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At least 19 recordsLinked to original sources
PubMed2000

Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture--primary culture cells markedly differ from fourth-passage cells.

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

Arthritis, Rheumatoid↗
PubMed1990

Antigenic staining patterns of human glioma cultures: primary cultures, long-term cultures and cell lines.

The immunocytochemical staining patterns of cultured glioma cells were investigated. Fifty nine individual cases were stained at different in vitro ages for glial fibrillary acidic protein, fibronectin, galactocerebroside, HNK-1/Leu 7, A2B5, vimentin, factor VIII and A4. Histologically, the cases were composed of eight low-grade astrocytomas, 11 high-grade astrocytomas, four low-grade oligodendrogliomas, seven high-grade oligodendrogliomas and 29 glioblastomas. The 45 cases were analysed within the first 3 weeks of culture, many of them as primary cultures. In 11 cases stainings were performed repeatedly at intervals of up to 6 months. Glial fibrillary acidic protein staining was positive in most of the early cultures of astrocytomas (low and high grade) and glioblastomas; expression in more than 50% of the cells was found in 1 of 5 low-grade astrocytomas, 5 of 11 high-grade astrocytomas and 14 of 29 glioblastomas. Two of the high-grade astrocytomas were stained once more after 6 weeks in culture and were found to be only 1% positive for glial fibrillary acidic protein but strongly positive for fibronectin. The same was true for five of the glioblastoma cases. Two of these cases remained glial fibrillary acid protein positive and developed into stable permanent cell lines. Only one case started with 1% of glial fibrillary acidic protein positive cells and later developed into a 99% glial fibrillary acidic protein positive cell line. Neither HNK-1/Leu 7 expression nor A2B5 staining appeared to have a relationship to the glial fibrillary acidic protein staining. It was observed that glial fibrillary acidic protein and HNK-1/Leu 7 were both 100% in some cases but that later one of the two antigens disappeared but not the other. The amount of glial fibrillary acidic protein staining does not allow the prediction of A2B5 staining. The study shows that initiation of primary cultures on an extracellular matrix yields more glial fibrillary acidic protein positive cells in primary cultures than have been found in other studies. It is concluded that only a rigid standardization of culture conditions will ensure the validity of comparisons of in vitro data obtained in primary cultures.

Antigens, Neoplasm↗
PubMed1990

Toxicological studies on a benzofuran derivative. III. Comparison of peroxisome proliferation in rat and human hepatocytes in primary culture.

Primary cultures of rat and human hepatocytes were used in our in vitro studies for investigating species differences in the response to a peroxisome proliferating benzofuran derivative, benzbromarone. Cyanide-insensitive palmitoyl coenzyme A oxidation (a marker of peroxisome fatty acid beta-oxidation) and electron microscopy were used to assess peroxisome proliferation. Hepatocytes were cultured essentially as described by Mitchell et al. (1984, Arch. Toxicol. 55, 239-246); clofibric acid and mono(2-ethylhexyl) phthalate (MEHP) were used as reference compounds, as they are well known to cause peroxisome proliferation in rat hepatocytes in primary culture. The benzofuran derivative, tested at drug concentrations ranging from 2.37 to 59.20 microM in rat hepatocyte primary cultures, induced, after 96 hr, a dose-related increase of the peroxisomal beta-oxidase activity correlated with an increased number of peroxisomes; this increase was much less marked than that obtained with clofibric acid or MEHP. By contrast, using the same range of concentrations, human hepatocytes in primary culture treated with benzbromarone revealed no enhancement of enzymatic activity and no concomitant statistically significant increase in the number of peroxisomes; the same observations were reported with clofibric acid and MEHP. These results demonstrate clearly that species differences in sensitivity to peroxisome proliferation with the benzofuran derivative do exist.

Animals↗
PubMed1986

Differentiation of astrocytes and oligodendrocytes from germinal matrix cells in primary culture.

Primary cultures derived from neonatal rat forebrain grow almost entirely as glial cultures, with a large astrocytic preponderance and smaller numbers of oligodendrocytic cells. Although both astrocytic and oligodendrocytic characteristics are acquired in vitro, the origins of both types of glia in primary cultures have not been determined. We tested the hypothesis that glia differentiate in vitro from immature neuroectodermal cells by following the fate of germinal zone cells in primary cultures. A monoclonal antibody that binds GD3 ganglioside was used as a marker for cells of the subventricular zone (SVZ), since antibody binding in newborn rat forebrain could be detected by immunofluorescence only in the SVZ of newborn rats (Goldman et al., 1984). We followed the expression of glial fibrillary acidic protein (GFAP), an astrocytic marker; galactocerebroside (GC), an oligodendrocytic marker; and GD3 during the first several weeks of culture. Both GFAP and GC expression were first detected in cells that bound the GD3 antibody. Astrocytes developed during the first week in vitro; eventually, they lost the ability to bind the GD3 antibody and most became GD3-/GFAP+ cells. In high-density cultures, a population of small cells that resided on top of the astrocytic monolayer retained GD3 expression. GC-antibody binding was first observed in these cells of the upper layer, although it was not readily apparent until the second week of culture. Few GC+ cells were seen in cultures grown at low density, however.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Proximal tubular cells in primary culture.

