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Results for “Serial Passage”

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PubMed1999

Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate.

Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspirates of the iliac crest of normal volunteers. The cells were isolated by their adherence to plastic and then passed in culture. Some of the samples expanded through over 15 cell doublings from the time frozen stocks were prepared. Others ceased replicating after about four cell doublings. The replicative potential of the cells in culture was best predicted by a simple colony-forming assay in which samples from early passages were plated at low densities of about 10 cells per cm2. Samples with high colony-forming efficiency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology. The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in culture and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocytes. The loss of multipotentiality following serial passage in culture may have important implications for the use of expanded MSCs for cell and gene therapy.

Bone Marrow Cells↗

PubMed1976

Alteration of the in vitro host range of rabies virus after serial chick embryo cell passage using alkaline maintenance medium.

HEP Flury strain of rabies virus maintained by 7-day chicken egg passage (parent line) and the same strain serially passaged in primary chick embryo (CE) cells using alkaline maintenance medium (AM line) were inoculated to cells of various species. Growth was negative in primary mouse embryo, L and HeLa cells, and positive in primary hamster kidney and BHK21 cells with both lines. An all-or-none difference between the two lines was observed in primary monkey kidney and Vero cells. The parent line did not multiply in these monkey cells, whereas the AM line grew to high titers. In the case of Vero cells a unique cytopathic effect (CPE) was induced by the AM line. After five consecutive passages in Vero cells, the CPE-inducing agent was identified as rabies virus by a neutralization test. It was infective to intracerebrally inoculated suckling mice but not to adult mice, and its Vero cell-infective titer determined by CPE induction was about 1 log lower than the baby mouse-infective and CE plaque-forming titers. In contrast to the AM line, HEP Flury strain receiving 150 CE cell passages under neutral maintenance medium and three other strains receiving similar CE cell passages all failed to grow in Vero cells.

Animals↗

PubMed1982

Somatostatin-reduced proliferation of murine aplastic carcinoma conditioned to diabetes.

Murine mammary aplastic carcinoma, when it is passed from one alloxan-diabetic animal to another, after several passages becomes conditioned to hypoinsulinemia by secreting its own substance(s) immunologically cross-reactive with insulin (SICRI). Although not cytotoxic, the total daily dose of 2.5 mg of synthetic linear somatostatin per one kg body mass, administered in three or more portions, retards tumor growth in normoinsulinemic mice and the proliferation of tumors preconditioned by three serial passages in diabetics. After the first passage, somatostatin treatment has no effect on the already slow growth of the unconditioned tumor. Somatostatin-reduced tumor proliferation is accompanied by the strong suppression of insulin and SICRI levels, respectively; this effect is completely abolished by the administration of modest doses of crystalline insulin. It is concluded that somatostatin retards tumor growth by diminution of plasma levels of insulin and SICRI, respectively.

Animals↗

PubMed1975

Characteristics of proliferative cells from young, old, and transformed WI 38 cultures.

Mitotic cells were obtained by a shake off procedure from cultures of normal WI 38 cells at various passage levels, and from SV-40 virus transformed cells. The size of all mitotic WI 38 cells was similar regardless of in vitro age, whereas cells from the monolayer displayed an age-related increase in size. Mitotic transformed cells were similar to normal in size, but no size changes were observed in transformed monolayer cells during serial passage. Ultrastructural studies of mitotic WI 38 cells revealed no consistent change in the numbers of mitochondria or lysosome-like bodies during aging in culture. Mitotic transformed cells displayed numbers of mitochondria comparable to normal cells, but lysosome-like bodies occurred less frequently. Size distribution and structural characteristic are presented in relation to the ability of cells to synthesize DNA and to divide. These results support the contention that aging in WI 38 cultures is characterized by a declining fraction of homogeneous, actively dividing cells, and an increasing fraction of heterogenous nondividers that display senescent changes.

Cell Biology↗

PubMed2000

Conditionally immortalized mouse hepatocytes for use in liver gene therapy.

