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Results for “Serial Passage”

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PubMed1983

Culture of retinal capillary cells using selective growth media.

Capillary fragments are isolated from the microvasculature of the bovine retina using limited collagenase digestion and sieving. The endothelial cells obtained from these capillary fragments are cultured in media containing platelet-poor plasma supplemented with retinal extract, which contains an endothelial cell mitogen. These cells form typical monolayers in vitro, contain Factor VIII antigen, and can be passaged serially. The pericytes obtained from these capillary fragments are cultured in media containing calf serum. These cells can also be passaged serially, are not contact-inhibited, and do not stain for Factor VIII antigen. Cultures of capillary cells such as these will allow the study of the individual cells from specific microvascular beds involved in various normal and pathological processes.

Animals↗

PubMed1999

Analysis of vancomycin population susceptibility profiles, killing activity, and postantibiotic effect against vancomycin-intermediate Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus strains with decreased vancomycin susceptibility have been isolated from patients in the United States and Japan. The impact of decreased vancomycin susceptibility on the drug's pharmacodynamic parameters has not been addressed. We studied the activity of vancomycin against three clinical strains of vancomycin intermediate-susceptible Staphylococcus aureus (VISA) under high- and low-inoculum conditions, with stationary- and logarithmic-growth-phase kill curves, and in postantibiotic effect (PAE) experiments. We also investigated the stability of the decreased vancomycin susceptibility by using population susceptibility profiles. The respective vancomycin microdilution MICs and MBCs for VISA strains HIP5836, 14379, and Mu50 were 8 and 8, 8 and 8, and 8 and 16 microg/ml. HIP5836 had the most homogeneous elevation of vancomycin MICs, because the MIC for nearly all bacteria in the inoculum was 8 microg/ml. The population MICs (defined as the lowest vancomycin concentration inhibiting 99. 9% of growth) for the first serial passages of HIP5836, Mu50, and 14379 were 8, 4, and 2 microg/ml, respectively. After 10 passages, they decreased to 4, 2, and 1 microg/ml, respectively. The Mu50 population MIC increased to 12 microg/ml after five serial passages on vancomycin agar. In the low- and high-inoculum kill curves, time to 99.9% killing was significantly (P < 0.05) longer for both Mu50 and HIP5836 than that for 14379 and a control strain. However, colony counts at 24 h were similar to those of the vancomycin-sensitive strain for all VISA strains. The PAE (at 4x MIC) ranged from 1.3 h for 14379 to 2.0 h for HIP5836 and was similar to or greater than the PAE against the vancomycin-sensitive strain. In conclusion, we found that the decreased vancomycin susceptibility increased during persistent exposures to the drug and decreased upon removal of the selective pressure. The decreased vancomycin susceptibility decreased the rate of vancomycin killing, but did not affect the extent of killing or the PAE.

Anti-Bacterial Agents↗

PubMed2005

Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers.

Adipose tissue represents an abundant and accessible source of multipotent adult stem cells and is used by many investigators for tissue engineering applications; however, not all laboratories use cells at equivalent stages of isolation and passage. We have compared the immunophenotype of freshly isolated human adipose tissue-derived stromal vascular fraction (SVF) cells relative to serial-passaged adipose-derived stem cells (ASCs). The initial SVF cells contained colony-forming unit fibroblasts at a frequency of 1:32. Colony-forming unit adipocytes and osteoblasts were present in the SVF cells at comparable frequencies (1:28 and 1:16, respectively). The immunophenotype of the adipose-derived cells based on flow cytometry changed progressively with adherence and passage. Stromal cell-associated markers (CD13, CD29, CD44, CD63, CD73, CD90, CD166) were initially low on SVF cells and increased significantly with successive passages. The stem cell-associated marker CD34 was at peak levels in the SVF cells and/or early-passage ASCs and remained present, although at reduced levels, throughout the culture period. Aldehyde dehydrogenase and the multidrug-resistance transport protein (ABCG2), both of which have been used to identify and characterize hematopoietic stem cells, are expressed by SVF cells and ASCs at detectable levels. Endothelial cell-associated markers (CD31, CD144 or VE-cadherin, vascular endothelial growth factor receptor 2, von Willebrand factor) were expressed on SVF cells and did not change significantly with serial passage. Thus, the adherence to plastic and subsequent expansion of human adipose-derived cells in fetal bovine serum-supplemented medium selects for a relatively homogeneous cell population, enriching for cells expressing a stromal immunophenotype, compared with the heterogeneity of the crude SVF.

