transferring isolated mitochondria into tissue culture cells  

PubMed Central

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.

Yang, Yi-Wei; Koob, Michael D.



[Cell culture of human gingival epithelium].  


Gingival explants were taken from 8 volunteers in order to find a method to grow gingival epithelial cells. We succeeded in what we regarded as optimal conditions in achieving a monolayer cell thickness in the culture by means of BHK medium and foetal calf serum. PMID:271582

Heidemann, D; Lampert, F



Microfluidics cell culture with sensing and SqueezeFluidics  

Microsoft Academic Search

Many biological studies, drug screening methods, and cellular therapies require culture and manipulation of living cells outside of their natural environment in the body. The gap between the cellular microenvironment in vivo and in vitro, however, poses challenges for obtaining physiologically relevant responses from cells used in basic biological studies or drug screens and for drawing out the maximum functional

Shuichi Takayama



Optically micropatterned culture of adherent cells  

NASA Astrophysics Data System (ADS)

We used a liquid-crystal spatial light modulator to project 473 nm light patterns surrounding a region of adherent cells and achieved an arbitrarily micropatterned cell culture. For a group of ~60 cells, the cell boundaries fit the pattern of light within 15% deviation of the side length. We also demonstrated a wound-healing experiment with a definite starting temporal point by using this technique. While observing mitochondrial structures in the illuminated cells, we found that the 473 nm light damaged the integrity of mitochondria and thus prohibited cell proliferation in the illuminated region.

Xiao, Jian-Long; Pan, Huei-Jyuan; Lee, Chau-Hwang



A comparative study of aggrecan synthesis between natural articular chondrocytes and differentiated chondrocytes from adipose derived stem cells in 3D culture  

PubMed Central

Introduction: The main obstacle for tissue engineering is to find the most appropriate cell which is able to produce extracellular matrix (ECM) similar or better than natural chondrocytes in vitro. This study compared aggrecan synthesis's potential between differentiated chondrocytes (DCs) from adipose-derived stem cells (ADSCs) and natural articular chondrocytes (NCs) in 3D culture in vitro. Materials and Methods: Human ADSCs were isolated from sub-cutaneous adipose tissue and then the surface markers including CD 14, 45 CD105, CD90, CD44 were analyzed by flow cytometry. Also human articular chondrocytes were yielded of non-weight bearing area of Knee cartilage. Both types of the cells were encapsulated in alginate scaffolds and cultured in chondrogenic medium with and without TGF?3 for 3 weeks. Then the extent of aggercan (AGC) production was evaluated by ELISA on days 14 and 21. Results: Our findings indicated that differentiated chondrocytes (DCs) with and without TGF?3 synthesized more AGC than natural chondrocytes (NCs) on day 14. But DCs without TGF?3 had higher production than other groups on day 21. Application of TGF?3 resulted in an increase of amount of AGC in DCs on day 14 but a decrease on day 21 than same group. Conclusion: Since, aggrecan is an important chondrogenic marker, it was concluded that ADSCs can be possible reliable alternative cell source for cartilage tissue engineering in future.

Ansar, Malek Masoud; Esfandiariy, Ebrahim; Mardani, Mohmmad; Hashemibeni, Batool; Zarkesh-Esfahani, Sayeed Hamid; Hatef, Masoud; Kabiri, Azadeh



Biosynthesis of highly enriched 13C-lycopene for human metabolic studies using repeated batch tomato cell culturing with 13C-glucose.  


While putative disease-preventing lycopene metabolites are found in both tomato (Solanum lycopersicum) products and in their consumers, mammalian lycopene metabolism is poorly understood. Advances in tomato cell culturing techniques offer an economical tool for generation of highly-enriched (13)C-lycopene for human bioavailability and metabolism studies. To enhance the (13)C-enrichment and yields of labelled lycopene from the hp-1 tomato cell line, cultures were first grown in (13)C-glucose media for three serial batches and produced increasing proportions of uniformly labelled lycopene (14.3±1.2%, 39.6±0.5%, and 48.9±1.5%) with consistent yields (from 5.8 to 9 mg/L). An optimised 9-day-long (13)C-loading and 18-day-long labelling strategy developed based on glucose utilisation and lycopene yields, yielded (13)C-lycopene with 93% (13)C isotopic purity, and 55% of isotopomers were uniformly labelled. Furthermore, an optimised acetone and hexane extraction led to a fourfold increase in lycopene recovery from cultures compared to a standard extraction. PMID:23561155

Moran, Nancy Engelmann; Rogers, Randy B; Lu, Chi-Hua; Conlon, Lauren E; Lila, Mary Ann; Clinton, Steven K; Erdman, John W



Studies on oxygen and volume restrictions in cultured cardiac cells. I. A model for ischemia and anoxia with a new approach.  


A novel incubation unit is described that is highly suitable for thorough studies of oxygen deprivation states. Its application with cultured heart cells is experimentally demonstrated. The release of enzymes, taken as a marker for cell damage, has clearly shown that restriction of the volume of extracellular medium combined with oxygen plus glucose deprivation caused greatest cellular damage. It may be considered as an experimental ischemia-like state. Furthermore, the onset of cellular damage followed a time table very much like that occurring in vivo under similar conditions, more so than any other previously described studies. A time lag between the release of cytoplasmic enzymes and lysosomal enzymes and other observations made in the present study suggests a sequential order of events in which the release of cytoplasmic enzymes occurs at a stage of reversible damage due to oxygen deprivation, whereas the release of lysosomal enzymes may point at irreparable damage. PMID:4044471

Vemuri, R; Yagev, S; Heller, M; Pinson, A



Immunodissection and culture of rabbit cortical collecting tubule cells  

SciTech Connect

A mouse monoclonal antibody designated IgG3 (rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG3 (rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10W rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10X RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of approx.32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D2, or prostaglandin I, and 2) to release PGE2 in response to bradykinin but not arginine vasopressin or isoproterenol. The results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia.

Spielman, W.S.; Sonnenburg, W.K.; Allen, M.L.; Arend, L.J.; Gerozissis, K.; Smith, W.L.



The consensus mechanics of cultured mammalian cells  

PubMed Central

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.

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



Human Primary Lung Endothelial Cells in Culture  

PubMed Central

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.

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



Comparative evaluation of maintenance of cell viability of an experimental transport media "coconut water" with Hank's balanced salt solution and milk, for transportation of an avulsed tooth: An in vitro cell culture study  

PubMed Central

The purpose of this study was to evaluate the efficiency of a new storage medium, coconut water, in comparison with other traditional storage media like Hank's balanced salt solution (HBBS) and milk, in maintaining the viability of an established cell line BHK-21/C13 (baby hamster kidney fibroblasts) using the direct suspension cell culture technique. The storage media tested in the study were divided into three major groups and two control groups - Group A: HBBS, Group B: milk, and Group C: coconut water. The positive and negative controls corresponded to 0-minute and 24-hour dry times respectively. The three groups were then divided into five subgroups, each denoting the storage time periods 15 min, 30 min, 45 min, 60 min and 120 min respectively. The cell line BHK-21/C13 was subcultured and the number of cells was standardized by making a cell suspension using Minimal Essential Medium in five culture plates. One ml of each experimental group (HBBS, milk and coconut water) was added to eight wells of each culture plate. The culture plates containing the cells and the experimental groups were incubated for the respective time periods. The cells were then counted with a Neubauer counting chamber, under light microscope. The results were statistically analyzed using One-way ANOVA and Multiple Range Test using the Tukey-HSD procedure to identify the significant groups at p ? 0.05. Within the parameters of this study, it appears that coconut water may be a better alternative to HBSS or milk, in terms of maintaining cell viability. Coconut water can be used as a superior transport medium for avulsed teeth.

Thomas, Toby; Gopikrishna, Velayutham; Kandaswamy, Deivanayagam



Comparative evaluation of maintenance of cell viability of an experimental transport media "coconut water" with Hank's balanced salt solution and milk, for transportation of an avulsed tooth: An in vitro cell culture study.  


The purpose of this study was to evaluate the efficiency of a new storage medium, coconut water, in comparison with other traditional storage media like Hank's balanced salt solution (HBBS) and milk, in maintaining the viability of an established cell line BHK-21/C13 (baby hamster kidney fibroblasts) using the direct suspension cell culture technique.The storage media tested in the study were divided into three major groups and two control groups - Group A: HBBS, Group B: milk, and Group C: coconut water. The positive and negative controls corresponded to 0-minute and 24-hour dry times respectively.The three groups were then divided into five subgroups, each denoting the storage time periods 15 min, 30 min, 45 min, 60 min and 120 min respectively. The cell line BHK-21/C13 was subcultured and the number of cells was standardized by making a cell suspension using Minimal Essential Medium in five culture plates.One ml of each experimental group (HBBS, milk and coconut water) was added to eight wells of each culture plate. The culture plates containing the cells and the experimental groups were incubated for the respective time periods. The cells were then counted with a Neubauer counting chamber, under light microscope. The results were statistically analyzed using One-way ANOVA and Multiple Range Test using the Tukey-HSD procedure to identify the significant groups at p study, it appears that coconut water may be a better alternative to HBSS or milk, in terms of maintaining cell viability. Coconut water can be used as a superior transport medium for avulsed teeth. PMID:20142880

Thomas, Toby; Gopikrishna, Velayutham; Kandaswamy, Deivanayagam



Cell Culture Imaging Using Microimpedance Tomography  

Microsoft Academic Search

We present a novel, inexpensive, and fast microimpedance tomography system for two-dimensional imaging of cell and tissue cultures. The system is based on four-electrode measurements using 16 planar microelectrodes (5 mum x 4 mm) integrated into a culture chamber. An Agilent 4294A impedance analyzer combined with a front-end amplifier is used for the impedance measurements. Two-dimensional images are obtained using

Pontus Linderholm; Laurent Marescot; Meng Heng Loke; Philippe Renaud



The impact of cell culture equipment on energy loss.  


