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Results for “Serial Passage”

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At least 847 records · Page 47Linked to original sources

PubMed1990

Improved culture conditions for Cowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants.

The causal agent of heartwater disease of domestic ruminants, Cowdria ruminantium, can, with difficulty, be isolated and passaged in lines of bovine endothelial cells grown in the presence of the Glasgow modification of Eagle's minimal essential medium. However, when Leibovitz's L-15 medium supplemented with 0.45% glucose at pH 6.0-6.5 is used as maintenance medium for these cells, isolation and serial passage may routinely be achieved.

Animals↗

PubMed2005

Combination of a mutagenic agent with a reverse transcriptase inhibitor results in systematic inhibition of HIV-1 infection.

Mutagenic treatments resulted in occasional, not systematic, human immunodeficiency virus type 1 (HIV-1) extinction. To study the possibility that a combination of an antiretroviral inhibitor, to reduce the viral replicative load, and a mutagenic agent could be more effective in producing viral extinction than a mutagenic agent alone, we have compared the efficiency of extinction of HIV-1 by the mutagenic deoxyribonucleoside analogue 5-hydroxydeoxycytidine (5-OHdC) alone and in combination with the HIV-1 nucleoside reverse transcriptase (RT) inhibitor AZT. Serial passages in peripheral mononuclear cells (PBMC) or MT-4 cells of primary HIV-1 isolates or HIV-1 NL4-3 in the presence of a single drug (AZT 0.01 microM or 5-OHdC 2 mM) failed to systematically extinguish high fitness HIV-1 replication after 16 serial transfers. However, systematic extinction of HIV-1 was observed when a combination of the mutagenic agent 5-OHdC and AZT was used. These results demonstrate that combinations of mutagenic agents and antiretroviral inhibitors have the potential to drive HIV-1 into extinction.

Cell Line↗

PubMed1994

Phenotypic and cytogenetic characterization of human bladder urothelia expanded in vitro.

A simple method for the harvest of bladder cell types from surgical specimens was used to generate strains of normal human urothelial cells that could be reproducibly cultivated, passaged and extensively expanded in serum-free medium. Immunostaining of the bladder epithelial cells with broadly reacting anti-cytokeratin antibodies and with an anti-cytokeratin antibody specific to cytokeratin 7, a transitional cell marker, indicated that they expressed a stable epithelial phenotype with serial passage. Low levels of immunostaining for E-cadherin and low levels of E-cadherin messenger ribonucleic acid, as determined by Northern blot analysis, and strongly positive immunostaining with an anti-vimentin antibody indicated collectively that the uroepithelial cells express a nonbarrier-forming phenotype under these culture conditions. However, when the urothelial cells were implanted subcutaneously into athymic mice on biodegradable synthetic polymers, they formed multilayered structures, suggesting that they retain the capability to differentiate in a living host. The urothelial cells proliferated in an epidermal growth factor independent manner and expressed high levels of transforming growth factor-alpha and amphiregulin messanger ribonucleic acids, suggesting the possibility of autocrine regulation of growth by epidermal growth factor-like factors. Cytogenetic analysis indicated that urothelial cells cultured for 6 passages possessed a normal chromosomal complement. These results demonstrate that primary cultures of autologous human bladder epithelial cells can be extensively expanded in vitro and, consequently, might be used in cell transplantation strategies for genitourinary reconstruction.

Amphiregulin↗

PubMed1986

[Experiments to culture the bovine pestivirus in heterologous cell cultures].

Attempts were made to adapt a strain of the bovine pestivirus to cell cultures of swine kidney in a series of 15 passages. The adaptation of the virus was followed up through the production of a cytopathic effect, through comparative investigations, and by means of cytologic preparations. It was found that the cytopathic effect induced and the titer of the virus grew with increasing the number of the serial passages. Immunofluorescence and cytologic data showed the progressive development of the virus in cell cultures of swine kidney. It was also established that the time of incubation of the infected cultures to produce an overall cytopathic effect was shorter.

Animals↗

PubMedDate not supplied

Passaging protocols for mammalian neural stem cells in suspension bioreactors.

Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders. As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large-scale expansion of mammalian NSC. Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media. The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time. We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter. When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 x 10(3) was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 microm) below levels at which necrosis would occur. Moreover, cell densities of 1.0 x 10(6) cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80%. The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates. We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.

Animals↗

PubMed1984

Isolation and propagation of nephropathia epidemica virus in bank voles.