Adipose Tissue↗

PubMed1973

Infectivity and RNA patterns as functions of high- and low-dilution passage of murine sarcoma-leukemia virus: evidence for autointerference within an oncornavirus population.

Heterogeneity of buoyant density and RNA content of virions of Moloney murine leukemia-sarcoma complex [MSV (MLV)] was the result of passage at low dilution. Heterogeneous stocks revealed two major RNA components in the population, with the smaller component, apparent mol wt 4 x 10(6) to 5 x 10(6), becoming predominant upon serial passage at low dilution. Concomitantly, infectivity titers of both MLV and MSV decreased upon serial passage at low dilution. MSV (MLV) passaged at high dilution retained high titers and a rather homogeneous high-molecular-weight RNA population characteristic of high-buoyant-density virions. Interference of both MLV and MSV replication was demonstrated by employing mixed inocula containing both low- and high-dilution passage stocks of MSV (MLV). In contrast to results with MSV (MLV), MLV freed of MSV by limit dilution did not show heterogeneity of buoyant density or of RNA when propagated at low dilution.

Animals↗

PubMed1975

Porcine parvovirus: frequency of naturally occurring transplacental infection and viral contamination of fetal porcine kidney cell cultures.

The frequency of naturally occurring transplacental infection of swine with porcine parvovirus (PPV) and one of the possible consequences of such infection--the presence of PPV in cell cultures prepared from fetal tissues--were investigated. Transplacental infection was indicated by the presence of high titers of hemagglutination inhibiting (HI) antibody for PPV in serums of 0-day-old, hysterectomy-derived, colostrum-deprived pigs of 3 of 82 litters. All letters were farm-raised dams. Moreover, cell cultures prepared from 3 of 49 lots of fetal porcine kidneys (FPK) collected from an abattoir during an interval of 14 months were found contaminated with PPV. Because each lot was usually comprised of kidneys from 2 litters, the latter finding suggests that 3 of approximately 98 litters were infected. Prior infection of FPK cell cultures with PPV resulted in only slight interference of replication of other selected viruses; i.e., porcine enterovirus (PEV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), and hemagglutinating encephalomyelitis virus (HEV). Moreover, PPV and HEV were propagated in the same cell cultures during 5 serial passages of the viruses. In contrast, when copropagation of PPV and VSV was attempted, PPV was not detected after the 2nd serial passage.

Animals↗

PubMed1997

Virulent isolates and mutants of Blastomyces in mice: a legacy for studies of pathogenesis.

This article provides information on the history of Blastomyces dermatitidis isolates and spontaneous mutants, the relationship with maintenance of cultures, and their virulence as quantified in murine models. Virulent isolates have been obtained from soil or from patients. Regardless of origin, mutants attenuated in virulence have sometimes arisen from storage as frozen yeast, frozen mycelia, or refrigerated yeast or serial passage in vitro at 35 degrees to 37 degrees C. Once virulence is lost, reversion to the virulent phenotype (even after serial passage in animals) has not been seen. These data, methods, and isolates provide a basis for future studies of virulence factors.

Animals↗

PubMed1985

Mechanisms of human cell neoplastic transformation: X-ray-induced abnormal clone formation in long-term cultures of human diploid fibroblasts.

Early passage cultures of a strain of normal human diploid fibroblasts were exposed to various doses of X-rays. The cells were serially passaged and followed throughout their life span in vitro. G-banded metaphase chromosome preparations were examined at each subculture to determine the presence of abnormal clones, i.e., groups of cells bearing identical chromosomal rearrangements. It was found that X-irradiation induced random chromosomal rearrangements which persisted throughout the life span of the cells. No abnormal clones were observed among the progeny of four nonirradiated cultures, nor in seven of nine cultures exposed to single radiation doses. On the other hand, multiple abnormal clones emerged among the progeny of cells in all five cultures exposed to multiple sequential radiation doses (three doses of 400 or 600 rads each). Evidence of clonal expansion and attenuation and of clonal succession during serial passaging occurred in these populations. In several cases, these clones expanded to include most of the cell population before the cultures became senescent. These findings are discussed in terms of their possible role in the transformation of human diploid cells by radiation.

Cell Transformation, Neoplastic↗

PubMed2001

Derivation and characterization of a live attenuated equine influenza vaccine virus.