Primary cultures of mammalian proximal tubules (PT) can be established in serum-free, hormone-supplemented media. These cells attain confluence, demonstrate morphological polarity and exhibit vectorial fluid transport. Primary cultures of rabbit PT cells retain the transport properties of the parent cells and exhibit Na+-H+ exchange, Na+-Ca2+ exchange and Na-glucose co-transport, the latter process having kinetic properties comparable to those described for the late proximal tubule. Glucose which enters the cell via the luminal Na-glucose co-transport system undergoes metabolism to CO2; it is not clear whether this glucose oxidation, which is minimal in PT cells under physiological conditions, is due to selection of a specific cell type or to the nature of the culture conditions. Confluent, quiescent PT cells in culture can be induced to hypertrophy by addition of supraphysiological concentrations of insulin, PGE, and hypertonic NaCl; each stimulus also leads to an early increase in Na+-H+ antiport suggesting that this transport process may be causally related to the process of cell growth. The primary culture thus represents a promising model for future studies on the cellular events which govern the process of cell hypertrophy.

Adenylyl Cyclases↗
PubMed1980

Glucocorticoid receptors and inhibition of neonatal mouse dermal fibroblast growth in primary culture.

Primary cultures of dermal fibroblasts from neonatal mice were used to investigate some of the anti-inflammatory effects of glucocorticoids in vitro as influenced by the genetic background of two different strains of mice (A/J and C47 Bl/6J). Fibroblasts were cultured in the absence or presence of various glucocorticoids for 2-7 days. After 4-7 days in the presence of steroid, DNA synthesis was reduced by 50-85% while protein synthesis was inhibited by 50-60%. Corticosterone produced a dose-dependent inhibition of DNA synthesis in these cells with a 50% reduction occurring at 10 nM. Specific, high affinity, low capacity binding proteins for [3H]dexamethasone or [3H]triamcinolone acetonide were identified in the cytoplasm of neonatal dermal fibroblasts which had an apparent Kd of 9 nM and approximately 5,200-6,400 binding sites/cell. Sedimentation analysis of the [3H]triamcinolone acetonide-receptor complexes on low salt glycerol gradients exhibited binding in the 7 to 8 S region of the gradients. These studies demonstrate that inhibition of growth of primary cultures of mouse neonatal dermal fibroblasts by glucocorticoids is probably mediated by a receptor-mediated pathway, and that this primary culture system might be useful in delineating other anti-inflammatory effects of glucocorticoids in vitro.

Animals↗
PubMed2001

Assessment of tissue-level kidney functions with primary cultures.

Primary cultures of renal proximal tubule have become important tools for examination of the mechanisms and control of transepithelial transport processes. The utility of the culture preparations for study of integrated tissue functions depends upon their accurate expression of in vivo transport processes. Maintenance of differentiation in culture is enhanced by contractible collagen substratum. Epithelial monolayer primary cultures of flounder and chicken proximal tubule, prepared by enzymatic and mechanical maceration with differential centrifugation, exhibit functional properties at the tissue level that generally resemble known properties of the freshly isolated or in vivo proximal tubule. Transepithelial electrical resistances and potential differences are very similar or identical to those of intact tubules. Na+-dependent glucose transport, a hallmark of proximal tubule function, has the same properties in culture as the tissue in vivo. Similarly, where appropriate comparisons are possible, amino acid, uric acid, and organic anion and cation transepithelial transport processes are qualitatively very similar in culture and in vivo. These two non-mammalian primary proximal tubule culture systems adequately reflect in vivo function, and thus provide opportunities for experimental manipulation otherwise not available.

Amino Acids↗
PubMed1985

Morphologic and metabolic development of human fetal epiphyseal chondrocytes in primary culture.