Light energy of discrete wavelengths supplied via lasers and broadband intense pulsed light have been used therapeutically for many years. In vitro models complement clinical studies, especially for the elucidation of underlying mechanisms of action. Clarification that light energy reaches the cells is necessary when developing protocols for the treatment of cells using in vitro models. Few studies report on energy loss in cell culture equipment. The ability of energy from light with therapeutic potential to reach cells in culture needs to be determined; this includes determining the proportion of light energy lost within standard cell culture media and cell culture vessels. The energy absorption of cell culture media, with/without the pH indicator dye phenol red, and the loss of energy within different plastics and glassware used typically for in vitro cell culture were investigated using intense pulsed light and a yellow pulsed dye laser. Media containing phenol red have a distinctive absorption peak (560 nm) absent in phenol red-free media and restored by the addition of phenol red. For both light sources, energy loss was lowest in standard polystyrene tissue culture flasks or multi-well plates and highest in polypropylene vessels or glass tubes. The effects of phenol red-free media on the absorption of energy varied with the light source used. Phenol red-free media are the media of choice; polystyrene vessels with flat surfaces such as culture flasks or multi-well plates should be used in preference to polypropylene or glass vessels. PMID:23568625

Davies, Lleucu B; Kiernan, Michael N; Bishop, Joanna C; Thornton, Catherine A; Morgan, Gareth



Canine coronavirus induces apoptosis in cultured cells  

Microsoft Academic Search

Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups.In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown.We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in

A. Ruggieri; L. Di Trani; I. Gatto; M. Franco; E. Vignolo; B. Bedini; G. Elia; C. Buonavoglia



Integration of porosity and bio-functionalization to form a 3D scaffold: cell culture studies and in vitro degradation.  


In this study, porous poly(lactide-co-glycolide) (PLGA) (50/50) microspheres have been fabricated by the gas-foaming technique using ammonium bicarbonate as a gas-foaming agent. Microspheres of different porosities have been formulated by varying the concentration of the gas-foaming agent (0%, 5%, 10% and 15% w/v). These microspheres were characterized for particle size, porosity and average pore size, morphology, water uptake ratio and surface area and it was found that the porosity, pore size and surface area increased on increasing the concentration of the gas-foaming agent. Further, the effect of porosity on degradation behavior was evaluated over a 12 week period by measuring changes in mass, pH, molecular weight and morphology. Porosity was found to have an inverse relationship with degradation rate. To render the surface of the microspheres biomimetic, peptide P-15 was coupled to the surface of these microspheres. In vitro cell viability, proliferation and morphological evaluation were carried out on these microsphere scaffolds using MG-63 cell line to study the effect of the porosity and pore size of scaffolds and to evaluate the effect of P-15 on cell growth on porous scaffolds. MTT assay, actin, alizarin staining and SEM revealed the potential of biomimetic porous PLGA (50/50) microspheres as scaffolds for tissue engineering. As shown in graphical representation, an attempt has been made to correlate the cell behavior on the scaffolds (growth, proliferation and cell death) with the concurrent degradation of the porous microsphere scaffold as a function of time. PMID:20539055

Mittal, Anupama; Negi, Poonam; Garkhal, Kalpna; Verma, Shalini; Kumar, Neeraj



Henrietta Lacks, HeLa cells, and cell culture contamination.  


Henrietta Lacks died in 1951 of an aggressive adenocarcinoma of the cervix. A tissue biopsy obtained for diagnostic evaluation yielded additional tissue for Dr George O. Gey's tissue culture laboratory at Johns Hopkins (Baltimore, Maryland). The cancer cells, now called HeLa cells, grew rapidly in cell culture and became the first human cell line. HeLa cells were used by researchers around the world. However, 20 years after Henrietta Lacks' death, mounting evidence suggested that HeLa cells contaminated and overgrew other cell lines. Cultures, supposedly of tissues such as breast cancer or mouse, proved to be HeLa cells. We describe the history behind the development of HeLa cells, including the first published description of Ms Lacks' autopsy, and the cell culture contamination that resulted. The debate over cell culture contamination began in the 1970s and was not harmonious. Ultimately, the problem was not resolved and it continues today. Finally, we discuss the philosophical implications of the immortal HeLa cell line. PMID:19722756

Lucey, Brendan P; Nelson-Rees, Walter A; Hutchins, Grover M



A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses  

Microsoft Academic Search

Neurons in primary cell cultures provide important experimental possibilities complementing or substituting those in the nervous system. However, Drosophila primary cell cultures have unfortunate limitations: they lack either a range of naturally occurring cell types, or of mature physiological properties. Here, we demonstrate a strategy which supports both aspects integrated in one culture: Initial culturing in conventional serum-supplemented Schneider's medium

Barbara Küppers-Munther; Johannes J. Letzkus; Karin Lüer; Gerhard Technau; Hartmut Schmidt; Andreas Prokop



Proliferation and differentiation of organoid hair follicle cells co-cultured with fat cells in collagen gel matrix culture.  


Using rat skin, we studied the influence of fat cells on the proliferation and differentiation of organoid hair follicle cells in a three-dimensional collagen gel matrix culture system. We cultured organoid hair follicles embedded in collagen gel under each of the following three conditions: cell-free collagen gel for control experiments (condition 1); co-culture with fat cells in close apposition (condition 2); and co-culture with fat cells in spatial separation (condition 3). Outgrowths of epithelial cells from the organoid hair follicles associated with perifollicular proliferation of fibroblasts were observed under conditions 1 and 3. Under condition 2, proliferation of both organoid hair follicle cells and fibroblasts was inhibited, but differentiation of the hair follicle cells appeared to be accelerated. Fat cells are considered to have an inhibitory effect on the proliferation of perifollicular fibroblasts, which might have resulted in the inhibition of hair follicle cell proliferation and also in the better maintenance of normal follicular structure and integrity, allowing for hair-type differentiation to proceed. A direct accelerating effect of fat cells on hair follicle differentiation may also have been responsible. In a physiological state (co-culture with keratinocytes on the collagen gel), similar results were observed under conditions 1 and 2. The different findings under conditions 2 and 3 may be due to either of two possibilities: either the concentration gradient of the soluble factors released from fat cells, acting on either the hair follicle cells or the perifollicular fibroblasts as an inhibitor of proliferation, caused the difference in the results, or direct contact between the organoid hair follicle cells and fat cells may have influenced the accelerating effect of fat cells on the differentiation of hair follicle cells. PMID:9764147

Misago, N; Toda, S; Sugihara, H; Kohda, H; Narisawa, Y



Cultural Studies in Indian Education. Position Paper.  

ERIC Educational Resources Information Center

A broad interpretation of cultural studies is used in this position paper. The need for insight and appreciation of cultural diversity between American Indians and non-Indians is described by cultural pluralism and its application in Indian education, and response to cultural pluralism--programs and activities in the Indian community. The…

Warren, Dave


Mutagenesis studies on cultured mammalian cells. The sensitivity of the asparagine-requiring phenotype to several chemical agents.  


The effect of several chemical agents on the mutation frequency from asparagine dependence to asparagine independence has been studied in Jensen sarcoma cells. It was found that ethylmethanesulfonate brought about a dramatic exponential increase, while nitrosoguanidine was not lighly effective as a mutagen, causing only a modest increase in mutation frequency, and quinacrine HCl was ineffective. The results presented here are compared with those obtained in other systems and with our previous work on the effects of UV on mutation induction in the asparagine system. They suggest that the basis of the asparagine requirement of mammalian cell lines resides in a specific genetic alteration in nuclear DNA which is corrected by the mutagenic action of the agents tested here. PMID:177865

Morrow, J; Prickett, M S; Fritz, S; Vernick, D; Deen, D



Tumor necrosis factor (cachetin) decreases adipose cell differentiation in primary cell culture  

Microsoft Academic Search

Cachetin has been shown to effect gene product expression in the established adipose cell line 3T3-L1. Expression of messenger RNA for lipoprotein lipase is suppressed in cultured adipocytes. The purpose of this study was to determine the effect of Cachetin on adipose cell differentiation in primary cell culture. Stromalvascular cells obtained from the inguinal fat pad of 4-5 week old

R. J. Martin; D. D. Jones; D. E. Jewell; G. J. Hausman



Production of Zebrafish Offspring from Cultured Female Germline Stem Cells  

PubMed Central

Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome.

Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul



Comparative SAXS and DSC study on stratum corneum structural organization in an epidermal cell culture model (ROC): Impact of cultivation time.  


Cell cultured skin equivalents present an alternative for dermatological in vitro evaluations of drugs and excipients as they provide the advantage of availability, lower variability and higher assay robustness compared to native skin. For penetration/permeation studies, an adequate stratum corneum barrier similar to that of human stratum corneum is, however, a prerequisite. In this study, the stratum corneum lipid organization in an epidermal cell culture model based on rat epidermal keratinocytes (REK organotypic culture, ROC) was investigated by small-angle X-ray scattering (SAXS) in dependence on ROC cultivation time and in comparison to native human and rat stratum cornea. In addition, the thermal phase behavior was studied by differential scanning calorimetry (DSC) and barrier properties were checked by measurements of the permeability of tritiated water. The development of the barrier of ROC SC obtained at different cultivation times (7, 14 and 21days at the air-liquid interface) was connected with an increase in structural order of the SC lipids in SAXS measurements: Already cultivation for 14days at the air-liquid interface resulted overall in a competent SC permeability barrier and SC lipid organization. Cultivation for 21days resulted in further minor changes in the structural organization of ROC SC. The SAXS patterns of ROC SC had overall large similarities with that of human SC and point to the presence of a long periodicity phase with a repeat distance of about 122Å, e.g. slightly smaller than that determined for human SC in the present study (127Å). Moreover, SAXS results also indicate the presence of covalently bound ceramides, which are crucial for a proper SC barrier, although the corresponding thermal transitions were not clearly detectable by DSC. Due to the competent SC barrier properties and high structural and organizational similarity to that of native human SC, ROC presents a promising alternative for in vitro studies, particularly as it can be obtained under overall rather straightforward cell culture conditions and thus low assay costs. PMID:23770376

Kuntsche, Judith; Herre, Angela; Fahr, Alfred; Funari, Sérgio S; Garidel, Patrick



ES-like cell cultures derived from early zebrafish embryos.  


