The continuous passage of agents of trachoma in cell culture. I. Characteristics of TW-3 and Bour strains of trachoma cultivated in serial passage in HeLa 229 cells.
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Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract.
A serum-free medium with bovine pituitary extract as the only undefined supplement has been developed for long-term culture of human mammary epithelial cells. This medium supports serial subculture of normal cells for 10-20 passages (1:10 splits) without conditioning or special substrates, and it supports rapid clonal growth with plating efficiencies up to 35%. It consists of an optimized basal nutrient medium, MCDB 170, supplemented with insulin, hydrocortisone, epidermal growth factor, ethanolamine, phosphoethanolamine, and bovine pituitary extract. Replacement of pituitary extract with prostaglandin E1 and ovine prolactin yields a defined medium that supports rapid clonal growth and serial subculture for three or four passages. Cultures initiated in these media from normal reduction mammoplasty tissue remain diploid and maintain normal epithelial morphology, distribution of cell-associated fibronectin, expression of keratin fibrils, and a low level of expression of milk fat globule antigen. Large cell populations can now be generated and stored frozen, permitting multiple experiments over a period of time with cells from a single donor. These media greatly extend the range of experiments that can be performed both conveniently and reproducibly with cultured normal and tumor-derived human mammary epithelial cells.
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The reversion of temperature-sensitive mutants of encephalomyocarditis virus upon serial passage in three continuous cell lines.
Several temperature-sensitive mutants of encephalomyocarditis virus were serially passed numerous times in HeLa, L929 and BHK-21 cells. The reversion of most of the mutants occurred most rapidly in BHK-21 cells and least rapidly in HeLa cells.
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Establishment of an Epstein-Barr virus-negative B-cell lymphoma line from a Japanese Burkitt's lymphoma and its serial passage in hamsters.
An Epstein-Barr virus (EBV)-negative lymphoma line (JBL) was established in vitro from pleural effusion of an EBV-seropositive 29-year-old Japanese female with Burkitt's lymphoma. JBL cells as well as her original lymphoma cells bore monoclonal surface IgM with lambda light chains. The JBL line grew in single cell suspension with a doubling time of 30 hours. Attempts were made to serially transplant JBL cells in antilymphocyte serum-treated newborn hamsters; intraperitoneal implantation of 1-3 X 10(7) cells gave rise to invasive tumors in all recipients with death after 10 to 14 days. The hamster-passage line, now in the 9th passage, has been converted to an ascitic form with progression to leukemia in some animals. A "starry sky" pattern closely resembling the human tumor material was preserved in every tumor through serial animal passage.
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Effects of serial passage on the endocrine response and steroid metabolism of a rat mammary carcinoma.
A rat mammary carcinoma induced by 7-12 dimethylbenzanthracene was serially transplanted into successive generations of thymectomized host animals. After its 2nd passage, the growth of the tumour appeared hormone-dependent, regressing after oophorectomy and regrowing with administration of oestradiol 17 beta to the host. Third-generation transplanted tumours, however, showed only a transient regression after oophorectomy, and the growth of tumours after further passages appeared ovary-independent. Loss in hormone dependency was not accompanied by histological changes. There was however a progressive increase with successive transplantation in the ability of tumours to metabolize 7 alpha [3H] testosterone in vitro. This was accounted for by raised conversion to 5 alpha androstanediol.
9,10-Dimethyl-1,2-benzanthracene↗
Chemically induced murine T lymphomas: continued rearrangement within the T-cell receptor beta-chain gene during serial passage.
The first constant region of the Tcrb gene was completely deleted from the DNA of 8/10 mouse cell lines established from 3-methylcholanthrene-induced RF/J thymic lymphomas, but 6/7 primary lymphomas contained the first constant region sequences. DNA from RF/J thymic lymphomas induced by N-methyl-N-nitrosourea was then examined serially as the tumors were passaged in vivo and adapted to growth in culture as uncloned and, in some cases, cloned lines. Patterns of Tcrb-specific restriction fragments from most tumors changed extensively during continued propagation. Analysis of the patterns often suggested that initial DNA rearrangements within the Tcrb complexes of monoclonal tumors had been followed by further rearrangements within the same genes. However, these different patterns may alternatively have represented successive outgrowth of separate lineages from lymphomas that were polyclonal in origin.