Primary chondrocyte culture was carried out after enzymatic digestion of femoral and tibial epiphyseal cartilage of human fetuses, collected with informed parental consent within 12 h postmortem. Chondrocytes were cultured in HAM F-12 medium with penicillin and 15% serum. Three types of serum were used: human placental cord serum (HPS), fetal calf serum, and human male adult serum. Chondrocytes cultured with HPS grew as monolayers, formed abundant colony groups with a highly metachromatic pericellular matrix, and floating round cells were observed in the culture medium. By the 10th day of culture the great majority of proteoglycans present in the culture medium were found as aggregates. Chondrocytes cultured with fetal calf serum or human male adult serum grew as monolayers, were polygonal in shape, and the pericellular matrix was far less developed than in HPS cultures. By the confluent phase of growth, only approximately a third of the proteoglycans present in the culture medium were found as aggregates. Chondrocytes cultured with HPS proliferated significantly more rapidly than those cultured with fetal calf serum or human male adult serum. The results suggest that certain, as yet unidentified, factors are present in sufficient amount in HPS to allow chondrocytes in culture to retain phenotypic morphological and biochemical characteristics. HPS also facilitates growth of human fetal epiphyseal chondrocytes in culture. Primary human fetal epiphyseal chondrocyte culture could be a suitable experimental tool for the in vitro study of biochemical characteristics of cartilage and factors involved in fetal cartilage metabolism.

Blood↗
PubMed1987

Characterization of cell types in human breast tumor primary cultures.

Primary cultures of cells from breast carcinomas were attempted in 74 cases. Growth was observed in 46 cases. Using immunochemical demonstration of keratin proteins (KER), epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA), three morphologically distinct cell populations were characterized and described. Two cell types (E- and E'-cells) were identified as epithelial by their positive staining for KER and EMA. The third type (F cells) displayed a negative staining for these two epithelial markers; they were considered as stromal cells (fibroblasts). More than 50% of the cultures consisted of pure epithelial cells. Positive CEA staining was observed only in KER- and EMA-positive cells. Out of the 30 cultures, 15 displayed positive staining for CEA. In 7 of these, 30-50% of cells displayed positive, diffuse staining for CEA. The other 8 cultures consisted of more than 50% CEA-positive cells. Strong and homogeneous positivity was restricted to the E-cells, while in the same cultures, E'-cells displayed heterogeneous and diffuse staining. Efficiency and value of this cell culture system are discussed, in comparison with other human breast tumor cell (HBTC) culture techniques. Identification of growing cells and cellular composition of primary cultures are emphasized.

Breast Neoplasms↗
PubMed1980

Multiple hormone requirement for the synthesis of alpha 2u-globulin by monolayers of rat hepatocytes in long term primary culture.

Primary cultures of adult rat hepatocytes were prepared by releasing liver cells through fractional trypsinization and by a novel nonenzymatic procedure involving the imprinting of liver explants on the surface of the culture flask (explant imprinting). The comparative properties of these two primary cultures and their abilities to synthesize alpha 2u-globulin under multihormonal stimulation were investigated. The hepatocytes isolated from male rats by fractional trypsinization were found to divide rapidly and form a confluent monolayer within 5-7 days of culture. However, these cells failed to synthesize alpha 2u-globulin even after treatment with 5 alpha-dihydrotestosterone, GH, T3, and dexamethasone, hormones with known stimulatory effects on the synthesis of alpha 2u-globulin in vivo. On the contrary, hepatocytes prepared from male rats by the nonenzymatic procedure of explant imprinting were found to synthesize alpha 2u-globulin for more than 2 weeks and responded to GH, T3, and dexamethasone with enhanced production of this protein. Fortification of the culture medium with all of the above three hormones caused an approximately 9-fold enhancement of alpha 2u production within 4 days of culture compared to production in control culture without these hormones. The above results substantiate earlier findings concerning the multiple hormonal requirement for hepatic synthesis of alpha 2u-globulin in vivo and show that isolation and culture of hepatocytes by nonenzymatic explant imprinting may serve as a useful procedure for studying endocrine regulation of gene activity in vitro.

Alpha-Globulins↗
PubMed1995

Homologous conditioned medium enhances expression of TRH in hypothalamic neurons in primary culture.

Primary cultures of hypothalamic cells maintained in the presence of serum were either kept with homologous conditioned medium (CM) (i.e. only half of the medium was removed at each medium change) or without (total medium change). In cultures with homologous CM, TRH levels were increased. The effects of CMs from various intervals of the primary culture were tested. The strongest increases of TRH levels were obtained with CM from cultures enriched with hypothalamic glia.

Amino Acid Sequence↗
PubMed1993

Appearance and evolution of calcium currents and contraction during the early post-fusional stages of rat skeletal muscle cells developing in primary culture.