Pluripotent embryonic stem (ES) cell cultures provide an efficient method for genome manipulation with many applications in marine biotechnology. To develop this technology we have been working to derive fish ES cell lines for in vitro studies of embryo cell growth and differentiation and for the generation of transgenic fish. Zebrafish embryonal cell cultures were derived from blastula-stage embryos in LDF medium supplemented with fetal bovine serum, trout serum, trout embryo extract, selenium, insulin, and leukemia inhibitory factor. Cultures derived under these conditions on feeder layers of zebrafish embryonic fibroblasts possessed a diploid karyotype and exhibited an ES-like morphology with elevated levels of alkaline phosphatase enzyme activity. Injection of primary cell cultures derived from embryos of transgenic fish carrying neo produced chimeric fish detected by polymerase chain reaction analysis. Embryo cells cultured on poly-D-lysine substrate in the presence of retinoic acid or Buffalo rat liver cell-conditioned medium (BRL-CM) and a reduced serum concentration differentiated into neuronal cell types exhibiting elevated levels of acetylcholinesterase enzyme activity and expression of neurofilament and glial fibrillary acidic protein. PMID:7670594

Sun, L; Bradford, C S; Ghosh, C; Collodi, P; Barnes, D W



Milk Stimulates Growth of Prostate Cancer Cells in Culture  

Microsoft Academic Search

Concern has been expressed about the fact that cows’ milk contains estrogens and could stimulate the growth of hormone-sensitive tumors. In this study, organic cows’ milk and two commercial substitutes were digested in vitro and tested for their effects on the growth of cultures of prostate and breast cancer cells. Cows’ milk stimulated the growth of LNCaP prostate cancer cells

Patricia L. Tate; Robert Bibb; Lyndon L. Larcom



Autonomous replication in Drosophila melanogaster tissue culture cells  

Microsoft Academic Search

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and tested them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA fragments were

Jessica G. Smith; Michele P. Calos



Changes in cytoplasmic and lysosomal enzyme activities in cultured rat heart cells: The relationship to cell differentiation and cell population in culture  

Microsoft Academic Search

Summary  Postnatal rat heart cells in culture enriched with respect to muscle cells were obtained by either high density seeding or\\u000a by the replating technique. [3H]Thymidine incorporation to DNA and the enzymatic pattern of cytoplasmic and lysosomal enzymes have been studied as a function\\u000a of the culture’s age, of seeding density, and replating. It was shown that (a) replating maintains predominance

Shu’a Yagev; Michael Heller; Arié Pinson



Anatomical location and culture of equine corneal epithelial stem cells.  


OBJECTIVE: To identify morphologically the locations of equine corneal epithelial stem cells (CESCs) and to culture these cells. ANIMALS STUDIED: We studied the eyes of 12 adult thoroughbred horses. PROCEDURES: Eye tissues were immunostained for two positive stem cell markers (p63, CK14) and one negative marker (CK3) to identify the locations of CESCs, so we could compare their immunostaining patterns with those of human stem cells previously reported. We compared the proliferation rates and morphological features of epithelial cells isolated from the corneal limbus and central cornea. RESULTS: Undifferentiated cells expressing the same immunostaining pattern as human CESCs were present in the equine corneal limbus. Cultured epithelial cells isolated from the limbus expressed the same immunostaining pattern that CESCs show histologically, but cells isolated from the central cornea did not proliferate and could not be evaluated. CONCLUSIONS: Equine CESCs were localized in the epithelial basal layer of the corneal limbus, where melanocytes reside. They could be cultured without loss of their undifferentiated nature. When collecting such stem cells, it may be useful to harvest and culture corneal epithelial tissues in the limbus where melanocytes serve as an indicator of the collecting area. PMID:23710670

Moriyama, Hidekazu; Kasashima, Yoshinori; Kuwano, Atsutoshi; Wada, Shinya



Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen.  


Different culture systems were evaluated for their ability to support attachment and proliferation of the somatic cells obtained from ovine semen. Ejaculates (n=14) were collected from eight rams representing three breeds, Dorper, Suffolk and Hampshire. All samples were processed immediately and somatic cells were obtained from 11 of the 14 ejaculates. These cells had classic epithelial morphology and expressed cytokeratin, indicating they were of epithelial origin. Cells from four rams with the greatest growth rates were used for subsequent studies. Cells were cultured in four different media for 5 days and total numbers of attached cells vs. total numbers of seeded cells were counted and compared each day. Four media were evaluated: (1) a supplemented medium composed of DMEM/F12, 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 30 microg/ml bovine pituitary extract, 5 microg/ml insulin, 10 ng/ml cholera toxin, and 50 microg/ml gentamycin; (2) sheep fetal fibroblast (SFF)-conditioned medium; (3) swiss 3T3 fibroblast-conditioned medium; and (4) basic medium composed of DMEM/F12, 10% FBS, and 50 microg/ml gentamycin. Cell proliferation was greater in the supplemented medium, SFF-conditioned medium, and 3T3 fibroblast-conditioned medium compared to the basic medium by day 2 of culture (p<0.05, n=24), and greater in supplemented medium compared to the SFF-conditioned medium and 3T3 fibroblast-conditioned medium by day 4 of culture (p<0.05, n=24). Three different surfaces: (1) Matrigel basement membrane matrix-coated plastic; (2) collagen I-coated plastic; and (3) uncoated plastic were evaluated for their ability to support proliferation and attachment of the cells obtained from semen. Cell proliferation was greater when cells were cultured on the Matrigel-coated compared to the collagen I-coated and uncoated plastic by day 2 of culture (p<0.05, n=16). Cell attachment was greater when cells were plated on the Matrigel-coated and collagen I-coated plastic compared to the uncoated plastic (p<0.05, n=16). These studies describe an effective system for the culture and proliferation of epithelial cells obtained from ovine semen samples. The system may increase the likelihood of obtaining cells from frozen semen, which could be used for cloning to recover animals of genetic value in which semen is the only material that is available. PMID:19108960

Liu, Jie; Westhusin, Mark; Johnson, Gregory; Raudsepp, Terje; Chowdhary, Bhanu; Burghardt, Robert; Long, Charles; Kraemer, Duane



Ultrastructural and tissue-culture studies on the role of fibronectin, collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo.  


The initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15-40nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin (greater than 3nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker (greater than 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen. Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates. PMID:7034954

Newgreen, D F; Gibbins, I L; Sauter, J; Wallenfels, B; Wütz, R



Rice suspension cultured cells are evaluated as a model system to study salt responsive networks in plants using a combined proteomic and metabolomic profiling approach.  


Salinity is one of the major abiotic stresses affecting plant productivity but surprisingly, a thorough understanding of the salt-responsive networks responsible for sustaining growth and maintaining crop yield remains a significant challenge. Rice suspension culture cells (SCCs), a single cell type, were evaluated as a model system as they provide a ready source of a homogenous cell type and avoid the complications of multicellular tissue types in planta. A combination of growth performance, and transcriptional analyses using known salt-induced genes was performed on control and 100 mM NaCl cultured cells to validate the biological system. Protein profiling was conducted using both DIGE- and iTRAQ-based proteomics approaches. In total, 106 proteins were identified in DIGE experiments and 521 proteins in iTRAQ experiments with 58 proteins common to both approaches. Metabolomic analysis provided insights into both developmental changes and salt-induced changes of rice SCCs at the metabolite level; 134 known metabolites were identified, including 30 amines and amides, 40 organic acids, 40 sugars, sugar acids and sugar alcohols, 21 fatty acids and sterols, and 3 miscellaneous compounds. Our results from proteomic and metabolomic studies indicate that the salt-responsive networks of rice SCCs are extremely complex and share some similarities with thee cellular responses observed in planta. For instance, carbohydrate and energy metabolism pathways, redox signaling pathways, auxin/indole-3-acetic acid pathways and biosynthesis pathways for osmoprotectants are all salt responsive in SCCs enabling cells to maintain cellular function under stress condition. These data are discussed in the context of our understanding of in planta salt-responses. PMID:23661342

Liu, Dawei; Ford, Kristina L; Roessner, Ute; Natera, Siria; Cassin, Andrew M; Patterson, John H; Bacic, Antony



Expression of cyclins in high-density cultured cells and in vivo tumor cells.  


It is widely assumed that during the mammalian cell cycle, four major cyclins, including G1 cyclins (D, E) and mitotic cyclins (A, B1), are expressed in an orderly scheduled pattern in exponentially cultured cells. In high-density cultured cells and in vivo growing cells, whether these cyclins are expressed in the same pattern remains unknown. In this study, we investigated the expression of cyclins by flow cytometry and western-blotting in cultured MOLT-4 and HepG2 cells at high-density. We found that, in high-density cultures, the expression levels of cyclin D, E, and A within each cell cycle phase was less than their levels in low-density cultures. Cyclin B1 was slightly reduced in G2/M phase and unexpectedly expressed in G1 phase. The cyclin B1 expressed in G1 phase was localized mainly in the cytoplasm, and its level was positively correlated with apoptosis. Moreover, we obtained similar cyclins expression pattern in inoculated HepG2 tumor cells from immune-deprived mice and leukemic cells from leukemia patients. These findings describe an altered pattern of cyclin expression in high-density cultured cells as well as in vivo tumor cells. We suggest that high-density cultures might mimic the in vivo growth conditions and such experimental situation may be useful for cell cycle research. PMID:22899539

Min, Jiang; Wang, Xiangyang; Tong, Yixin; Li, Xiaolan; Tao, Deding; Hu, Junbo; Xie, Daxing; Gong, Jianping



Growth of Plant Cell Cultures. Iii. Growth Kinetics and Mass Culture.  

National Technical Information Service (NTIS)

Suspension cultures of bean and lettuce cells have been maintained by serial transfer for over three years. Such cultures may show exponential growth, although growth rates are low with doubling times of three to four days. These suspension cultures have ...

M. Mandels R. O. Matthern H. M. El-Bisi



Phenotypic and Functional Characterization of Human Mammary Stem\\/Progenitor Cells in Long Term Culture  

Microsoft Academic Search

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures.