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Serial passages of larval Echinococcus granulosus from equine origin in mice. I. Infection with protoscolices.
Starting with protoscolices from Echinococcus granulosus cysts of equine origin it was possible to isolate and maintain several "strains" of the parasite in mice through successive transfers of protoscolices. The most advanced strain is now in its 7th generation. Several criteria to evaluate the results are presented and discussed. It is concluded that serial echinococcosis of E. granulosus through passages with protoscolices is a practical method to maintain different strains for fundamental and applied comparative studies.
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Increased response to cisplatin after long-term serial passage of a squamous cell carcinoma xenograft.
We have retrospectively investigated the response to cisplatin of a squamous cell carcinoma of the head and neck xenografted to nude mice during nine years of serial transplantation. Tumour growth rate decreased gradually. After nine years and over 100 passages, there was a sudden increase in cisplatin sensitivity. Histopathological examination showed that, of two histopathologically different subpopulations present in earlier passages, the predominant one was no longer detectable. The DNA-index did not change.
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Temperature-sensitive alteration in fusion activity of subacute sclerosing panencephalitis virus during serial passages in vitro and expression of hemagglutinin on the infected cells.
The Biken strain of subacute sclerosing panencephalitis (SSPE) virus, a maturation-defective variant of measles virus, was serially passed in human embryonic lung (HEL) cells at 37 C. The strain formed syncytial giant cells (GC) at both 37 C and 39 C, but the surface of infected cells did not show hemadsorption at early passages of the strain. However, GC-forming activity of the strain diminished at 39 C after 25 passages or more and hemadsorption on the infected cells became positive at both 37 C and 39 C after 40 passages or more of the strain. Hardly any infectious cell-free virus was detected in the culture fluid even after hemadsorption became positive. Possible mechanisms for the defect of SSPE virus were discussed.
Cell Fusion↗
Duck virus hepatitis: serial passage of attenuated virus in ducklings.
The safety of three attenuated virus vaccines of proven efficacy against duck virus hepatitis was assessed by controlled laboratory studies which involved the serial transmission of the virus through groups of two-day-old ducklings known to be susceptible to the disease. Each vaccine was initially derived from a different source. Enhancement of virulence which resulted in deaths from the disease in test groups of ducklings occurred in each instance.
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Studies on the transmission of human viral hepatitis to marmoset monkeys. I. Transmission of disease, serial passages, and description of liver lesions.
Inoculation of human serums or plasmas obtained during the early acute phase of viral hepatitis induced chemical and morphological hepatic disease in marmosets in two out of five experimental series. The disease was transmissible in series from marmoset to marmoset with an apparent increased virulence of the causative agent in later marmoset passages. The chemical evidence for the disease was elevation of the activity of SGOT and SICD and of serum bilirubin. In serial liver biopsy specimens interpreted under code, a hepatitis, exhibiting some of the characteristics of human viral hepatitis, was readily distinguishable from nonspecific changes. The morphological changes preceded the biochemical alterations and persisted after them. The data reported in these studies indicate that marmosets may be susceptible to human hepatitis. If these observations are confirmed, these animals may provide good experimental models for this disease. Final proof that the hepatitis observed in marmosets is caused by agents of human viral hepatitis is still lacking.
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Growth characteristics and metastatic potential of seven intestinal carcinoma lines serially passaged in syngeneic rats.
Transplantable tumour lines were obtained from one duodenal carcinoma induced by N-methyl-N'-nitro-N-nitrosoguanidine in the Lewis rat and from six colonic carcinomas induced by 1,2 dimethylhydrazine in BDIX or Fisher rats. The tumours were serially transplanted by the subcutaneous route into homologous syngeneic rats. The seven tumours differ from one another in their histological structure, five of them being well or moderately differentiated adenocarcinomas, and in their capacity to produce neutral or acidic mucins. The seven tumours also differ in their growth rate. The seven lines produced metastases; the metastatic potential and the location of the metastases differed from one line to another. The seven lines kept their original differentiation characteristics through multiple passages, representing several years of transplantation into syngeneic hosts. These tumours represent a useful and diversified model of metastatic intestinal carcinoma, available for basic research and therapeutic trials.
