Morphology and growth potential of stromal cell cultures derived from human endometrium

Summary Propagable cell cultures derived from human endometrial tissue were determined to contain cells predominantly of stromal cell origin based on their morphologic resemblance to endometrial stromal cells. These features included nexi, solitary cilia, and predecidual cytology. In addition to morphology the cell cultures retained a normal karyotype and responded to steroid hormones as evidenced by cellular aggregation. The stromal cells were evaluated for a variety of characteristics associated with transformed cells and seemed to be biologically normal without neoplastic phenotypes. Growth potential of the stromal cell cultures was also characterized in normal maintenance medium, in nutritionally depleted medium with reduced levels of calcium or serum, and in medium with increased levels of serum. The prolonged survival of the stromal cells in vitro coupled with the retention of in vivo characteristics and an absence of neoplastic phenotype provides a human cell system that is amenable to a variety of long-term experimental analyses. This work was supported by contract CP75956 and grant CA31733 from the National Cancer Institute. B. Hugh Dorman was the recipient of a predoctoral scholarship from the Chemical Industry Institute of Toxicology. Jill M. Siegfried was supported by National Research Service Award CA09156. David G. Kaufman is the recipient of a Research Career Development Award (CA00431) from the National Cancer Institute.     

Monolayer culture of human endometrium: Methods of culture and identification of cell types

Summary Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture. This work was supported by contract N01-CP75956 and grant R01-CA31733 from the National Cancer Institute. V. A. Varma is a recipient of an American Cancer Society fellowship; B. H. Dorman, a predoctoral fellowship from the Chemical Industry Institute of Toxicology; J. M. Siegfried, a training grant (CA09156) from the National Cancer Institute; and D. G. Kaufman, a Research Career Development Award (K04-CA-00431) from the National Cancer Institute.     

Histochemical identification of cultured cells from human endometrium

Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, gamma-glutamyltranspeptidase, peroxidase, and beta-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelia in sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma.     

Identification of new cryoprotective agents for cultured mammalian cells

Summary Thirty-one compounds have been identified that act as cryoprotective agents for cultured mammalian cells. Eight compounds were comparable to dimethylsulfoxide (DMSO) in cryprotective effectivenes. Many of the cryoprotective compounds studied also (a) promote cell fusion and (b) induce cell differentiation in erythroleukemia and other cell systems. Thus previously unrecognized effects on the differentiated state of cells may occur when cells are treated with cryoprotective agents. This study was supported, in part, by grants CA 33074 from the National Cancer Institute, Bethesda, MD, and GM 31056 from the National Institutes of Health, Bethesda, MD.     

Establishment and characterization of a cell line (SS78) from a human renal cell carcinoma

Summary A new cell line, SS78, was established from a primary renal cell carcinoma of a Caucasian male. The tissue was dispersed with collagenase, and viable cells were separated by flotation on a Ficoll-Hypaque gradient. In culture, the SS78 cells retained a distinct epithelial morphology, and no fibroblastlike cells were seen. The cultured cells were aneuploid with a modal chromosome number of 80 and had several marker chromosomes. Inoculation of the cultured cells into athymic nude mice caused tumors at the sites of inoculation. This research was supported in part by Grants CA 15972 and CA 14930 from the National Cancer Institute through the National Bladder Cancer Project and by the Medical Research Service of the Veterans Administration.     

Presence of retrovirus in the B95-8 Epstein-Barr virus-producing cell line from different sources

Summary The B95-8 cell line, a widely used source of highly transforming Epstein-Barr virus (EBV), obtained from the laboratory of origin, harbored an infectious retrovirus. This retrovirus generally resembled the Type D retroviruses structurally and developmentally and like the Type D retroviruses preferred Mg²⁺ to Mn²⁺ in its RNA-directed DNA polymerase reaction. Evidence for the presence of retrovirus was found in B95-8 cultures from two other sources within the United States, either by assay for polymerase or by electron microscopy. Comparison of two B95-8 cell lines showed cytogenetic differences as well as differences in retroviral activities. The results suggest that any B95-8 culture should be tested for the presence of retrovirus before its use as a source of EBV. This research was supported through the National Research and Demonstration Center (HL-17269-07) awarded to Baylor College of Medicine by the National Heart, Lung, and Blood Institute, Bethesda, MD, by RD-125 from the American Cancer Society, by K06 CA14219, CA16781, CA25465, and CA16672 from the National Cancer Institute, Bethesda, MD, and by G-429 from the Robert A. Welch Foundation. G. E. G. was supported by Public Health Service training Grant CA-09299.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Characterization of cells cultured from early lactation milks

Summary Two major types of cells can be cultured from early lactation human milks: a colony-forming epithelial cell and an adherent nondividing cell referred to as a foam cell The epithelial cells show a positive reaction with a specific antiserum reactive against membrane components of the milk fat globule, whereas the foam cells do not. The nondividing foam cells are phagocytic and can be killed by silica particles; they produce lysozyme, are resistant to trypsinization, and have Fc receptors. These properties, together with the lack of reaction with antiserum to the milk fat globule membrane, suggest that the foam cells are not terminally differential epithelial cells, but tissue macrophages. R. L. C. was supported by Grant No. Ca 19455 from the National Cancer Institute, a Yamagiawa-Yoshida Memorial International Cancer Study Grant, and the Imperial Cancer Research Fund. J. A. P. was supported by Grant No. CA 19455 from the National Cancer Institute.     