OBJECTIVE: To develop and characterize a cold-adapted live attenuated equine-2 influenza virus effective as an intranasal vaccine. ANIMALS: 8 ponies approximately 18 months of age. PROCEDURES: A wild-type equine-2 virus, A/Equine/Kentucky/1/91 (H3N8), was serially passaged in embryonated chicken eggs at temperatures gradually reduced in a stepwise manner from 34 C to 30 C to 28 C to 26 C. At different passages, infected allantoic fluids were tested for the ability of progeny virus to replicate in Madin-Darby canine kidney (MDCK) cells at 34 C and 39.5 C. Virus clones that replicated at 26 C in eggs and at 34 C in MDCK cells, but not at 39.5 C in MDCK cells, were tested for stability of the cold-adapted, temperature-sensitive (ts), and protein synthesis phenotypes. A stable clone, P821, was evaluated for safety, ability to replicate, and immunogenicity after intranasal administration in ponies. RESULTS: Randomly selected clones from the 49th passage were all ts with plaquing efficiencies of < 10(-6) (ratio of 39.5 C:34 C) and retained this phenotype after 5 serial passages at 34 C in either embryonated eggs or MDCK cells. The clone selected as the vaccine candidate (P821) had the desired degree of attenuation. Administered intranasally to seronegative ponies, the virus caused no adverse reactions or overt signs of clinical disease, replicated in the upper portion of the respiratory tract, and induced a strong serum antibody response. CONCLUSION AND CLINICAL RELEVANCE: A candidate live attenuated influenza vaccine virus was derived by cold-adaptation of a wild-type equine-2 influenza virus, A/Equine/Kentucky/1/91, in embryonated eggs.

Animals↗

PubMedDate not supplied

Human embryonic stem cell (hES) colonies display a higher degree of spontaneous differentiation when passaged at lower densities.

Human embryonic stem (hES) cells require cooperative interactions with each other for their survival. Previously, the size of hES cell clumps has been reported to be an important factor in determining their viability during routine serial passage. However, the effects of seeding density of the hES cell clumps per se have not yet been investigated. Therefore, this study attempted to compare the level of spontaneous differentiation of hES colonies passaged at two different split ratios (1:3 and 1:8) of a single confluent well of a six-well dish. After 7 d of in vitro culture following serial passage, hES colonies were assigned into three grades according to their degree of spontaneous differentiation: (1) Grade A, which was completely or mostly undifferentiated; (2) grade B, which was partially differentiated; and (3) grade C, which was mostly differentiated. Assessment of the degree of spontaneous differentiation was based on morphological observations under bright-field and phase-contrast microscopy, as well as on immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. We observed that, at a split ratio of 1:3, the percentages of grade A, B, and C colonies were 89.5, 8.8, and 1.7%, respectively. This was significantly different from the corresponding values of 52.7, 31.3, and 16.0%, respectively, obtained at a split ratio of 1:8. Hence, our results indicated that a lower passage density led to a higher degree of spontaneous differentiation of hES colonies.

Cell Culture Techniques↗

PubMed1985

Analysis of stable and unstable viral forms in SV40-infected human keratinocytes.

We analyzed the state of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral-infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full-length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the "free" viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bg1 II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bg1 I site and contains a large deletion in the SV40 early region at one of the viral-host junctions.

Cell Line↗

PubMed2004

Type II topoisomerase mutations in Bacillus anthracis associated with high-level fluoroquinolone resistance.

OBJECTIVES: To identify and characterize the mechanisms of high-level fluoroquinolone resistance in two strains of Bacillus anthracis following serial passage in increasing concentrations of fluoroquinolones. METHODS: Fluoroquinolone-resistant isolates of the Sterne and Russian Anthrax Vaccine STi strains were obtained following serial passage in the presence of increasing concentrations of four different fluoroquinolones. The quinolone-resistance-determining regions of the type II topoisomerase genes from the resistant strains were amplified by PCR and characterized by DNA sequence analysis. The MICs in the presence and absence of reserpine were determined using broth microdilution as a means of detecting active efflux. RESULTS: Single and double amino acid substitutions in the GyrA (Ser-85-Leu; Glu-89-Arg/Gly/Lys) and GrlA (Ser-81-Tyr; Val-96-Ala; Asn-70-Lys) were most common. A single amino acid substitution in GyrB (Asp-430-Asn) was also identified. Efflux only applied to isolates selected for by either levofloxacin or ofloxacin. CONCLUSIONS: Specific amino acid substitutions in the type II topoisomerase enzymes significantly contributed to the development of high-level fluoroquinolone resistance in B. anthracis. However, notable differences between the strains and the drugs tested were identified including the role of efflux and the numbers and types of mutations identified.