Devaveena Dey; Meera Saxena; Anurag N. Paranjape; Visalakshi Krishnan; Rajashekhar Giraddi; M. Vijaya Kumar; Geetashree Mukherjee; Annapoorni Rangarajan



Phenotypic and Functional Characterization of Human Mammary Stem\\/Progenitor Cells in Long Term Culture  

Microsoft Academic Search

BackgroundCancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since

Devaveena Dey; Meera Saxena; Anurag N. Paranjape; Visalakshi Krishnan; Rajashekhar Giraddi; M. Vijaya Kumar; Geetashree Mukherjee; Annapoorni Rangarajan; Gianni Parise



Date Palm Cell and Protoplast Culture  

Microsoft Academic Search

\\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

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


Improved transfection efficiency of cultured human cells  

Microsoft Academic Search

We simultaneously tested the transfection efficiency of NT2\\/D1 and HeLa cells with Lipofectamine™ (Life Technologies) and Effectene™ (Qiagen) transfection reagents using the pCH110 eukaryotic assay vector, which contains the lacZ reporter gene. Under our culture conditions for NT2\\/D1 and HeLa cells, Effectene™ transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5–3 times above the recommended

Gordana Nikcevic; Natasa Kovacevic-Grujicic; Milena Stevanovic



Shape Memory Polymers for Active Cell Culture  

PubMed Central

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture. Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

Davis, Kevin A.; Luo, Xiaofan; Mather, Patrick T.; Henderson, James H.



Shape memory polymers for active cell culture.  


Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat(1-5). In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, T(trans;) [either the melting temperature (T(m;)) or the glass transition temperature (T(g;))]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its T(trans;) while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through T(trans;) under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment(6). The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces(7-14). These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture. Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions. PMID:21750496

Davis, Kevin A; Luo, Xiaofan; Mather, Patrick T; Henderson, James H




EPA Science Inventory

This chapter familiarizes the reader with the need to develop, validate and utilize in vitro models to test chemicals for neurotoxic potential. he major advantages and disadvantages of using cell and tissue culture, factors which have stimulated and hampered the promulgation of i...


Wound Coverage by Cultured Skin Cells.  

National Technical Information Service (NTIS)

The major purpose of our work is to refine the technology for growth of human epidermal cells to achieve more rapid growth in vitro and easier handling of tissue cultured materials in clinics. It is also to evaluate the possibilities for the use of alloge...

M. Eisinger E. M. Duffy



Intelligent Control for Cell Culture Processes  

Microsoft Academic Search

An Integrated Intelligent System is being developed for cell culture process control, which consists of several symbolic reasoning systems and numerical computation packages. These software programs are written in different languages and can be used independently. They are under the control of a supervisory expert system, namely, the Meta-System. The key issue to construct the Integrated Intelligent System is to

Ming Rao; Rafael Crus; Tao Yang; Tsung-Shann Jiang; Shaw S. Wang; Ik-K Wan Kim



A novel closed cell culture device for fabrication of corneal epithelial cell sheets.  


Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25?µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300?µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets. Copyright © 2012 John Wiley & Sons, Ltd. PMID:23239605

Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu



21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2010 CFR

... Tissue culture media for human ex vivo tissue and cell culture processing applications... Tissue culture media for human ex vivo tissue and cell culture processing applications...maintenance of tissues and cells of human origin. The solutions...



21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2010 CFR

... Tissue culture media for human ex vivo tissue and cell culture processing applications... Tissue culture media for human ex vivo tissue and cell culture processing applications...maintenance of tissues and cells of human origin. The solutions...



Three-Dimensional Culture Alters Primary Cardiac Cell Phenotype  

PubMed Central

The directed formation of complex three-dimensional (3D) tissue architecture is a fundamental goal in tissue engineering and regenerative medicine. The growth of cells in 3D structures is expected to influence cellular phenotype and function, especially relative cell distribution, expression profiles, and responsiveness to exogenous signals; however, relatively few studies have been carried out to examine the effects of 3D reaggregation on cells from critical target organs, like the heart. Accordingly, we cultured primary cardiac ventricular cells in a 3D model system using a serum-free medium to test the hypothesis that expression profiles, multicellular organizational pathways, tissue maturation markers, and responsiveness to hormone stimulation were significantly altered in stable cell populations grown in 3D versus 2D culture. We found that distinct multi-cellular structures formed in 3D in conjunction with changes in mRNA expression profile, up-regulation of endothelial cell migratory pathways, decreases in the expression of fetal genes (Nppa and Ankrd1), and increased sensitivity to tri-iodothyronine stimulation when compared to parallel 2D cultures comprising the same cell populations. These results indicate that the culture of primary cardiac cells in 3D aggregates leads to physiologically relevant alterations in component cell phenotype consistent with cardiac ventricular tissue formation and maturation.

Rockwood, Danielle; Robinson, Karyn G.; Sandusky, Daniel; Rabolt, John; Pizarro, Christian



Prevention and Detection of Mycoplasma Contamination in Cell Culture  

PubMed Central

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.

Nikfarjam, Laleh; Farzaneh, Parvaneh



Characterisation and Germline Transmission of Cultured Avian Primordial Germ Cells  

PubMed Central

Background Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds. Principal Findings We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring. Conclusions The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.

Macdonald, Joni; Glover, James D.; Taylor, Lorna; Sang, Helen M.; McGrew, Michael J.



Cell Culture Assay for Human Noroviruses [response  

SciTech Connect

We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

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



Region specific response of intervertebral disc cells to complex dynamic loading: an organ culture study using a dynamic torsion-compression bioreactor.  


The spine is routinely subjected to repetitive complex loading consisting of axial compression, torsion, flexion and extension. Mechanical loading is one of the important causes of spinal diseases, including disc herniation and disc degeneration. It is known that static and dynamic compression can lead to progressive disc degeneration, but little is known about the mechanobiology of the disc subjected to combined dynamic compression and torsion. Therefore, the purpose of this study was to compare the mechanobiology of the intervertebral disc when subjected to combined dynamic compression and axial torsion or pure dynamic compression or axial torsion using organ culture. We applied four different loading modalities [1. control: no loading (NL), 2. cyclic compression (CC), 3. cyclic torsion (CT), and 4. combined cyclic compression and torsion (CCT)] on bovine caudal disc explants using our custom made dynamic loading bioreactor for disc organ culture. Loads were applied for 8 h/day and continued for 14 days, all at a physiological magnitude and frequency. Our results provided strong evidence that complex loading induced a stronger degree of disc degeneration compared to one degree of freedom loading. In the CCT group, less than 10% nucleus pulposus (NP) cells survived the 14 days of loading, while cell viabilities were maintained above 70% in the NP of all the other three groups and in the annulus fibrosus (AF) of all the groups. Gene expression analysis revealed a strong up-regulation in matrix genes and matrix remodeling genes in the AF of the CCT group. Cell apoptotic activity and glycosaminoglycan content were also quantified but there were no statistically significant differences found. Cell morphology in the NP of the CCT was changed, as shown by histological evaluation. Our results stress the importance of complex loading on the initiation and progression of disc degeneration. PMID:24013824

Chan, Samantha C W; Walser, Jochen; Käppeli, Patrick; Shamsollahi, Mohammad Javad; Ferguson, Stephen J; Gantenbein-Ritter, Benjamin



Use of Organ Explant and Cell Culture in Cancer Research  

Microsoft Academic Search

\\u000a The culture of human cells and tissues has become an established discipline since the pioneering work of individuals like\\u000a Carrel, Hayflick, Gey and Ham (Freshney 3rd edition 1994). Our first edition chapter concentrated on qualitative and biochemical\\u000a approaches to the use of invitro culture to study human pathology and in particular cancer. This edition describes molecular\\u000a and new microscopic approaches

Eric J. Kort; Christine R. Moore; Eric A. Hudson; Brandon Leeser; James H. Resau; G. M. Yerushalmi; R. Leibowitz-Amit; Galia Tsarfaty; Ilan Tsarfaty; Sharon Moskovitz


Cytokinin-regulated transcripts in tobacco cell culture  

Microsoft Academic Search

To study early responses to the hormone cytokinin we identified transcripts in cell culture of Nicotiana tabacum L. Representational difference analysis (RDA) was used to amplify partial cDNAs of genes that were induced or repressed 45 after addition of 5?×?10-7 to the culture medium. Ten different mRNAs whose abundance changed rapidly after cytokinin treatment were identified. Transcripts coding for osmotin,

S. Schäfer; S. Krolzik; G. A. Romanov; T. Schmülling



Properties of muscarinic acetylcholine receptors in heart cell cultures  

PubMed Central

The binding of acetylcholine to receptors in the intact heart causes a decrease in the frequency (chronotropic effect) and force (ionotropic effect) of contraction. The studies reported here demonstrate a chronotropic response of cultured embryonic chicken heart cells to the muscarinic agonist carbamoylcholine. This response is markedly decreased after a 3-hr incubation with 0.1 mM carbamoylcholine. In order to determine whether agonist-induced alterations in muscarinic receptors were responsible for this decrease, we studied the effects of incubation with carbamoylcholine on the binding of the 3H-labeled muscarinic antagonist quinuclidinyl benzilate (QNB) to homogenates of heart cell cultures. [3H]QNB binding to homogenates of cultures of embryonic hearts of chicks 9 days in ovo was characterized and shown to have properties similar to those of muscarinic receptors in intact hearts. Binding was both specific and saturable. [3H]QNB was displaced by muscarinic agonists and antagonists in concentrations consistent with their known potency. Binding was poorly inhibited by the nicotinic antagonist D-tubocurarine. Kinetic analysis of the binding of QNB by muscarinic receptors showed that initially the reaction proceeds by formation of a rapidly reversible complex with a Kd of 1.8 nM, which is converted to a slowly reversible form. These properties of muscarinic receptors in heart cell cultures are strikingly similar to those observed in homogenates of intact hearts. Homogenates of heart cell cultures bound 84 ± 6 fmol (mean ± SD) of QNB per mg of protein. The number of receptors remained stable from day 4 to day 8 in culture. Incubation of cultures with 0.1 mM carbamoylcholine for 3 hr decreased QNB binding by 55%, to 38 ± 5 fmol/mg protein. When cell cultures were first homogenized and then incubated with carbamoylcholine, no decrease in QNB binding sites could be detected. Thus, incubation with carbamoylcholine causes loss of muscarinic binding sites as well as decreased physiologic responsiveness to muscarinic agonists.