BACKGROUND AND AIMS: The use of hepatocytes for gene therapy is limited by the difficulty of maintaining and altering primary liver cells in culture. A conditionally immortalized mouse hepatocyte cell line has been developed which can be passaged indefinitely at the permissive temperature (33 degrees C), but fails to proliferate and dies at the non-permissive temperature (39 degrees C) in vitro. METHODS: Hepatocytes were harvested from a 6 week-old male transgenic mouse ('immortomouse') carrying a thermolabile SV40 Large T gene, using a modified two-step collagenase perfusion method, and serially passaged at 33 degrees C for more than 1 year. To assess the ability of immortohepatocytes to engraft and populate mouse liver, cells were infused into partially hepatectomized congenic mice via the portal vein (n = 10) or the spleen with (n = 2) and without (n = 2) partial hepatectomy. The ability to transfect immortohepatocytes was assessed using the reporter gene enhanced green fluorescent protein (EGFP). RESULTS: All immortohepatocytes in culture stained positive by immunohistochemistry for the hepatocyte markers albumin, AFP, CK8 and CK18. In early cultures a proportion of cells also stained strongly for the biliary epithelial markers CK7 and CK19. Late cell cultures were negative for M2PK and CK7 and stained variably with anti-CK19 antibodies. Cells transferred to the non-permissive temperature of 39 degrees C ceased proliferation and died within 1 week in vitro. Large T DNA was detected in the liver of all postoperative mice up to 2 weeks post-hepatocellular transplantation via PCR and Southern blot analysis. The immortohepatocytes were easily transfected with a reporter gene. CONCLUSIONS: Immortohepatocytes can survive in vivo after transfer to liver, and will be useful as a model for hepatic gene therapy.

Animals↗

PubMed2003

[Passage adaptability of candidate strains for hemorrhagic fever with renal syndrome purified vaccine in Vero cells and their immunogenicity].

OBJECTIVE: To adapt the candidate strains of hemorrhagic fever with renal syndrome (HFRS) purified vaccine to Vero cells and to study their antigenicity and immunogenicity. METHODS: The viral strains H8207 (Hantaan virus, HTN) and Y86013 (Seoul virus, SEO) were continuously propagated in Vero cell by the terminal dilution method and studied the characteristics of virus multiplication, viral titers and the amounts of virus antigen after serial passages. Three batches of crude monovalent inactivated vaccine were developed using the different passages of these 2 viral strains. RESULTS: The strains H8207 and Y86013 adapted to Vero cells and stably grew on the cells with high titers. Rabbits immunized with the crude vaccines of H8207 and Y86013 showed 100% sero-conversion and the neutralizing antibody titers of the rabbit immune sera reached 1?10 at 4 weeks after 2 times of immunization. CONCLUSIONS: The results suggest that these 2 candidate strains had adapted to Vero cells, possessed high titers and good immunogenicity and be feasible to prepare the HFRS purified vaccine in Vero cells.

Animals↗

PubMed1979

Incomplete genomes of the parvovirus minute virus of mice: selective conservation of genome termini, including the origin for DNA replication.