Amino Acid Substitution↗

PubMed1992

Possible approaches to develop vaccines against hepatitis A.

More than a decade ago, successful replication of hepatitis A virus (HAV) in cell culture opened the way to the development of live attenuated and inactivated vaccine candidates. Serial passages of HAV in cell culture led to attenuation as demonstrated by experiments in non-human primates. Several live vaccine candidates obtained through serial passages have been evaluated in volunteers. Significant improvements in the yield of viral antigen from infected cell cultures stimulated the development of killed vaccine candidates. These formalin-inactivated vaccines contain the viral capsid antigens assembled into viral particles. The immunogenic potential of the vaccine candidates depends strongly on the preservation of the configuration of the capsid proteins. Synthetic peptides covering immunogenic sequences of VP1 as well as soluble capsid proteins expressed as fusion proteins in Escherichia coli were therefore only weakly immunogenic when injected at high concentrations in rabbits. On the other hand, tamarin monkeys immunized with a live recombinant vaccinia expressing P1 were protected against virulent challenge. There are, however, considerable drawbacks related to the use of live vaccinia as a carrier virus. Chimeric polio-HAV VP1 viruses have been constructed. These hybrid viruses were not able to induce an immune response, probably because of configurational constraints of poliovirus on the inserted HAV epitopes. More recently, encouraging data on empty virus particles expressed in baculovirus and vaccinia virus systems have been reported.

Animals↗

PubMed1988

Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells.

The influence of extracellular folate concentration on cellular levels of the folate transport protein and its soluble product was studied directly in cultured human nasopharyngeal carcinoma (KB) cells. As determined by radioimmunoassay, levels of the folate transport protein and the soluble folate-binding protein were 58 +/- 17 (mean +/- SD) and 5 +/- 2 pmol/mg cell protein, respectively, in KB cells maintained in standard medium (containing 2,300 nM folic acid). These levels significantly increased to 182 +/- 34 and 26 +/- 6 pmol/mg cell protein, respectively, in KB cells serially passaged in low folate medium (containing 2-10 nM 5-methyltetrahydrofolate). Increases in folate-binding protein levels occurred more rapidly in KB cells serially passaged in very low folate medium containing less than 2 nM folate and were prevented by the addition of 100 nM 5-methyltetrahydrofolate or 0.1-1 microM 5-formyltetrahydrofolate to this medium. When KB cells which had been passaged in low folate medium were passaged back into either standard medium or low folate medium supplemented with reduced folates, the levels of both folate-binding proteins fell linearly towards the levels in KB cells continuously maintained in standard medium. The folate transport protein was identified in and underwent similar changes in human and mouse mammary tumor cells. These studies indicate that the folate transport system is probably regulated by the extracellular folate concentration through changes in intracellular metabolite levels.

Animals↗

PubMed1985

Infection of AtT20 murine pituitary tumour cells by mouse hepatitis virus strain A59: virus budding is restricted to the Golgi region.

AtT20 cells, a line of murine pituitary tumour cells that secrete adrenocorticotropic hormone (ACTH), have been infected with the coronavirus mouse hepatitis virus strain A59 (MHV-A59). Between 5% and 10% of AtT20 cells are susceptible to the infection. Unlike infections of fibroblastic sac- and 17Cl 1 cells, the infection of AtT20 cells does not lead to cell fusion, despite the production of the fusogenic E2 viral spike glycoprotein. Within infected AtT20 cells the second viral envelope glycoprotein, E1, is located in a perinuclear region; at least until very late in the infection it fails to accumulate to detectable levels in the rough endoplasmic reticulum (RER). By contrast to infection of sac- and 17Cl 1 cells, where the RER is a major site of assembly of progeny virions, in AtT20 cells budding of progeny virions is restricted to the Golgi cisternae, which eventually vesiculate, and peri-Golgi smooth membraned vesicles. Apparently, therefore, the intracellular compartments into which wild-type MHV-A59 buds are determined not by the virus but by the host cells. MHV-A59 infected cultures of AtT20 cells can be serially passaged without loss of the infection or increase in the proportion of infected cells; they become persistently infected carrier cultures. The progeny virus from serially passaged, infected AtT20 cells is apparently wild-type. It infects sac- cells and induces them to form syncitia. Within the sac- syncitia the viral E1 glycoprotein accumulates in the RER and many virions assemble there.