Galper, Jonas B.; Smith, Thomas W.



Phenotypic diversity in cultured cerebral microvascular endothelial cells  

Microsoft Academic Search

Summary  Diversity exists in both the structure and function of the endothelial cells (EC) that comprise the microvasculature of different\\u000a organs. Studies of EC have been aided by our ability to first isolate and subsequently establish cultures from microvascularized\\u000a tissue. After the isolation of microvessel endothelial cells (MEC) derived from rat cerebrum, we observed morphologic differences\\u000a in colonies of cells that

Maria A. Rupnick; Andrew Carey; Stuart K. Williams



Survival of cultured mammalian cells exposed to ultrasound  

Microsoft Academic Search

Summary The colony-forming ability of cultured mammalian cells exposed to monochromatic ultrasonic vibrations of different frequencies (0.1, 0.5, 1.0, 2.0, 3.3 MHz) was studied. The combined effect of x rays and 1.0-MHz ultrasonic waves on the survival of M3-1 cells was also investigated. Split-dose experiments showed that cells exposed to ultrasound are “sensitized” to a subsequent exposure. Almost twice as

B. I. Martins; M. R. Raju; T. L. Hayes; C. A. Tobias



Cultural studies of science education  

NASA Astrophysics Data System (ADS)

In response to Stetsenko's [2008, Cultural Studies of Science Education, 3] call for a more unified approach in sociocultural perspectives, this paper traces the origins of the use of sociocultural ideas in New Zealand from the 1970s to the present. Of those New Zealanders working from a sociocultural perspective who responded to our query most had encountered these ideas while overseas. More recently activity theory has been of interest and used in reports of work in early childhood, workplace change in the apple industry, and in-service teacher education. In all these projects the use of activity theory has been useful for understanding how the elements of a system can transform the activity. We end by agreeing with Stetsenko that there needs to be a more concerted approach by those working from a sociocultural perspective to recognise the contribution of others in the field.

Higgins, Joanna; McDonald, Geraldine



Cultural Studies in the English Classroom.  

ERIC Educational Resources Information Center

|This book opens up ways of teaching and devising programs which place the students' cultural experiences at the center of language production and consumption. It provides concrete models of cultural studies programs and classrooms for high school and college teachers who would like to try the "cultural studies approach." It also offers a…

Berlin, James A., Ed.; Vivion, Michael J., Ed.


Cultural Studies in Turkey: Education and Practice  

Microsoft Academic Search

It would be difficult to pinpoint when the instruction of cultural studies began in Turkey, as humanities and social science scholars with ties to academic circles in various parts of the world where cultural studies has been practised reflected the methodology in the various courses they taught as soon as they themselves became acquainted with it. The Crossroads in Cultural

Gonul Pultar; Ayse Lahur K?rtunc



Characterization and Culture of Fetal Rhesus Monkey Renal Cortical Cells  

PubMed Central

The renal glomerulus is composed of endothelial and mesangial cells with podocytes contributing to glomerular filtration. Podocyte damage is associated with renal disorders, thus there is interest in these cells for regenerative medicine. These studies investigated the use of extracellular matrix (ECM) to grow third trimester fetal monkey renal cortical cells and to assess mature podocytes in culture. Immunohistochemistry provided a profile of podocyte differentiation with metanephric mesenchyme and developing podocytes nestin positive and synaptopodin negative, whereas mature podocytes were positive for both markers. Primary cell cultures devoid of mature podocytes were established on plastic and renal ECM. A cell population (nestin+/synatopodin?) cultured on renal ECM showed greater proliferative potential compared to plastic with limited podocytes developing in culture over time. Further investigation of individual components of ECM (laminin, fibronectin, collagen I or IV) indicated that collagen I supported the greatest proliferation similar to renal ECM, whereas a greater number of mature podocytes (nestin+/synaptopodin+) were observed on fibronectin. These results suggest: (1) culture of fetal monkey podocytes can be accomplished, (2) renal ECM and collagen I can support renal cortical cells in vitro which may recapitulate the developing kidney in vivo, and (3) fibronectin can support podocyte differentiation in vitro.

Leapley, Alyssa C.; Lee, C. Chang I.; Batchelder, Cynthia A.; Yoder, Mervin C.; Matsell, Douglas G.; Tarantal, Alice F.



Nitrogen Metabolism in Plant Cell Suspension Cultures  

PubMed Central

Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea.

Behrend, Josef; Mateles, Richard I.



Retinitis pigmentosa: a quantitative study of the apical membrane of normal and dystrophic human retinal pigment epithelial cells in tissue culture in relation to phagocytosis  

Microsoft Academic Search

The phagocytic capabilities of both normal and dystrophic human retinal pigment epithelial cells were assessed by challenging cultures with both non-biological and biological particles. Cells from either source ingested both carbon particles and latex microspheres. Quantification of the phagocytic process in relation to microspheres showed that uptake was linear over an 8 h period and the rate was dose-dependent. All

M. Boulton; J. Marshall; J. Mellerio



Calcium deposition in osteoarthritic meniscus and meniscal cell culture  

PubMed Central

Introduction Calcium crystals exist in the knee joint fluid of up to 65% of osteoarthritis (OA) patients and the presence of these calcium crystals correlates with the radiographic evidence of hyaline cartilaginous degeneration. This study sought to examine calcium deposition in OA meniscus and to investigate OA meniscal cell-mediated calcium deposition. The hypothesis was that OA meniscal cells may play a role in pathological meniscal calcification. Methods Studies were approved by our human subjects Institutional Review Board. Menisci were collected during joint replacement surgeries for OA patients and during limb amputation surgeries for osteosarcoma patients. Calcium deposits in menisci were examined by alizarin red staining. Expression of genes involved in biomineralization in OA meniscal cells was examined by microarray and real-time RT-PCR. Cell-mediated calcium deposition in monolayer culture of meniscal cells was examined using an ATP-induced 45calcium deposition assay. Results Calcium depositions were detected in OA menisci but not in normal menisci. The expression of several genes involved in biomineralization including ENPP1 and ANKH was upregulated in OA meniscal cells. Consistently, ATP-induced calcium deposition in the monolayer culture of OA meniscal cells was much higher than that in the monolayer culture of control meniscal cells. Conclusions Calcium deposition is common in OA menisci. OA meniscal cells calcify more readily than normal meniscal cells. Pathological meniscal calcification, which may alter the biomechanical properties of the knee meniscus, is potentially an important contributory factor to OA.



Portable microcontact printing device for cell culture.  


In this work, we developed a portable device to perform microcontact printing in a safety cabinet for cell culture. The device was designed to be small and non-bulky, easy to sterilize, while not requiring the use of electricity, and which requires very little manual handling. Moreover, the portable microcontact printer is reproducibly fabricated with a rapid prototyping system, and allows for the easy micropatterning of biomolecules with a resolution ranging from 20 to 500 ?m. This opens new horizons in the direct and simple micropatterning of culture dishes and the mimicking and biofabrication of complex architectures of tissues. PMID:20817249

Elloumi-Hannachi, Imen; Maeda, Masanori; Yamato, Masayuki; Okano, Teruo



[Serum-free Culture of Umbilical Cord Mesenchymal Stem Cells].  


This study was purposed to observe the culture of umbilical cord mesenchymal stem cells (UC-MSC) with serum-free medium, and compared it with the medium containing 10% fetal bovine serum (FBS) . The normal umbilical cords were acquired during cesarean section, and then were cultured with MesenCult-XF serum-free medium or medium containing 10% fetal bovine serum(FBS). The monphology, immunophenotype, cell cycle, proliferation and differentiation potential of mesenchymal stem cells and the inhibition of mixed lymphocyte reaction were observed through different medium culture method. The results showed that the MSC cultured with serum-free MesenCult(-)XF medium could transfer and multiply for average of 6.57 ± 0.7 times, and the serum medium-cultured MSC could transfer and multiply for average of 4.59 ± 0.45 times (P < 0.05). Two kinds of medium cultured MSC all expressed CD44, CD90, CD73, CD105 antigen, but did not expressed CD31, CD45, HLA-DR and CD34 antigen, and their expression levels were not significantly different. The serum-free medium-cultured MSC (65 ± 5.2%) were all at Go/G1 phase, and the serum-contained medium-cultured MSC (62+3.1%) were at Go/G1 phase(P > 0.05); the 2 kinds of media-cultured MSC all could differentiate into fat and ossification; when serum-free medium cultured umbilical cord MSC were innoculated at the the density of 10(3), 5×10(3), 10(4), and 2×10(4) cells/well, then co-cultured with the reactant and stimulating cells, the CPM were (6.43 ± 0.47)×10(4), (4.30 ± 0.38)×10(4), (1.97 ± 0.13)×10(4) and (0.24 ± 0.03)×10(4), respectively, and the serum-containing medium-cultured MSC were incubated with different density of mixed lymphocyte, displaying CPM that were (7.85 ± 0.07)×10(4), (5.64 ± 0.12)×10(4), (3.09 ± 0.18)×10(4) and (1.73 ± 0.05)×10(4). It is concluded that the serum-free medium has been confirmed to culture MSC, which have potential of transfer and differentiation with count for clinical application, and can avoid foreign protein sensitization. PMID:24156445

Zhou, Ping; Li, Dan; Chen, Guang-Hua; Wang, Yi



Plant cell cloning and culture products.  


Biotechnological developments in the use of plant cells as sources of biochemicals have now brought several applications within commercial range. Plant propagation has developed faster commercially, but now requires similar biotechnology to enable the automated production of large numbers of plants at low cost. Although some of the enabling technology has been developed for pregerminated seeds this has not been coupled to production of plantlets in bioreactors. Our present lack of knowledge of how to control gene expression in plant cells, which stems from our ignorance of the organization and regulation of the plant genome, is a critical factor limiting all applications of plant cell cultures. PMID:6443753

Jones, L H



Protective effects of tetramethylpyrazine on rat retinal cell cultures.  