Deletion mutants of minute virus of mice arising during a single high-multiplicity passage and after serial undiluted passage have been isolated, and the incomplete viral genomes contained therein have been analyzed. The DNA isolated from incomplete virions derived from a single high-multiplicity passage was heterogeneous, ranging in size from 15 to 70% of the intact viral genome, with an average molecular length of approximately, 2,000 nucleotides. Two distinct types of molecules, designated as type I D-DNA and type II D-DNA, could be distinguished on the basis of their degree of secondary structure, and these were present in roughly equal amounts. Type I D-DNAs were predominantly single-stranded, recombinant molecules in which the self-complementary sequences derived from both genomic termini were conserved. The 5' terminus was modified relative to the analogous wild-type structure. Although virtually all of the wild-type genome sequence was seen in the total type I D-DNA population, sequences which map between coordinates 47.3 and 87.1 were clearly underrepresented. However, the extent and position of the deletions in individual molecules varied significantly. The shortest molecules in the population lacked between 90 and 95% of the internal wild-type genome sequence and consisted of sequences derived almost exclusively from within 5.0 map units (250 nucleotides) at both ends of the viral genome. Moreover, these miniature recombinant molecules were selectively amplified during serial undiluted passage and were therefore believed to contain all of the critical recognition sites necessary for the replication of minute virus of mice viral DNA. Type II D-DNAs were virus-specific, double-stranded hairpin molecules whose complementary strands were covalently continuous at variable sites distal to the 5' end of the viral minus strand. In sharp contrast to the type I genomes, these hairpin molecules consisted of sequences which mapped entirely at the 5' end of the viral genome between positions 85.0 and 100. Furthermore, type II molecules were gradually lost from the total D-DNA population during serial undiluted passage, suggesting that these molecules are not competent for DNA Replication but arise as the result of fatal replication errors. Deletion mutants of the type described here for minute virus of mice should be valuable generally as aids to future studies on parvovirus DNA replication, transcription, and cell-virus interactions.

Base Sequence↗

PubMed1989

Evolution of the 5'-end of genomic RNA of murine coronaviruses during passages in vitro.

The 5'-ends of the genomic RNA and subgenomic mRNAs of murine coronavirus (MHV) have a stretch of approximately 70 nucleotides of leader sequences. The 3'-region of this leader sequence contains several repeats of a pentanucleotide (UCUAA), whose number varies among different MHV strains. It has been demonstrated that this UCUAA repeat plays crucial roles in the discontinuous transcription of MHV mRNAs. In the present study, we demonstrate that the number of UCUAA repeats in the leader sequence of MHV genome rapidly decreases during serial passages of viruses on susceptible cells. The downward evolution of the number of UCUAA repeats was not due to a higher growth rate of the viruses with fewer repeats, but seemed to be due to homologous interference between viruses with different numbers of UCUAA repeat. The ease with which these variant viruses arose suggests the high frequency of the occurrence of this deletion during RNA replication. This finding is in agreement with the proposed discontinuous and nonprocessive mode of coronavirus RNA synthesis. Analysis of the intracellular subgenomic mRNA species of viruses with different numbers of UCUAA repeats and of MHV recombinant viruses suggests that the number of this pentanucleotide repeat at the 3'-end of the leader sequence may regulate the synthesis of certain mRNA species, in agreement with the leader-primed transcription mechanism.

Animals↗

PubMed1981

Infection of cultured human pancreatic B cells with reovirus type 3.

The capacity of reovirus type 3 to infect insulin-producing B cells was studied in human pancreatic cell cultures. Antibody to reovirus was labelled with fluorescein isothiocyanate and antibody to insulin was labelled with tetramethyl rhodamine isothiocyanate. By using a double-labelled immunofluorescent antibody technique, it was shown that only about 6% of the insulin-containing human B cells in culture became infected when inoculated with unpassaged reovirus type 3. However, by repeated passage of the virus in human pancreatic B cell cultures, the percentage of infected B cells increased to 27%, and the virus titre in cultures rose from 8.0 X 19(4) pfu/ml in the first passage, to 4.9 X 10(6) pfu/ml in the 5th passage. As measured by radioimmunoassay, the intracellular immunoreactive insulin began to decrease at 24 h after infection. This decrease roughly paralleled the increase in virus titre. In contrast, there was relatively little change in immunoreactive insulin in cultures inoculated with unpassaged reovirus type 3. These studies show that the ability of reovirus type 3 to infect human B cells is enhanced by serial passage in human pancreatic cell cultures and that the infection resulted in the destruction of B cells and release of insulin.

Cells, Cultured↗

PubMed1999

Infection of human endothelial cells with Chlamydia pneumoniae stimulates transendothelial migration of neutrophils and monocytes.