Three strains of nephropathia epidemica (NE) virus were isolated from lung tissues of bank voles (Clethrionomys glareolus) and a grey-sided vole (C. rufocanus) trapped in Västerbotten county, Sweden. Two of these isolates were serially passaged in seronegative laboratory-bred bank voles. Experimentally infected animals developed a subclinical infection characterized by virus persistence, particularly in lung tissue. Attempts to infect other species of colonized rodents with NE virus and to isolate NE virus from acute phase patient blood were unsuccessful. The serial propagation of NE virus in colonized bank voles provides opportunities to study experimental infection in its reservoir rodent host.

Animals↗

PubMed1976

Temperature-sensitive mutants of rabies virus in mice: a mutant (ts2) revertant mixture selectively pathogenic by the peripheral route of inoculation.

Analysis of the pathogenic potential in mice of a variety of rabies and rabies serogroup viruses revealed that an apparently revertant population of virus derived from CVS mutant ts 2 had a unique capacity to selectively induce paralytic disease when given by a peripheral [intraplantar (i.pl.)] route of inoculation. Little paralytic disease was induced by high concentrations of virus administered by the intracerebral (i.c.) route, whereas paralytic disease and death were characteristically induced in mice given only a few infectious doses of virus i.c. Disease induced by i.pl. inoculation was dose dependent. Mice frequently survived paralytic disease induced by i. pl. inoculation, with clinical signs often persisting indefinitely; mice surviving i.c. inoculation of high concentrations of virus frequently exhibited chronic nonspecific signs of minor debility. Analysis of the ts 2 virus population indicated that it was composed of a mixture of ts and revertant virions, each with characteristic pathogenic (or nonpatholgnic) propensities, none of which was identical to the original composite ts 2 virus populations. Despite the heterogeneity of the ts 2 virus population, the typical pathogenic pattern of selective pathogenic capicity after i. pl. inoculation at high doses was retained during 11 ocnsecutive passages in suckling mouse brain. ts 2 virus was demonstrated to interfere with the disease-producing capacity of CVS fixed rabies virus when ts 2-CVS mixtures were inoculated i.c. However, attempts to demonstrate a particular propensity for induction in vitro of "autointerference" by ts 2 in serial passage in BHK-21 cell culture inoculated at high multiplicity were unsuccessful.

Animals↗

PubMed1986

Radiation-induced anchorage-independent growth and collagenase production in diploid human fibroblasts.

Early-passage human foreskin fibroblasts were exposed to X-ray doses ranging from 100 to 600 rad. The X-ray treatments resulted in cell killing in a dose-dependent manner as judged by the colony-forming efficiency of the cells. When cultures exposed to radiation were serially passaged and checked at various times for growth in semi-solid medium they showed the presence of cells with the ability to grow under anchorage-independent conditions (in agarose) at 24 population doublings. The frequencies of colonies with the ability to grow in agarose increased with increasing doses of X-rays above the levels observed for control cultures under similar conditions. When assayed for plasminogen activator levels, the X-ray-treated cultures at various passages showed insignificant differences from levels observed in control cultures. However, the amounts of collagenase (type IV collagen-specific) increased significantly by 32 population doublings in the X-ray-treated cultures compared with control cultures. Our results suggest that the production of type IV collagen-specific collagenase could be useful as an in vitro marker for the transformation of human diploid fibroblasts by X-irradiation.

Cell Survival↗

PubMed2003

Hyperoxia augments pulmonary lipofibroblast-to-myofibroblast transdifferentiation.

Bronchopulmonary dysplasia (BPD) remains a major cause of morbidity and mortality in premature infants, and despite many advances, its pathophysiology remains incompletely understood. Exposure of the premature lung to hyperoxia is commonly implicated in its pathogenesis. However, the exact link between hyperoxia and BPD, particularly its role in the generation of myofibroblasts, the signature cell-type for lung fibrosis, is undetermined. There is increasing evidence that lipid interstitial fibroblasts play an important role in injury-repair mechanisms in various organ systems. This study demonstrates that exposure to hyperoxia augments the transdifferentiation of pulmonary lipofibroblasts to myofibroblasts. Fetal rat lung fibroblasts (FRLF) from embryonic (e) (term = e22) 18 and e21 gestation were studied. After initial culture in minimum essential medium (MEM) and 10% fetal bovine serum (FBS) in 21% O2 / 5% CO2 at 37 degrees C, FRLF were maintained in MEM and 10%FBS at 37 degrees C under control (21% O2 / 5% CO2) and under experimental conditions (24-hour exposure to 95% O2 /5% CO2) at passage (P) 1 and 5. At each passage, cells were allowed to attach to 100 cm2 culture dishes and grow in 21% O2 before being subjected to the experimental conditions. Passage 1 and 5 cells were analyzed for the expression of well-characterized lipogenic and myogenic markers based on semiquantitative competitive RT-PCR (for parathyroid hormone-related protein receptor [PTHrPR]), adipose differentiation related protein (ADRP), and alpha smooth muscle actin (alphaSMA), triglyceride uptake, and leptin assay. Serial passage and maintenance of cells in 21% O2 resulted in a significant decrease in the expression of the lipogenic markers from P1 to P5, spontaneously. This decrease was greater for e18 than for e21 FRLF. However, exposing cells to 95% O2 augmented the loss of the lipogenic markers and gain of the myogenic marker from P1 to P5 in comparison to cells maintained in 21% O2. These changes were also greater for e18 vs e21 lipofibroblasts. These changes in mRNA expression were accompanied by decreased triglyceride uptake and leptin secretion on exposure to hyperoxia. These results suggest that exposure to hyperoxia (95% O2) augments the transdifferentiation of pulmonary lipofibroblasts to myofibroblasts.