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Loss on serial passage of rhesus monkey kidney cells of proteolytic activity required for Sendai virus activation.
Primary and secondary cultures of rhesus monkey kidney cells supported multiple-cycle replication of Sendai virus, but later passages lost this ability, and this was reflected in decreased plaque formation. Multiple-cycle replication also did not occur in LLC-MK2 cells, a continuous line of RMK cells. Failure of replication in serially passed cells was correlated with a decrease in proteolytic cleavage of a viral surface glycoprotein (Fo), and the ability of cells to support multiple-cycle replication and plaque formation could be restored by the addition of trypsin (0.3 microgram/ml) to the overlay medium. The use of wild-type virus, which requires trypsin, and protease activation mutants that require chymotrypsin or elastase for activation has provided evidence that the activating protease supplied by primary or secondary cells has trypsin-like activity. Inactive virus, with uncleaved Fo glycoprotein, absorbed to primary or secondary cells but did not infect them, even though such cells possess the enzyme that is capable of cleaving the Fo glycoprotein of virus synthesized in these cells. The inability of these cells to activate adsorbed virus indicates that the activating protease that they possess is inacessible to adsorbed virus, although it can act on the Fo glycoprotein during virus maturation in these cells. These data provide a biochemical explanation for the failure of later passages of a cell strain or a continuous cell line to support the replication of a paramyxovirus.
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Primary culture and serial passage of normal and carcinogen-treated rat mammary epithelial cells in vitro.
A newly developed culture system was used to examine the proliferative potential of rat mammary epithelial (RME) cells in vitro. RME cells were obtained by enzymatic dissociation of mammary tissues of 45- to 50-year-old virgin female LEW rats. The tissues were dissociated to small aggregates (10-50 cells per aggregate) separated from stromal cells and plated at a density of 10(5) cells per 60-mm tissue culture dish. The cells were grown in Ham's medium F12 supplemented with 5% fetal bovine serum, insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, and cholera toxin. Plating of 10(5) cells as small aggregates resulted in the attachment of 1,000-1,500 aggregates per plate. When grown on tissue culture plastic, approximately 1-2% of these aggregates gave rise to rapidly proliferating epithelial colonies. Individual colonies expanded with a population-doubling time of 24-34 hours and grew for about 3 weeks. Although these cells grew well in primary culture, they were not subculturable. When RME cells were plated onto dishes coated with type I collagen, the number of rapidly proliferating epithelial colonies per dish increased fivefold to tenfold. Cells grown on type I collagen-coated dishes expanded with a population-doubling time of approximately 27 hours and after 2 weeks in primary culture were nearly confluent. Unlike cells grown on plastic, RME cells grown on type I collagen were readily subculturable and serial subculture resulted in the cells undergoing 15-20 population doublings (5-6 passages) before exhibiting any loss of growth potential. Continued feeding of senescent cultures resulted in the emergence of discrete RME cell foci that retained proliferative potential and that eventually developed into rapidly growing cell strains. Exposure of primary cultures to the carcinogen N-methyl-N'-nitro-N'-nitrosoguanidine (CAS: 70-25-7) enhanced the proliferative potential of RME cells in early passages and in later passages either delayed or eliminated the "senescent" phase of cell growth. Carcinogen treatment of RME cells also facilitated the establishment of rapidly growing cell strains with long-term growth potential (greater than 20 passages).
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Neuroendocrine cells in serially passaged rat stomach cancers induced by MNNG.