We have previously shown that different isolates of Chlamydia pneumoniae display heterogeneity in the in vitro stimulation of chemokines and adhesion molecules from infected human endothelial cells. In the present study, we examined the ability of different isolates of C. pneumoniae to promote transendothelial migration of neutrophils and monocytes. Human umbilical vein endothelial cells (HUVEC) were infected with low (<15)-passage C. pneumoniae isolates A-03, PS-32, and BR-393 and high (>40)-passage isolates BAL-16, TW-183, and T-2634, and levels of neutrophil and monocyte transendothelial migration were determined following 24 h of infection. Compared to mock-infected controls, significant increases in neutrophil migration were observed in response to most C. pneumoniae isolates examined (P < 0.001). Levels of monocyte migration were significantly increased in response to TW-183 and T-2634 (P < 0.001). Serial passage (>40 times) of the three low-passage isolates in HEp-2 cell cultures prior to infection of HUVEC generally resulted in the promotion of higher levels of neutrophil and monocyte transendothelial migration. These findings were compatible with differences observed in the extent of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) stimulation between low- and high-passage A-03, PS-32, and BR-393. As opposed to C. pneumoniae, infection with C. trachomatis L2 caused only a slight increase in neutrophil transendothelial migration, which correlated with the lack of measurable IL-8 levels by this species. However, significant levels of monocyte migration were induced in response to C. trachomatis L2 despite a lack of measurable MCP-1 stimulation. C. trachomatis serovars A and E also failed to induce IL-8 and MCP-1 production in HUVEC. Results from this study indicate that the passage history of C. pneumoniae may play a role in the divergence of stimulatory activities observed among isolates in human endothelial cells. In addition, the differences observed between this organism and C. trachomatis suggest that the upregulation of IL-8 and MCP-1 in endothelial cells may be unique to C. pneumoniae.

Adult↗

PubMed2002

Caenorhabditis elegans DNA mismatch repair gene msh-2 is required for microsatellite stability and maintenance of genome integrity.

Mismatch repair genes are important in maintaining the fidelity of DNA replication. To determine the function of the Caenorhabditis elegans homologue of the MSH2 mismatch repair gene (msh-2), we isolated a strain of C. elegans with an insertion of the transposable element Tc1 within msh-2. Early-passage msh-2 mutants were similar to wild-type worms with regard to lifespan and meiotic chromosome segregation but had slightly reduced fertility. The mutant worms had reduced DNA damage-induced germ-line apoptosis after genotoxic stress. The msh-2 mutants also had elevated levels of microsatellite instability and increased rates of reversion of the dominant unc-58(e665) mutation. In addition, serially passaged cultures of msh-2 worms died out much more quickly than those of wild-type worms. These results demonstrate that msh-2 function in C. elegans is important in regulating both short- and long-term genomic stability.

Alleles↗

PubMed1978

Synthesis and packaging of herpes simplex virus DNA in the course of virus passages at high multiplicity.

Herpes simplex virus type 1 (HSV-1), strain ANG, was passaged serially at high MOI or undiluted on RC-37 cells. The yields of infectious virus decreased and increased periodically with a maximum of about 3 log units variation in titer. The amount of newly synthesized HSV-1 DNA, however, did not vary by more than a factor of 2. On the other hand, the fraction of total HSV-1 DNA associated with virions was reduced at passage numbers coinciding with the minima in the yields of infectious progeny virus. Apparently, low yields of infectious virus mainly reflected a reduced efficiency of packaging of viral DNA into mature virus particles. One step in the process of virus maturation which appeared to be impaired was the assembly of nucleocapsids. HSV-1 ang was shown to be capable of generating different classes of defective particle in independent series of virus passages at high MOI.

Capsid↗

PubMed1997

[Transmission of rabies virus: importance of the species barrier].