Actins↗

PubMed1975

Origin of lymphoid lines established from mixed cultures of cord-blood lymphocytes and explants from infectious mononucleosis, Burkitt lymphoma and healthy donors.

Lymphocytes were explanted from EBV-seropositive donors including peripheral blood of infectious mononucleosis patients, healthy donors and EBV-genome-carrying cells from Burkitt lymphoma (BL) biopsies or nude mouse-passaged, BL-biopsy-derived lines. The explanted cells were mixed with fresh cord-blood lymphocytes from mice of the opposite sex. In all categories of derived lines, cord-blood cell progeny was predominant, as judged by the sex marker and other associated markers. Only one BL biopsy line, serially passaged in nude mice, gave rise to a monoclonal lymphoma line.

Animals↗

PubMed1987

Immortality of cell cultures derived from brains of mice and hamsters infected with Creutzfeldt-Jakob disease agent.

Isolates from six patients with Creutzfeldt-Jakob disease (CJD) were injected into various strains of hamsters and mice, and the infective agent was propagated. Serially passaged cultures were established from these CJD agent-infected brains and from uninfected control brains. All healthy cultures (21 out of 21) from CJD agent-infected brains became immortal and/or transformed. In contrast only 3 out of 13 normal brain cultures became immortal, and the rest died out with serial propagation in vitro. The fact that permanent cell lines were readily derived from multiple rodent strains and all CJD isolates tested suggests that a transforming capability is an intrinsic property of CJD agents. This conclusion is supported by demonstrations of in vitro cell transformation by CJD infectious brain fractions. Although the molecular mechanism of transformation events associated with the CJD agent is not presently known, a provocative possibility is that the CJD agent has a direct effect on the host genome by mechanisms analogous to those known for slowly oncogenic retroviruses.

Animals↗

PubMed1995

Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.

PURPOSE: These studies were designed to develop an orthotopic model for human bladder cancer and to isolate variant metastatic cell lines. MATERIALS AND METHODS: The human bladder cancer cell line 253J was implanted into the muscular wall of the bladder of athymic nude mice. By in vivo recycling, we selected for 2 variant cell lines: 253J B-V, a bladder line isolated after 5 serial passages in the bladder, and 253J lung-IV, established from a lung tumor nodule that was recycled through the bladder. RESULTS: These 2 cell lines showed enhanced tumorigenicity, as measured by a decreased latent period, and rapid growth as compared with the parental cell line. Moreover, orthotopic implantation of these cell lines resulted in metastasis to the lungs. These in vivo-selected, metastatic cell lines exhibited unique karyotypic alterations, increased anchorage-independent growth, overexpression of basic fibroblast growth factor, altered expression of adhesion molecules and the ability to migrate through Matrigel. CONCLUSIONS: This reproducible model of human bladder cancer offers the opportunity to study cellular properties associated with tumor progression and metastasis and is suitable for the evaluation of new therapeutic strategies for invasive bladder cancer.

Animals↗

PubMedDate not supplied

Human fibroblasts from normal and malignant breast tissue grown in vitro show a distinct senescence profile and telomerase activity.

The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle alpha-actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.

Actins↗

PubMed1990

Oligonucleotide fingerprint analysis of a wild strain & its attenuated variant of Japanese encephalitis virus.

A strain of Japanese encephalitis (JE) virus was passaged serially through primary chick kidney cell cultures (45 times) and primary baby hamster kidney cell cultures (21 times). The resultant virus lost its lethal effect to 3 wk old mice by the ic route and 10 day old mice by the ip route. The oligonucleotide fingerprint analysis of the parent and the passaged strains showed 64 spots in common; 17 spots were present in the parent strain which were absent in the passaged virus, while the latter had acquired 9 spots which were not present in the parent virus.