Primary cultures from enzymatically dissociated satellite cells of newborn rat skeletal muscles enabled developmental in vitro studies of mechanical and electrical properties during the first steps of myogenesis. The present work focused on the appearance, evolution and roles of two types of calcium currents (ICa,T and ICa,L) and of depolarization-induced contractile activity during the early stages of muscle cell development in primary culture. Prefusional mononucleated cells (myoblasts), young myotubes of 1 day (with less than 10 nuclei) or 2-3 days (more than 9 nuclei) and myoballs from 4-6, 7-9, 10-12 and 13-16 days cultures were patch-clamped (whole-cell configuration), and calcium currents and contraction simultaneously recorded. Sodium but not calcium currents could be recorded at the myoblast stage. In young myotubes (1 day), ICa,L was present with high incidence as compared to ICa,T, which was poorly expressed. Contractile responses appeared at the next stage (2-3 days) while the occurrence of ICa,T progressively increased. This developmental evolution of the calcium currents and contraction expression was accompanied by some changes in their characteristics: the ICa,T/ICa,L amplitudes ratio progressively increased and the time-to-peak of contraction progressively decreased with the age of myoballs. Physiological functions for calcium currents in developing muscle are suggested and discussed: ICa,T, which is transiently expressed, could be involved in the pacemaker-like activity while ICa,L could serve as an early contraction triggering mechanism and/or initially to fill and then to maintain the intracellular calcium stores.

Animals↗
PubMedDate not supplied

Cytotoxicity of xenobiotics in adult rat hepatocytes in primary culture.

Primary cultures of hepatocytes isolated by collangenase perfusion of adult rats were used as an in vitro system for assessing cytotoxicity of xenobiotics. The leakage of two intracellular enzymes, lactate dehydrogenase (LDH) and glutamic oxaloacetic transaminase (GOT) were compared as indicators of cell damage. Although some differences in sensitivity were detected, either enzyme could be used to evaluate the cytotoxic potential of a chemical. Release of LDH was also compared with determination of cell viability by trypan blue uptake. For cultures exposed to 2-aminofluorene or alpha-naphthylisothiocyanate both endpoints gave comparable results. The cytotoxicities of benzo(a)pyrene, 2-aminofluorene, 3,2'-dimethyl-4-aminobiphenyl, methylene-bis-2-chloroaniline, aflatoxin B1, pyrene, aflatoxin G2, fluorene, methapyrilene, and alpha-naphthylisothiocyanate were monitored by the release of LDH and were found to be related to the length of exposure and concentration of the chemical. For chemicals that elicited DNA repair, little or no cytoxicity was observed at genotoxic concentrations in the hepatocyte primary culture/DNA repair test. Thus, measurement of enzyme release provides a means of quantifying cytotoxicity. Moreover, the use of hepatocyte primary cultures permits identification of genotoxic effects versus general cytotoxic effects of chemicals.

1-Naphthylisothiocyanate↗
PubMed1989

Ethanol in clinically relevant concentrations enhances expression of oligodendroglial differentiation but has no effect on astrocytic differentiation or DNA synthesis in primary cultures.

Primary cultures of cerebral glia derived from newborn rat brain were utilized to evaluate the effects of ethanol on DNA synthesis and cellular differentiation. Glutamine synthetase was employed as a marker for astrocytic differentiation and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), for oligodendroglial differentiation. Concentrations of ethanol of 17-86 mM, i.e., a concentration range comparable to that observed in humans, were utilized. No effect of ethanol on DNA synthesis was observed, despite the use of synchronized cultures. Similarly, no effect of ethanol on the developmental increase of glutamine synthetase activity was seen, despite the use of astrocytes purified by the selective detachment technique and a prolonged duration of exposure to ethanol (17 days). In contrast, however, a striking enhancement of CNP activity was demonstrable in both mixed glial cultures and in oligodendroglia purified by the selective detachment technique. In the latter cells, the stimulatory effect of ethanol was evident within 9 days of exposure, and after 17 days of exposure, ethanol-treated cells exhibited a two-fold higher CNP activity than did control cells. This peak effect was observed at an ethanol concentration of only 17 mM. Thus, these data indicate that (1) clinically relevant concentrations of ethanol have no effect on either DNA synthesis or on at least one expression of astrocytic differentiation in glial primary cultures, but (2) these concentrations exert a striking enhancement of CNP activity, a marker of oligodendroglial differentiation. The findings have implications both for the effects of ethanol on the developing nervous system and the regulation of oligodendroglial differentiation.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1989

Electrophysiologic study of rabbit proximal tubular cell monolayers in primary culture.