The retinal degeneration characterized with death of retinal ganglion cells is a pathological hallmark and the final common pathway of various optic neuropathies. Thus, there is an urgent need for identifying potential therapeutic compounds for retinal protection. Tetramethylpyrazine has been suggested to be neuroprotective for central neurons by acting as an antioxidant and a calcium antagonist. In this study, we tested the effects of tetramethylpyrazine on the viability of both neuronal and non-neuronal cells in mixed rat retinal cell cultures during a long-term cultivation or following hydrogen peroxide treatments. Cellular and biochemical analyses demonstrated that 50 microM tetramethylpyrazine significantly preserved neuronal morphology and survival in retinal cell cultures following 4-week in vitro cultivation as well as lethal exposures to hydrogen peroxide (10 microM or 50 microM for 24h). Hydrogen peroxide treatments induced remarkable increases in lipid peroxidation and mitochondrial reactive oxygen species (ROS) generation paralleled by the loss of mitochondrial membrane potential, microtubule-associated protein-2 (MAP-2) in neuronal soma and rattin peptide in cultured cells. Addition of tetramethylpyrazine in the cultures efficiently attenuated the signs of oxidative stress and retained abundance of MAP-2 and rattin in association with cell survival. In addition, siRNA-mediated downregulation of MAP-2 or rattin significantly increased the vulnerability of retinal neurons or the number of degenerating cells in the cultures, respectively, whereas exogenous humanin peptide, an analog of rattin, promoted cell survival in cultures under hydrogen peroxide attacks. These results suggest that tetramethylpyrazine protect retinal cells through multiple pathways and might be a potential therapeutic candidate for retinal protection in certain optic neuropathies. PMID:18261827

Yang, Zhikuan; Zhang, Qingjiong; Ge, Jian; Tan, Zhiqun



Paracrystal Formation in Cell Cultures Infected with Adenovirus Type 2  

PubMed Central

Large rod-shaped structures corresponding to paracrystals were seen in the nucleus, cytoplasm, or both of adenovirus type 2 (Ad2)-infected cells by immunofluorescence staining with antibody prepared against purified Ad2. In exception to this, Ad2-induced crystals did not stain with either hexon or fiber antibody. The crystalline structures were first observed in Ad2-infected Vero cells at 28 hr with a maximum number at 70 hr postinoculation. The kinetics of paracrystalline formation closely paralleled the experimental synthesis of infectious progeny virus. Acridine-orange staining revealed the lack of nucleic acids associated with the crystal. Also, the paracrystals stained intensely with phenanthrenequinone, suggesting that they are composed of basic proteins. Interferon induced by Newcastle disease virus from African green monkey kidney cell cultures was used to pretreat Vero cells prior to Ad2 infection. This resulted in inhibiting the formation of viral-induced paracrystals in 97% of the cells and reduced virus yields by 95%. The African green monkey kidney cell culture interferon did not reduce Ad2 yields in HeLa cell cultures or display any virus inhibitory activity in rabbit kidney cell cultures. Staining procedures, fluorescent-antibody tests with whole virus, hexon or fiber antibody, and interferon studies suggested that the paracrystals were viral-directed and composed of basic proteins (possibly core proteins). Images

Henry, Claude J.; Atchison, Robert W.



Hydroxyapatite incorporated into collagen gels for mesenchymal stem cell culture.  


Collagen gels could be used as carriers in tissue engineering to improve cell retention and distribution in the defect. In other respect hydroxyapatite could be added to gels to improve mechanical properties and regulate gel contraction. The aim of this work was to analyze the feasibility to incorporate hydroxyapatite into collagen gels and culture mesenchymal stem cells inside it. Human bone marrow mesenchymal stem cells (hMSC-BM) were used in this study. Gels were prepared by mixing rat tail type I collagen, hydroxyapatite microparticles and MSCs. After polymerization gels were kept in culture while gel contraction and mechanical properties were studied. In parallel, cell viability and morphology were analyzed. Gels became free-floating gels contracted from day 3, only in the presence of cells. A linear rapid contraction phase was observed until day 7, then a very slow contraction phase took place. The incorporation of hydroxyapatite improved gel stability and mechanical properties. Cells were randomly distributed on the gel and a few dead cells were observed all over the experiment. This study shows the feasibility and biocompatibility of hydroxyapatite supplemented collagen gels for the culture of mesenchymal stem cells that could be used as scaffolds for cell delivery in osteoarticular regenerative medicine. PMID:23798652

Laydi, F; Rahouadj, R; Cauchois, G; Stoltz, J-F; de Isla, N



Initiation, growth and cryopreservation of plant cell suspension cultures.  


Methods described in this paper are confined to in vitro dedifferentiated plant cell suspension cultures, which are convenient for the large-scale production of fine chemicals in bioreactors and for the study of cellular and molecular processes, as they offer the advantages of a simplified model system for the study of plants when compared with plants themselves or differentiated plant tissue cultures. The commonly used methods of initiation of a callus from a plant and subsequent steps from callus to cell suspension culture are presented in the protocol. This is followed by three different techniques for subculturing (by weighing cells, pipetting and pouring cell suspension) and four methods for growth measurement (fresh- and dry-weight cells, dissimilation curve and cell volume after sedimentation). The advantages and disadvantages of the methods are discussed. Finally, we provide a two-step (controlled rate) freezing technique also known as the slow (equilibrium) freezing method for long-term storage, which has been applied successfully to a wide range of plant cell suspension cultures. PMID:21637194

Mustafa, Natali R; de Winter, Ward; van Iren, Frank; Verpoorte, Robert



Ten commandments for preventing contamination of primary cell cultures  

Microsoft Academic Search

Procedures for preventing contamination in primary cell cultures must be carefully defined and strictly followed in order to obtain healthy cells. Protocols have been developed and refined in our laboratory for establishing primary cultures of muscle and fat stem cells without contamination from a variety of animals. Contamination of cell cultures is not only frustrating, but is also very expensive

Janet L. Vierck; Katherine Byrne; Priya S. Mir; Michael V. Dodson



Cultured bovine brain capillary endothelial cells (BBCEC) - a blood-brain barrier model for studying the binding and internalization of insulin and insulin-like growth factor 1  

SciTech Connect

Cultured bovine brain capillary endothelial cells (BBCEC) have previously been reported by their laboratory as a working model for studying nutrient and drug transport and metabolism at the blood-brain barrier. In the present study, they have utilized this culture system to investigate the binding and internalization of (/sup 125/I)-labelled insulin (INS) and insulin-like growth factor 1(IGF-1) by BBCEC. After 2 hrs at 23/sup 0/C, the specific binding of INS and IGF-1 was 1.6% and 13.6%, respectively. At 37/sup 0/C, the maximum specific binding was 0.9% for INS and 5.8% for IGF-1. Using an acid-wash technique to assess peptide internalization, it was observed that, at 37/sup 0/C, approximately 60% of the bound INS rapidly became resistant to acid treatment, a value which was constant over 2 hr. With IGF-1, a similar proportion of the bound material, 62%, became resistant by 30 min, but subsequently decreased to 45% by 2 hr. Scatchard analysis of competitive binding studies indicated the presence of two binding sites for each protein, having K/sub d/'s of 0.82 nM and 19.2 nM for INS and 0.39 nM and 3.66 nM for IGF-1. Little change in the amount of INS binding was observed over a four-day interval as the cultures became a confluent monolayer. The present report of binding and internalization of these proteins suggests that the BBCEC may utilize a receptor-mediated process to internalize and/or transport (transcytosis) INS and IGF-1 from the circulation.

Keller, B.T.; Borchardt, R.T.



Microcystin-LR induced cellular effects in mammalian and fish primary hepatocyte cultures and cell lines: A comparative study  

Microsoft Academic Search

The impact of microcystin-LR, one of the most common cyanobacterial toxins, on liver and gut cells originating from mammals and fish was compared. Upon exposure of human and rainbow trout (Oncorhynchus mykiss) cell lines up to 2.5?M microcystin-LR, no alteration in cell viability was observed as assessed with three fluorescent indicators dyes, CFDA-AM, Alamar Blue and neutral red. The lack

Daniela Alina Boaru; Nicolae Drago?; Kristin Schirmer



A comparative study of arachidonic acid metabolism in rabbit cultured astrocytes and human astrocytoma cells (1321N1)  

Microsoft Academic Search

1.1. ATP, bradykinin (BK), and A-23187 activated the generation of prostaglandin (PG) E2 and thromboxane (TX) B2 in rabbit astrocytes, but not in human astrocytoma cells (1321N1).2.2. In human astrocytoma cells, ATP, BK, and A-23187 could not release [3H]arachidonic acid (AA) from [3H]AA-labeled cells and exogenous AA was not converted to TXB2 and PGE2, suggesting the lack of phospholipase (PL)

Hiromi Ishimoto; Isao Matsuoka; Hironori Nakanishi; Norimichi Nakahata



Murine mesenchymal stem cell isolated and expanded in low and high density culture system: surface antigen expression and osteogenic culture mineralization.  


Marrow culture from mice has been reported to be overgrown by non-mesenchymal cells. In almost all protocols for isolation of murine mesenchymal stem cells (MSCs), high density culture systems have been employed. Since MSCs are colonogenic cells, the initiating cell seeding density may have significant impact on their cultures. This subject was explored in this study. For this purpose, the bone marrow cells from NMRI mice were plated at 2.5 x 10(6) cells/cm(2) and upon confluency were reseeded as either low density (50 cells/cm(2)) or high density (8 x 10(4) cells/cm(2)) cultures. The cells were expanded through an additional subculture and the passage 2 cells as a product of two culture systems were statistically compared with respect to their surface antigen profiles and osteogenic culture mineralization. While low density culture grew with multiple colony formation, there were no distinct colonies in high density cultures. In contrast to high density cultures, passage 2 cells from low density system possessed typical homogenous fibroblastic morphology. Some cells from high density system but not the low density cultures expressed hematopoietic and endothelial cell markers including CD135, CD34, CD31, and Vcam surface antigens. Furthermore, osteogenic cultures from low density system displayed significantly more mineralization than those from high density system. Taken together, it seems that low density culture system resulted in more purified MSC culture than its counterpart as high density culture system. PMID:19452230

Eslaminejad, Mohamadreza Baghaban; Nadri, Samad



Culture, Development, and Social Theory: On Cultural Studies and the Place of Culture in Development  

Microsoft Academic Search

Debates in development theory have recently swung back to taking seriously the relationship of culture to development, especially in the face of manifest failures of conventional approaches to economic growth and social transformation. This has happened at a moment when, especially within anthropology, the concept of culture itself is undergoing critical examination, and when cultural studies has emerged as a

John Clammer



Kinetics of Thallium Exchange in Cultured Rat Myocardial Cells  

Microsoft Academic Search

SUMMARY. The kinetics of thallium exchange in cultured rat myocardial cells were studied and compared to those of potassium in the same tissue. Studies were carried out using low concentra- tions (10 nM to 5 HM) of thallium-204, approximating those likely to be encountered during clinical myocardial scintigraphy. Both thallium uptake and release could be described by a single exponential

David McCall; Lawrence J. Zimmer; Arnold M. Katz


Aluminum silicate toxicity in cell cultures.  