The authors describe experimental inoculations of different animal species with either the wild-type rabies virus or the modified one through in vivo or in vitro passages. With these experiments, it was possible to determine the 50% lethal dose of these viruses for these species, and to thus quantify the importance of the species barrier that opposes, in particular, the transmission of vulpine rabies to cats and dogs (respectively 10(5) and 10(6) times more resistant than foxes). Studies were also undertaken on the influence of the inoculation route and that of serial passages of the virus in vivo or in vitro on the importance of resistance to rabies. The epidemiological consequences of the existence of a species barrier, its nature and variability, are discussed.

Animals↗

PubMed1997

Transplantation analyses of the immunogenicity of epidermal tumors generated in murine skin two-stage carcinogenesis protocols.

SSIN mice are very sensitive to tumor promoters in two-stage skin carcinogenesis protocols. It was recently reported that SSIN mice have fewer CD8+ T-cells than other strains of mice and develop a weaker cytotoxic T-cell response upon challenge with an allogeneic tumor transplant. The significance of this muted immune response to processes involved in two-stage carcinogenesis depends on the immunogenicity of the tumors generated in such protocols. Although they have low CD8+ T-cell contents, SSIN rejected a variety of subcutaneously transplanted allogeneic murine tumors. Analyses of the growth of primary papillomas derived from 7,12-dimethylbenz[a]anthracene-initiated/12-O-tetradecanoylphorbol-13-ac etate-promoted SSIN mice and then subcutaneously transplanted into triple-deficient (bg-nu-xid), athymic nude and immune-competent and immunosuppressed SSIN mice revealed that few tumors took and tumor takes were not markedly influenced by the immumological status of the transplant recipient. Two tumor cell lines (RS1 and RS2) were derived from the transplantation studies and could be passaged in normal SSIN mice (H-2q haplotype). Both tumors were squamous cell carcinomas (SCCs) by the second in vivo passage and were rejected in allogeneic mice (BALB/c) but grew in FVB/N mice, a strain having the H-2q haplotype. Transplantation studies revealed that prior exposure to RS1 and RS2 did not prime SSIN mice to reject a subsequent tumor challenge. Three primary SCC tumors derived from SSIN mice in a two-stage carcinogenesis protocol also grew when subcutaneously transplanted in SSIN mice and could be serially passaged. Consequently, the epidermal SCCs that develop in two-stage carcinogenesis protocols appear to be nonimmunogenic.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1984

Correlation between loss of the temperature-sensitive phenotype and pathogenicity of fowl plague virus mutants in the chicken.

The reversion of temperature-sensitive (ts) mutants of fowl plague virus to the ts+ phenotype was correlated with pathogenicity for chicken. Two types of ts mutants were investigated: those obtained by mutagenesis with 5-fluorouracil and those obtained by undiluted passages at 33 degrees C. The reversion frequency of the former mutants depended on the RNA segment in which the ts defect was located, mutations in RNA segments 1 and 2 having the highest reversion frequency, those in the RNA segments coding for the glycoproteins the lowest. ts mutants obtained by undiluted passages behaved differently in this respect. There was an approximate correlation between frequency of reversion and pathogenicity for chicken. Double mutants induced by 5-fluorouracil, having one tight and one leaky mutation, reverted easily without loss of the leaky mutation. These double mutants were still to a limited extent pathogenic for the chicken. Only one double mutant with two tight mutations (ts 293) was completely nonpathogenic after intramuscular inoculation. Two ts mutants with multiple tight defects (ts 1/1 and ts 3/18) obtained by undiluted passage did not revert to wild-type after injection into embryonated eggs and incubation at 33 degrees C, but they were still slightly pathogenic for the chicken. There was no obvious correlation between the shut-off temperature and pathogenicity of mutants carrying a single ts defect. However, for mutants with multiple tight mutations a high shut-off temperature seemed to be essential for reversion during serial passages as well as for pathogenicity in the chicken, when different routes of inoculation were examined. ts mutants seem to be safe as live vaccines only, (1) if they carry at least two tight ts defects, (2) if they have a relatively low shut-off temperature, and (3) if they could be administered other than via the respiratory tract.