Five gastric carcinomas, induced in inbred Wistar rats by oral administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) dissolved in drinking water, were successfully transplanted to isologous rats. The transplants grew to a size of 10 to 35 mm in diameter within 8 to 25 weeks of implantation. In one case, serial transplantation were maintained up to the 11th generation, with occurrence of distant metastasis in the 3rd generation. Histological histochemical, and electron microscopical comparison of the original and transplanted tumors revealed that (1) the original tumors were quite well differentiated, forming either papillary or tubular structures, whereas the transplants were more anaplastic and pleomorphic showing often solid nests; and (2) tumor cells with gastrointestinal differentiation and cells with neuroendocrine differentiation were present and evenly distributed in both the original and the serially transplanted tumors. As it is unlikely that the normal and neoplastic neuroendocrine cells are growing side-by-side with and independently of the epithelial neoplastic components in the present series of transplants, the findings strongly suggest (1) the multidirectional potency of the inbred rat stomach carcinoma cells and (2) the common neoplastic origin of the epithelial and neuroendocrine components.
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Primary isolation and serial passage of hepatitis A virus strains in primate cell cultures.
Although several primate cell types have been reported to support replication of hepatitis A virus, optimal conditions for the isolation and production of quantities of virus have not been defined. We therefore examined seven different primate cell types for their ability to support replication of primate-passaged and wild-type virus as reflected by intracytoplasmic accumulation of viral antigen (direct immunofluorescence and radioimmunoassay) and propagation of cell culture-adapted virus. Of the cells tested, low-passage African green monkey kidney (AGMK) cells were most sensitive for initial isolation. Viral replication was documented after inoculation of AGMK cells with seven of nine hepatitis A virus antigen-positive fecal specimens (from seven epidemiologically distinct sources). With six inocula, virus was successfully passed in serial cultures. AGMK-adapted virus was readily propagated in continuous AGMK (BS-C-1) cells. The optimal temperature for the growth of virus in BS-C-1 cells was 35 degrees C. Viral release into supernatant fluids was documented in the absence of any cytopathic effect, and infectivity titers in supernatant fluids 21 days after inoculation (50% tissue culture infective does [TCID50], 10(6.0)/ml) equalled or exceeded those in the cell fraction (TCID50, 10(5.5)/ml). Cells maintained in serum-free media readily supported viral growth, with yields of virus (TCID50, 10(6.5)/ml) equal to or greater than those obtained with cells maintained in 2% fetal bovine serum.
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Growth and serial passage of Pneumocystis carinii in the A549 cell line.
Pneumocystis carinii obtained from infected rats and patients was cultured in the A549 cell line, a presumptive alveolar type 2 cell line derived from a human lung carcinoma. Standard criteria were established for organism sampling, quantitation, and growth. The trophozoite form of P. carinii was a more sensitive indicator of growth than was the cyst. Rat P. carinii increased 10-fold in primary culture and could be serially passed three additional times to new cultures; success in growing human P. carinii was limited and appeared to be related to the quality of the specimen received for culture. Growth pattern experiments suggested that close interaction of P. carinii with the cell monolayer is an important step in the life cycle of the organism. Thus, the A549 culture system should be useful for in vitro studies of the immunobiology of P. carinii.
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Effect of serial passage in female nude athymic mice on androgen dependency of Shionogi carcinoma 115.
When Shionogi carcinoma 115 (SC115, an undifferentiated medullary carcinoma showing a compact cell pattern and containing androgen receptor) was transplanted into male and female DS mice, it grew only in males. In contrast with this strict androgen dependency in DS hosts, SC115 tumors grew in male and female nude athymic (BALB/c-nu/nu) mice. Although most of the tumors developing in female nude mice were composed of spindle-shaped cells and did not contain androgen receptor, about 5% of tumors in female nude mice retained morphological and biochemical characteristics of the original SC115 tumor. Such a tumor was serially transplanted in female nude mice. Although no significant changes were detectable in histological and chromosomal features and in androgen receptor values, the growth speed in female nude mice accelerated and became comparable to the growth speed of the original SC115 tumor in intact male DS mice. However, this subline of SC115 tumor showed a marked androgen dependency when reinoculated into male and female DS mice after 14 passages in female mice nude mice in spite of its relative androgen independency in nude hosts. Therefore, the present results seem to suggest that the immunological status of the hosts may affect the hormone dependency of tumors.
Androgens↗