To assess the cytotoxicity of four clays containing an aluminum silicate--montmorillonite, bentonite, kaolinite and erionite--we used human umbilical vein endothelial, N1E-115 neuroblastoma, and ROC-1 oligodendroglial cells. Morphological examination, lactate dehydrogenase release and fatty acid release were used as indices of trauma. The clays were added in suspension to the cell cultures at concentrations of 0.1, 0.03 and 0.01 mg/ml of medium and the cells were incubated for 1, 6 and 24 h. The clays did not lyse ROC-1 and N1E-115 cells and did not cause a dose-dependent increase in fatty acid levels at 24 h. There were no significant increases in lactate dehydrogenase activity in N1E-115 neuroblastoma or ROC-1 oligodendroglial cells. In human umbilical vein endothelial cells, montmorillonite, kaolinite and bentonite caused a dose-dependent increase in fatty acids at 24 h. All three clays caused cell lysis. We postulate that the cytotoxicity of the clays containing an aluminum silicate towards endothelial cells may disrupt the blood-brain barrier in the affected areas, allowing the entry of the clay particle into the brain. Aluminum silicate clays caused a dose-dependent release of fatty acids in human umbilical vein endothelial cells. The clays also caused lysis of these cells. ROC-1 oligodendroglia and N1E-115 neuroblastoma cells were not lysed by the clays, suggesting that this is not a general phenomenon. PMID:8397348

Murphy, E J; Roberts, E; Horrocks, L A



Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 dalton range  

Microsoft Academic Search

Summary Cell-to-cell junctional permeability in mammalian cell cultures was probed with a series of fluorescent tracers ranging 300 to 800 in molecular weight, during treatment with metabolic inhibitors, Ca-transporting ionophore, and carbon dioxide. Treatment with the combination of cyanide and iodoacetic acid (1–2mm each), but not with either one alone, caused reversible junctional blockade to all tracer molecular species, large

J. Flagg-Newton; W. R. Loewenstein



Cell population evolution in tissue cultures from embryo barley ( Hordeum vulgare L.) after caffeine treatment  

Microsoft Academic Search

Summary Over a period of time a comparative study of the evolution of the different number of nuclei as well as the different chromosome number in the cells of severalin vitro tissue cultures of barley (Hordeum vulgare L.) was carried out. The cultures were obtained by culturing embryos in a control culture medium or in a media supplemented with 0.5

M. L. Ruiz; A. M. VikzQurz



Cell division modulates prion accumulation in cultured cells  

PubMed Central

The phenotypic effect of prions on host cells is influenced by the physical properties of the prion strain and its level of accumulation. In mammalian cell cultures, prion accumulation is determined by the interplay between de novo prion formation, catabolism, cell division, and horizontal cell-to-cell transmission. Understanding this dynamic enables the analytical modeling of protein-based heritability and infectivity. Here, we quantitatively measured these competing effects in a subline of neuroblastoma (N2a) cells and propose a concordant reaction mechanism to explain the kinetics of prion propagation. Our results show that cell division leads to a predictable reduction in steady-state prion levels but not to complete clearance. Scrapie-infected N2a cells were capable of accumulating different steady-state levels of prions, dictated partly by the rate of cell division. We also show that prions in this subline of N2a cells are transmitted primarily from mother to daughter cells, rather than horizontal cell-to-cell transmission. We quantitatively modeled our kinetic results based on a mechanism that assumes a subpopulation of prions is capable of self-catalysis, and the levels of this subpopulation reach saturation in fully infected cells. Our results suggest that the apparent effectiveness of antiprion compounds in culture may be strongly influenced by the growth phase of the target cells.

Ghaemmaghami, Sina; Phuan, Puay-Wah; Perkins, Beth; Ullman, Julie; May, Barnaby C. H.; Cohen, Fred E.; Prusiner, Stanley B.



Effects of carbon monoxide on cardiac muscle cells in culture  

SciTech Connect

Embryonic rat cardiac muscle cells grown in the presence of various tensions of CO (5-95%) without the presence of O{sub 2} survived and exhibited reduced cell growth, which was concentration dependent. When cardiac muscle cells were grown in the presence of a mixture of CO (10-20%) and O{sub 2} (10-20%), the growth rate of these cells was comparable to that of the control cells. Cardiac myocytes continued to beat when exposed to varying tensions of CO, except in the case of 95% CO. The cells exposed to different concentrations of CO contained fewer myofibrils of different stages of differentiation compared with the control and the culture exposed to a mixture of 20% O{sub 2} and 20% CO, with cells that contained abundant, highly differentiated myofibrils. There was no significant difference in the structural organization of mitochondria between the control and the surviving experimental cells. It is evident from the present studies that O{sub 2} is required for the optimum in vitro cellular growth of cardiac muscle. Furthermore, CO in combination with O{sub 2} at a concentration of 10 or 20% can produce optimal growth of cardiac muscle cells in culture. To determine maximum labeling index during the labeling period, cells were continuously labeled with ({sup 3}H)thymidine for 24 h before the termination of cultures.

Nag, A.C.; Chen, K.C.; Cheng, Mei (Oakland Univ., Rochester, MI (USA) General Motors Research Laboratories, Warren, MI (USA))



Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures.  


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

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



Hypoxic contraction of cultured pulmonary vascular smooth muscle cells  

SciTech Connect

The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (PO2 approximately 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the PO2 in the culture medium to 59 mmHg caused a 45% increase in the proportion of MLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32P and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells.

Murray, T.R.; Chen, L.; Marshall, B.E.; Macarak, E.J. (Univ. of Pennsylvania, Philadelphia (USA))



Splitting culture medium by air-jet and rewetting for the assessment of the wettability of cultured epithelial cell surfaces.  


This study found that the phenomenon of rewetting after squeezing culture medium varied in different culture conditions for rat oral mucosal epithelial cells. When culture medium covering over cultured cells was squeezed by an air-jet application, the motion of squeezed culture medium was able to be observed by using a commercially available movie camera. Squeezed width on cells cultured in keratinocyte culture medium (KCM), which contained with fetal bovine serum, was one-sixth of that in FBS-free KCM. This result corresponded to the mucous layer staining statuses of cultured cells in both cases; positive in KCM and negative in FBS-free medium. Furthermore, the gene expression of mucous glycoprotein MUC4 in KCM was 100 times higher than that in FBS-free medium, and the expression of MUC4 protein only showed on the apical surface of cells cultured in KCM. The relative gene expression levels of MUC1, 13, 15, and 16 in both the normal and FBS-free medium were found to be no more than one-thirtieth of that of MUC4 in KCM. The main factor of the wettability difference between KCM and FBS-free medium was speculated to be the difference of MUC4 expression between both media. This method can be a simple technique for testing not only the surface wettability but also the mucous formation of cultured cells. PMID:24008039

Tanaka, Nobuyuki; Kondo, Makoto; Uchida, Ryohei; Kaneko, Makoto; Sugiyama, Hiroaki; Yamato, Masayuki; Okano, Teruo



Use of irradiated human amnion as a matrix for limbal stem cell culture.  


Several ocular diseases affect the corneal surface; the development of effective technologies for the treatment of corneal lesions has brought about an improvement in the quality of life of affected patients. The aim of this study is to culture and characterize limbal stem cells cultured on gamma ((60)Co) radiosterilized human amnion (RHA). Limbal stem cells were isolated from ten preserved samples of corneal transplant. The cells were cultured since primary culture until expanded cells on RHA and stained with monoclonal antibodies to establish their immunophenotype, after which cytokeratin 12 and Vimentin were positive by immunohistochemistry. The immunophenotype remained constant since primary culture until expanded cells in RHA. The RHA and cells construct were structurally integrated. Immunohistochemistry was cytokeratin 12, Vimentin positive, and cytokeratin 19 negative. In vitro limbal cells maintain a constant epithelial transition immunophenotype in culture up to primary culture until expanded cells on RHA. PMID:22392228

Landa-Solís, Carlos; Vázquez-Maya, Leticia; Martínez-Pardo, María Esther; Brena-Molina, Ana M; Ruvalcaba, Erika; Gómez, Ricardo; Ibarra, Clemente; Velasquillo, Cristina



Application of 19F magnetic resonance to study the efficacy of fluorine labeled drugs in the three-dimensional cultured breast cancer cells.  