Animals↗

PubMed1976

Virulence of established vaccine strains and artificially passaged field strain of Mycoplasma mycoides subsp mycoides.

The virulence of Mycoplasma mycoides for mice, which appears to parallel virulence for cattle, was assessed in respect of four vaccine strains well known for their use in the control of contagious bovine pleuropneumonia. The T1 strain was slightly more virulent than V5, and both were much more virulent than F and KH3J, which could not be distinguished from each other. There was no significant difference between cultures of the T1 strain supplied by three different vaccine-producing laboratories, The mousevirulence test was also used to study the effect of serial passage in artificial systems on virulent field strains of M mycoides; two such strains underwent no appreciable change in virulence during many passages in chick embryos or in mice, although the same strains, and two others, were readily attenuated by repeated subculture in broth. With one strain of M mycoides there was evidence that the total number of subcultures in broth over a given period was more important than the duration of each subculture.

Animals↗

PubMed1996

In vitro changes in plasma membrane heparan sulfate proteoglycans and in perlecan expression participate in the regulation of fibroblast growth factor 2 mitogenic activity.

Fibroblast growth factor 1 (FGF1) and 2 (FGF2) bind to two classes of receptors: the high affinity receptors, a family of four known transmembrane tyrosine kinases (FGF R1-R4), and the low affinity receptors, cell surface and basement membrane heparan sulfate proteoglycan (HSPG). During early (first and second) passages of retinal pigmented epithelial (RPE) cells, both FGF1 and FGF2 exhibited low mitogenic activity, while in later (fifth to ninth) passages the activity of FGF1 remained constant but FGF2 activity increased two- to threefold. We have investigated aspects of FGF receptor interactions and the role of heparin/heparan sulfate which modulates FGF activity on RPE cells during in vitro senescence. Northern blot analysis demonstrated that FGF receptor type 1 (FGF R1) is the major high affinity receptor expressed in RPE cells and that its level of expression did not change during serially passage. Both the FGF R1 and the FGF low affinity receptors' binding characteristics (i.e., Kd and number of sites per cell) for FGF1 were unaffected by passage number, whereas the capacity of FGF2 binding to FGF R1 and to the low affinity receptors increased by two- and fivefold, respectively, in late passages, although the affinities were unchanged. This change in the capacity of FGF2 to bind to FGF R1 and to HSPG was not due to a switch of the IIIc splice form of FGF R1 to the IIIb splice form since the exon IIIc was the most predominant splice form of FGF R1 during RPE cell cultures. Furthermore the ratio of the IIIb to the IIIc splice form was not modified during cell subcultures. In parallel in the older RPE cell passages, expression of perlecan, the major FGF low affinity binding site localized on the extracellular matrix of RPE cells, was much elevated compared to early RPE cell passages. Moreover, the cell surface of late passage RPE cells had 79% more HSPG than early passage cells. Therefore, it is suggested that the increase in the number of FGF low affinity receptors present on the cell surface or basement membrane could account for a part of the greater proliferative response of aged RPE cells to FGF2.

Alternative Splicing↗

PubMed1995

Dramatic interstrain differences in the replication of human cytomegalovirus in SCID-hu mice.

The ability of a low-passage strain (Toledo) and laboratory strains (AD169 and Towne) of human cytomegalovirus to replicate in SCID-hu (thymus plus liver) mice were compared. At a time of peak replication, 14 days after inoculation, the Toledo strain grew 2-3 orders of magnitude better than any laboratory strain, a difference reflecting the number of infected thymic stromal cells in the implants. The growth property of the Toledo strain was stable through serial passage and plaque purification. The AD169-ATCC strain failed to grow at all, while an independently maintained stock of this strain obtained from the United Kingdom replicated to low levels, suggesting that divergence had occurred during propagation in different locations. This work predicts the existence of viral genetic determinant(s) for growth in tissues that are lost during propagation in culture.

Animals↗