Animals↗

PubMed1988

Topographical variation in growth in cultured bovine retinal pigment epithelium.

Cell cultures of adult bovine retinal pigment epithelium (RPE) were propagated from posterior and equatorial regions of the same eyes to study topographical differences in cell growth. To generate the cultures, the retina was removed, eyecups were incubated in collagenase followed by trephining three zones from the posterior area centralis and three zones from the equatorial region of the tapetal retina. Outgrowth in primary cultures and proliferation in passaged cultures were evaluated in paired cultures derived from the same eyes. Relative to cells from the more peripheral regions, RPE cells from the area centralis showed lower growth potential which was manifested as reduced cell numbers in primary outgrowth and a smaller fraction of serum-responsive cells when cultures were serially passaged. Cells from older donors have been shown to exhibit similar reductions in in vitro growth potential, making it appear as if, with regards to proliferation, RPE from the area centralis is more "aged" than RPE from more peripheral regions of the same eyes.

Animals↗

PubMed1986

Serial culture of single adult human prostatic epithelial cells in serum-free medium containing low calcium and a new growth factor from bovine brain.

Primary cultures of epithelial cells from human prostate acini proliferate in defined medium. However, the limited availability of human tissue and the lack of knowledge of the conditions required for clonal growth and serial culture of epithelial cells have limited progress in the study of human prostate cell biology. Here we report conditions that permit the proliferation of single epithelial cells from normal, benign hyperplastic, and carcinomatous prostate through three to four serial passages, which represents at least seven to nine cumulative doublings of the cell populations. Primary cultures were prepared from prostatic acini. Monolayers resulting from the outgrowth of epithelial cells from acini were harvested and dissociated into suspensions of single cells which gave rise to discrete colonies in subsequent culture. The requirements for successful serial culture were (a) a low calcium concentration, (b) the presence of a growth factor that is concentrated in bovine neural tissue, (c), detachment of the epithelial cells with collagenase, and (d) harvest of cells before the cell concentration reached 6000 cells/cm2 of culture surface. Suspensions of single cells were successfully stored between subcultures in 10% dimethylsulfoxide with 5% fetal bovine serum and revived after storage for up to 2 months in liquid nitrogen.

Animals↗

PubMed1978

Variations in biological properties of geographic strains of western equine encephalomyelitis virus before and after passage in Culex tarsalis and Culiseta melanura.

Western equine encephalomyelitis (WEE) virus strains from the eastern and western United States were compared using two biological markers: antigenic properties measured by cross complement fixation (CF) tests and the size of plaques produced in Vero cell cultures. Slight antigenic differences were discernible on a geographic basis, and all of the virus strains from the West produced significantly larger plaques than the eastern isolates. One virus strain from each region was serially passaged in Culex tarsalis and Culiseta melanura by intrathoracic inoculation. No change in plaque size was detected as a result of mosquito passage. After passage of the eastern strain of WEE virus in C. tarsalis, its mouse brain CF antigen titer was increased to resemble the high CF antigen titer produced by the western strain. However, the antisera titers did not change when tested with this antigen. A cross plaque reduction neutralization test (PRNT) with the two virus strains before and after passage in mosquitoes failed to reveal any antigenic changes.

Animals↗

PubMedDate not supplied

Mild stress-induced stimulation of heat-shock protein synthesis and improved functional ability of human fibroblasts undergoing aging in vitro.

Repeated mild heat-shock (RMHS) treatment has anti-aging hormetic effects on human fibroblasts undergoing aging in vitro. Since heat and various other stresses induce the transcription and translation of heat-shock proteins (Hsp), it was investigated if RMHS treatment affected the basal levels of four major stress proteins Hsp27, 70, 90 and Hsc70. The basal levels of Hsp27, Hsc70, and Hsp70 increased significantly in late passage senescent cells, which is indicative of an adaptive response to cumulative intracellular stress during aging. RMHS increased the levels of these Hsp even in early passage young cells and were maintained high throughout their replicative lifespan. In comparison, the amount of Hsp90 decreased both with aging and RMHS treatment in vitro. However, whereas the difference in the levels of Hsp70 and Hsp90 was statistically significant, the levels of Hsp27 and Hsc70 were statistically similar in normal and RMHS-treated serially passaged cells. These alterations were accompanied by an improved functional and survival ability of the cells in terms of increased proteasomal activities, increased ability to decompose H(2)O(2), reduced accumulation of lipofuscin and enhanced resistance to ethanol, H(2)O(2) and UV-A radiation.

Blotting, Western↗