The cellular monitoring of tumor response to treatments is important for drug discovery and drug development in cancer therapy. We studied efficacy of Herceptin, a common breast cancer drug conjugated with a fluorine organic compound, perfluoro-15-crown-5-ether (PFCE) which easily forms biocompatible emulsions. Three new pharmaceutical forms of Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink were synthesized for the ex vivo study of their efficacy in breast cancer treatment. The emulsions were administered to 10(9)cells mL(-1) of HER-2 positive human adenocarcinoma (MCF-7) cells and the same amount of human mammary epithelial cells (HMEC) cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. Following drugs administration ex vivo, fluorine-19 magnetic resonance imaging ((19)F MRI) was applied for cells imaging to measure their viability and to study drug efficacy over 72h. To ensure optimum drug tracking, HydraLink was used to provide stable binding affinity of emulsified Herceptin to receptor while cationic lipid (Lipofectamine) was used to enhance lipophilicity of the emulsions. After 72h of treatment with Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink the viability of cells was 54+/-2%, 49+/-3%, 43+/-5% and 42+/-1%, respectively, as compared with control 93+/-2%. The efficacy (EC(50)) of Herceptin conjugated with emulsions was found to be 970+/-13 microg mL(-1) for Herceptin/PFCE, 645+/-11 microg mL(-1) for Herceptin/PFCE/Lipoplex, 678+/-7 microg mL(-1) for Herceptin/PFCE/HydraLink and 1000+/-3 microg mL(-1) for Herceptin. The results show that fluorine emulsions improved the efficacy of Herceptin and (19)F signal intensity changes validated drug efficiency. The significant correlations between duration of treatments and MCF-7 cells viability were observed. While we studied breast cancer cells, the fluorine emulsions could be applied for treatment of other cancer cells overexpressing HER-2. PMID:19900396

Bartusik, Dorota; Tomanek, Boguslaw



Plant cell cultures: Chemical factories of secondary metabolites  

Microsoft Academic Search

This review deals with the production of high-value secondary metabolites including pharmaceuticals and food additives through plant cell cultures, shoot cultures, root cultures and transgenic roots obtained through biotechnological means. Plant cell and transgenic hairy root cultures are promising potential alternative sources for the production of high-value secondary metabolites of industrial importance. Recent developments in transgenic research have opened up

S Ramachandra Rao; G. A Ravishankar



Literary Studies and the Third Culture  

Microsoft Academic Search

This dissertation is predicated on the notion that the concept of the Third Culture describes a dominant intellectual force in contemporary society and that literary and cultural studies thinkers must learn to engage it while maintaining and understanding the rich and varied history of humanistic thought (even its most skeptical kind). The Third Culture describes a move beyond the traditional

Curtis D Carbonell



Method of determining the number of cells in cell culture  

SciTech Connect

This patent describes a color-sensitivity method for determining the number of cells in in vitro cell culture at a sensitivity as low as about 100 or about 500 cells. It comprises lysing the cells and incubating the lysate with p-nitrophenyl phosphate at acid pH for a predetermined period of time at a temperature of from about 35{degrees} to about 38{degrees}C. and then measuring the color development at 400 to 420 nanometers and correlating the color development with cell number by comparing with a control standard of known cell number.

Connolly, D.T.



Culture and characterisation of epithelial cells from human pterygia  

PubMed Central

BACKGROUND/AIMS—Pterygia are a common disorder of the ocular surface. The disease represents a chronic fibrovascular and degenerative process thought to originate at the conjunctival-corneal junction, where altered limbal stem cells are proposed to be the cell of origin. Extensive epidemiological evidence exists to implicate ultraviolet B irradiation in the pathogenesis of pterygia. To date no animal or in vitro culture model has been developed to test such an hypothesis. The aim of this study was to establish and characterise a pure population of epithelial cells derived from pterygium tissue.?METHODS—Tissue specimens were obtained from patients undergoing pterygium excision. Explants were cultured in either serum free or serum supplemented medium. Primary and passaged cells were processed for light microscopy, analysed by flow cytometry, and characterised immunohistochemically using specific antibodies.?RESULTS—In serum free culture, cuboidal cells with typical morphology of epithelial cells migrated from the pterygium explants from 3 days onwards and eventually formed a cohesive monolayer. Passaged cells consisted of 98.4% cytokeratin positive cells and demonstrated immunoreactivity for multiple cytokeratins, including AE1, AE3, AE5, but were negative for AE8. These cells also expressed an epithelial specific antigen, together with vimentin and mucin, as did epithelial cells in sections of pterygia.?CONCLUSIONS—A relatively simple method of isolating pterygium epithelial cells has been established. Cultured pterygium epithelial cells are phenotypically and functionally similar to their in vivo counterparts with respect to keratin, vimentin, and mucin expression. In vitro assays using these cells may aid in elucidating the pathogenesis of pterygia.??

Di, G; Tedla, N.; Kumar, R.; McCluskey, P.; Lloyd, A.; Coroneo, M.; Wakefield, D.



Effects of Homocysteine on Smooth Muscle Cell Proliferation in Both Cell Culture and Artery Perfusion Culture Models  

Microsoft Academic Search

Background. Hyperhomocysteinemia is associated with increased risk for vascular disease. However, the pathogenic mechanisms of homocysteine are largely unknown. We evaluated the effects of homocysteine on smooth muscle cell (SMC) and endothelial cell proliferation in cell culture and on SMC proliferation of balloon angioplasty-injured arteries in a perfusion culture model.Methods. Human and pig SMCs and endothelial cells were cultured with

Changyi Chen; Michael E. Halkos; Scott M. Surowiec; Brian S. Conklin; Peter H. Lin; Alan B. Lumsden



Aragonite precipitation by "proto-polyps" in coral cell cultures.  


The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (?(arag)~4), the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM) and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective) similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms. PMID:22514707

Mass, Tali; Drake, Jeana L; Haramaty, Liti; Rosenthal, Yair; Schofield, Oscar M E; Sherrell, Robert M; Falkowski, Paul G



Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells  

Microsoft Academic Search

These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly,

W. J. Wasilenko; A. C. Marchok



Characterization and Long-Term Maintenance of Rat Taste Cells in Culture  

Microsoft Academic Search

Taste cells have a limited life span and are replaced from a basal cell population, although the specific factors involved in this process are not well known. Short- and long-term cultures of other sensory cells have facilitated efforts to understand the signals involved in proliferation, differentiation, and senescence, yet few studies have reported successful primary culture protocols for taste cells.

Hakan Ozdener; Karen K. Yee; Jie Cao; Joseph G. Brand; John H. Teeter; Nancy E. Rawson



Adherence of Actinobacillus pleuropneumoniae to primary cultures of porcine lung epithelial cells  

Microsoft Academic Search

To study adherence of Actinobacillus pleuropneumoniae to porcine lower respiratory epithelium, a cell culture model was developed using primary cultures of porcine lung epithelial cells (LEC). Adherence assays were performed and results were compared with data obtained with swine kidney cells (SK6). A. pleuropneumoniae efficiently adhered to LEC with up to 62 bacteria per cell after 2h of incubation. Reference

Bouke K. H. L. Boekema; Norbert Stockhofe-Zurwieden; Hilde E. Smith; Elbarte M. Kamp; Jos P. van Putten; Jos H. Verheijden



Neonatal Rat Heart Cells Cultured in Simulated Microgravity.  

National Technical Information Service (NTIS)

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 cel...

R. E. Akins N. A. Schroedl S. R. Gonda C. R. Hartzell



Recombinant Protein Production and Insect Cell Culture and Process.  

National Technical Information Service (NTIS)

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 pr...

G. Spaulding T. Prewett T. Goodwin K. Francis A. Andrews



Nanopillar based electrochemical biosensor for monitoring microfluidic based cell culture  

NASA Astrophysics Data System (ADS)

In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes---Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the 'Enzyme electrode' was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the 'Working electrode' was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose monitoring devices. Third, the methods used to develop 3D electrodes incorporated with nanopillars can be used for other applications such as neural probes, fuel cells, solar cells etc., and finally, the knowledge obtained from the immobilization of enzymes onto nanostructures sheds some new insight into nanomaterial/biomolecule interactions.

Gangadharan, Rajan


Cell Culture-Derived Influenza Vaccines  

Microsoft Academic Search

\\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

Philip R. Dormitzer


The time course of synapse formation of mouse neuroblastoma cells in monolayer cultures  

Microsoft Academic Search

Neuroblastoma cells grown on substrates in culture develop long processes and assume the morphology of normal neurons as judged light microscopically. The development of synapses in the cultured tissue is studied by periodic electron microscopic examination of the areas of contact between cells. The initial expiants are free of any apparent synaptic contacts. After 48 h in culture, simple swellings

P. E. Spoerri; P. Glees; W. Dresp



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


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.

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



Clearance of Human-Pathogenic Viruses from Sludge: Study of Four Stabilization Processes by Real-Time Reverse Transcription-PCR and Cell Culture  

PubMed Central

Sludges derived from wastewater treatment are foul-smelling, biologically unstable substances. As well as containing numerous pathogenic microorganisms, they also consist of organic matter that can be used as agricultural fertilizer. Legislation nevertheless requires sludges to be virologically tested prior to spreading by the counting of infectious enterovirus particles. This method, based on culture of enterovirus on BGM cells, is lengthy and not very sensitive. The aim of this study was to propose an alternative method of genome quantification for all enteroviruses that is applicable to verifying the elimination of viruses in complex samples such as sludges. Our complete protocol was compared to the official method, consisting of enterovirus enumeration with the most probable number of cythopathic unit (MPNCU) assay through the study of four stabilization procedures: liming, composting, heat treatment, and mesophile anaerobic digestion. Enterovirus quantities at the start of the stabilization procedures were between 37 and 288 MPNCU/g on the one scale and between 4 and 5 log genome copies/g on the other. It was shown that all procedures except mesophile anaerobic digestion were highly effective in the elimination of enterovirus particles and genomes in wastewater sludges. Reduction of viruses by mesophile anaerobic digestion was by only 1 log (infectious particles and genomes). In conclusion, stabilization processes can indeed be checked by virological quality control of sludges with gene amplification. However, the infectivity of genomes needs to be confirmed with cell culture or a correlation model if the virological risk inherent in the agricultural use of such sludges is to be fully addressed.

Monpoeho, S.; Maul, A.; Bonnin, C.; Patria, L.; Ranarijaona, S.; Billaudel, S.; Ferre, V.



Cell identification in primary cell cultures from skin  

Microsoft Academic Search

Summary  Primary cell cultures can readily be obtained from human skin using the explant method. With special care it is possible to\\u000a obtain primary cultures consisting of epidermal keratinocytes without fibroblast contamination. By means of differences in\\u000a their growth patterns and retention of specific differentiative functions in vitro, keratinocytes and fibroblasts can easily\\u000a be distinguished. The high degree of confidence in

B. Allen Flaxman



Scalable expansion of human pluripotent stem cells in suspension culture  

Microsoft Academic Search

Routine commercial and clinical applications of human pluripotent stem cells (hPSCs) and their progenies will require increasing cell quantities that cannot be provided by conventional adherent culture technologies. Here we describe a straightforward culture protoc