Animals↗

PubMed2000

Live attenuated tetravalent dengue vaccine.

The development of a live attenuated tetravalent dengue vaccine is currently the best strategy to obtain a vaccine against dengue viruses. The Mahidol University group developed candidate live attenuated vaccines by attenuation through serial passages in certified primary cell cultures. Dengue serotype 1, 2 and 4 viruses were developed in primary dog kidney cells, whereas dengue serotype 3 was serially passaged in primary African green monkey kidney cells. Tissue culture passaged strain viruses were subjected to biological marker studies. Candidate vaccines have been tested as monovalent (single virus), bivalent (two viruses), trivalent (three viruses) and tetravalent (all four serotype viruses) vaccines in Thai volunteers. They were found to be safe and immunogenic in both adults and children. The Mahidol live attenuated dengue 2 virus was also tested in American volunteers and resulted in good immune response indistinguishable from those induced in Thai volunteers. The master seeds from the four live attenuated virus strains developed were provided to Pasteur Merieux Connaught of France for production on an industrial scale following good manufacturing practice guidelines.

Adolescent↗

PubMed1978

The appearance of bisporocytic oocysts of Eimeria maxima in drug-treated chicks.

A line of Eimeria maxima acquired resistance to Lerbek (a mixture of clopidol and methyl benzoquate) after serial passage against rising drug levels. Abnormal bisporocystic oocysts which appeared throughout this series were picked out individually and these produced infections in further groups of chicks. Serial passages of selected bisporocystic forms raised their proportion in oocyst yields to about 80% after 10-14 passages, but a few normal oocysts were still present.

Animals↗

PubMed1967

Studies on pneumonia virus of mice (PVM) in cell culture. I. Replication in baby hamster kidney cells and properties of the virus.

Pneumonia virus of mice (PVM) has been serially propagated in a line of baby hamster kidney (BHK21) cells. A maximum titer of 6.3 x 10(6) TCID(50) per ml was obtained, and there was little variation in yield on serial passage. PVM grown in BHK21 cells was antigenically similar to virus obtained from the mouse lung, but was somewhat less virulent for the mouse after 10 serial passages in these cells. Virus produced by BHK21 cells agglutinated mouse erythrocytes without prior heating or other treatment. Sedimentation of PVM in the ultracentrifuge or precipitation by ammonium sulfate resulted in a loss in infectivity but an increase in hemagglutinating activity, presumably due to disruption of the virus particle. In a potassium tartrate density gradient, the major portion of infective virus sedimented at a density of approximately 1.15, and noninfective hemagglutinin, at a density of approximately 1.13. Stock virus preparations appear to contain a large amount of noninfective hemagglutinin. The replication of PVM was not inhibited by 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. Infected cells contained eosinophilic cytoplasmic inclusions which showed the acridine orange staining characteristic of single-stranded RNA. Foci of viral antigen were observed in the cytoplasm of infected cells by fluorescent antibody staining. The results suggest that PVM is an RNA virus that replicates in the cytoplasm.

Animals↗

PubMed1981

Modulation of the shape of epithelial lens cells in vitro directed by a retinal extract factor. A model of interconversions and the role of actin filaments and fibronectin.

We have shown previously [1] that bovine epithelial lens cells can be stimulated to divide and elongate by a retinal extract (RE). In this report we show that the morphological response to the stimulatory factor is directly related to the target-cell shape, and we describe how the cell shape can be modulated into morphologically different types. If the cells are grown continuously from the explant in the presence of the RE factor, they keep a typical regular pavement-like epithelial shape (type I), even after serial passages. If the same cells are cultured in the absence of the factor, they become extremely irregular in shape and enlarge enormously (type II), and during serial passage elongate spontaneously to a fibroblast-like pattern. However, when type II cells are stimulated by RE, they elongate dramatically into type III cells as described in [1], provided they are stimulated at the optimal cell density. We show that the transformation of one type to another is directly under the control of RE, and we demonstrate that the changes in cell morphology are accompanied by alterations in cytoplasmic actin filaments. Type I cells contain few microfilaments, while type II cells display actin-tropomyosin polygonal fibre networks that reform during conversion to type III cells and then to elongated stress fibres. The change from type I to type II cells is also accompanied by massive accumulation of surface-associated fibronectin. We conclude that factors obtained directly from the eye have a direct ability to control morphology and proliferation of ocular cells like lens cells perhaps by modulation of cellular adhesiveness mediated by surface fibronectin and reorganization of cytoplasmic actin-based filaments.

Actins↗