Primary cultures of renal cortical cells prepared by selective sieves have been found to display some characteristics of renal proximal tubular epithelium but their site of origin has not been confirmed by electrophysiologic studies. Cells were cultured in a defined medium on collagen gels. Confluency was approached after 7-10 days but gels were found to have zero transepithelial resistance unless they were allowed to contract spontaneously. With the appearance of a nonzero resistance, there was a change in morphology to a more columnar cell with better developed microvilli. These structural features were particularly prominent in clusters of proliferating cells observed on and around remnants of original tubules embedded in the gel. In noncontracted cultures there was no focal cell clustering and cells were squamous-like with rudimentary microvilli, similar in appearance to cells grown on plastic culture dishes. Measurements made in contracted monolayers yielded an average transepithelial resistance of 6.5 omega cm2, a spontaneous transepithelial potential difference of +0.9 mV, measured with respect to the serosa, and an apical membrane potential of -75 mV when cells were bathed in 0.4 mM K and -49 mV when cells were bathed in 4 mM K media. Mucosal protamine (50 micrograms/ml) increased transepithelial resistance by 22%, suggesting that the epithelial cell tight junctions were responsive to external stimuli. Monolayers were anion selective, giving a dilution potential (lumen-directed NaCl gradient) of -2.6 mV with respect to the serosa. These experiments show that primary culture of rabbit renal cortical cells separated by differential sieves displays electrophysiologic and morphologic characteristics of a proximal renal tubular epithelium. Confluency and attainment of differentiated morphology and function are promoted when monolayer cells are not bound to an unyielding substrate.

Animals↗
PubMed1988

Mu and delta opiate receptors coupled negatively to adenylate cyclase on embryonic neurons from the mouse striatum in primary cultures.

Primary cultures of pure populations of neuronal or glial cells from the striatum, the cerebral cortex, and the mesencephalon of the mouse embryo were used to look for the presence of opiate receptors coupled to adenylate cyclase. Leu-enkephalin inhibited cAMP production in membranes of embryonic striatal neurons but not in those of other cell types examined. Mu and delta opiate receptors seemed to be coupled negatively to adenylate cyclase in embryonic striatal neurons. It was found that DTLET (a selective delta agonist), as well as DAGO (a selective mu agonist), inhibited cAMP production on these cells. DTLET but not, however, DAGO produced a similar effect on homogenates from the adult rat striatum and on membranes from the neuroblastoma x glioma hybrid cell line NG 108-15, two preparations known to possess only delta receptors negatively coupled to adenylate cyclase. The selective kappa agonist U 50.488 was ineffective on all types of membrane preparations used. The inhibitory effects of both DTLET and DAGO on basal adenylate cyclase activity in striatal neurons were reversed by naloxone with a similar efficacy. Two other selective mu agonists, trimu 5 and morphiceptin, inhibited cAMP production in membranes of striatal neurons as well. The nonadditivity of the inhibitory effects of DTLET and DAGO on basal or forskolin-induced activation of adenylate cyclase suggested that mu and delta receptors were colocalized on a similar subpopulation of striatal cells in primary culture. These cells possess dopaminergic receptors of the D1 subtype as well since the amplitude of the inhibitory effects of DTLET and DAGO on cAMP production was increased in the presence of dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1987

Maintenance of woodchuck hepatitis virus activity in woodchuck hepatocyte primary culture.

Primary cultures of non-proliferating hepatocytes isolated by the two-step collagenase perfusion method from woodchuck naturally infected with hepatitis virus (WHV) were used to study WHV propagation in vitro. Hepatocytes carrying WHV DNA exhibited a very high level of survival and retained their morphological characteristics for 2 to 3 months. Over this time, they were found to produce virus-specific proteins and release viral particles with DNA polymerase activity into the medium. Using Southern blot analysis and a recombinant hepatitis B virus DNA plasmid probe, intracellular and extracellular viral DNA was consistently detected. Only extrachromosomal forms of WHV DNA were observed and no integration could be demonstrated in the DNA of the cells. The WHV DNA patterns were repeatedly identical with a characteristic smear starting from 3.3 kb associated with other smaller DNA fragments which presumably represented intermediate replicative forms of viral DNA. Furthermore, dot blot hybridization of the total RNA revealed the presence of WHV-specific transcripts in cells after 3 weeks of culture. All these results are compatible with the maintenance of active WHV replication in vitro although it was somewhat reduced after the first day of culture. This provides a mammalian model for hepadnavirus replication studies in stable primary hepatocyte cultures.

Animals↗