In vitro characterization of estrogen induced syrian hamster renal tumors: Comparison with an immortalized cell line derived from diethylstilbestrol-treated adult hamster kidney

Summary Primary diethylstilbestrol-induced kidney tumors from Syrian hamsters were grown in vitro and maintained in culture for 6 mo. Combined immunohistochemical studies using antibodies to intermediate filaments and ultrastructural studies of tumor cells in culture exhibited characteristics similar to tumor cells in vivo. Furthermore, the cells manifested transformed properties in culture; they grew both as multilayered colonies attached to the tissue culture substrate and as floating multicellular colonies (spheroids). When cultured cells were injected into diethylstilbestrol-treated recipient hamsters, tumors developed at the injection sites. In contrast, renal tubules or whole kidney cortex from control hamsters cultured in the same medium underwent only short-term growth, with senescence developing after approximately 1 mo. However, cell cultures of kidney cortex from animals treated in vivo for 5 mo. with diethylstilbestrol formed a cell line. This diethylstilbestrol-induced cell line has been maintained in culture for 1.5 yr and has the following characteristics: a) it is anchorage-dependent, b) it is negative in in vivo tumorigenicity tests, and c) cultured cells are histochemically and ultrastructurally similar to cultured tumor cells. This culture system should prove to be of use in studying hormonal carcinogenesis in vitro. This study was supported by the Medical Research Service, Department of Veterans Affairs, Washington, DC, and by grant CA-22008 from the National Cancer Institute, NIH, DHHS, Bethesda, MD.     

Lymphoid cell line identification and the detection of cross contamination

Summary Four lymphoid cell lines, presumably recent derivatives from non-neoplastic human tonsils, were identified in actuality as established Burkitt, lymphoma and lymphoblastoid cell lines (LCL) when tested by chromosome banding and typing for HLA antigens. Additionally, the morphology, growth characteristics, tumorigenicity, expression of Epstein-Barr virus antigens and surface membrane immunoglobulins (SmIg) of the four lymphoid cell lines confirmed the correct identifications. In determining the possiblity of cross contamination, the most reliable criteria for the identification of lymphoid cell lines were found to be HLA antigenic profile, karyotype, cellular morphology, and growth characteristics. This work was supported by Contract N01 CP-53516 from the Virus Cancer Program of the National Cancer Institute, NIH, and by NIH Grants AI 13154, CA 16069, and CA 16071. Dr. Ferrone is a recipient of an American Heart Association Established Investigatorship Award. Dr. Pellegrino is a recipient of a Research Career Development Award. Dr. Glaser is the recipient of a Leukemia Society of America Scholar Award. This is publication 1786 from Scripps Clinic and Research Foundation.     

A cloned rat thymic epithelial cell line established from serum-free selective culture

Summary A serum-free system has been developed for selective growth and long-term culture of rat thymic epithelial cells. The growth media is a modification of McKeehan's WAJC 404, plus insulin, cholera toxin, dexamethasone, and epidermal growth factor. Cultures have been continuously passaged and maintained for over 6 mo., and a cloned cell line, TEA3A1, has been established. These cells are epithelial, judging by morphology and ultrastructure, and are positive for A2B5 and thymosin α markers for thymic endocrine cells. This work was partly supported by grant PCM-834 0582 from the National Science Foundation, Washington, DC, and grant P01 CA 37589-2 from the National Cancer Institute, Bethesda, MD.     

Growth of human-human hybridomas in serum-free media enhances antibody secretion

Summary Human-human hybridomas derived from fusing lymph node lymphocytes with UC 729-6 were adapted to grow in commercially available serum-free medium and were compared with serum-supplemented [10% fetal bovine serum (FBS)] cultures. Over a 6-d period, no significant changes occurred in the growth of the cells in 10% FBS or serum-free medium. In cultures supplemented with 10% FBS more total proteins were secreted than in serum-free cultures. However, there was an enhanced secretion of three- to four-fold of both immunoreactive human IgG and IgM under serum-free conditions compared to serum-supplemented conditions. Serum-free conditions may provide the appropriate milieu for the increased level of Ig secretion from human hybridomas derived from UC 729-6 in that there are no inhibitors that may be present in serum. The work described in this report was partially supported by grants from the National Institutes of Health (CA 32047, CA 37497), Bethesda, MD, and the University of California Cancer Research Coordinating Committee. R.E.P. was a postdoctoral fellow supported by the UCSD Cancer Center and M.C.G. was the recipient of an NIH New Investigator Research Award. A.M. was a visiting scientist from the Medical Academy of Bulgaria, Sofia.     

Mouse mammary cells ind-valine medium

Summary Cells of a mouse mammary epithelial cell line as well as fibroblasts from a mouse mammary explant were severely inhibited from proliferating in a medium in whichd-valine was substituted forl-valine. After the first few days ind-valine medium, the number of epithelial cells did not increase despite the fact that a few percent continued to synthesize DNA. The cells did recognize the presence of thed-valine in the medium because cells ind-valine increased in volume and their numbers remained stationary, whereas cells without valine shrank and the cell numbers decreased with time. The NMuMG cells were obtained from Mr. Robert Owens and were produced with support from the National Cancer Institute, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention under the auspices of the Office of Naval Research and the Regents of the University of California. This project was funded by the National Cancer Institute, Bethesda, MD, Contract N01-CB-74094.     

Ornithine decarboxylase activity in human endometrium and endometrial cancer cells

Summary Ornithine decarboxylase (ODC) activities were significantly higher in proliferative endometrium during the estrogen-dominated follicular phase of the menstrual cycle than in secretory endometrium after the formation of the progesterone-secreting corpus luteum. The enzymatic activity was increased about fivefold by renewal of the medium during incubations of endometrial fragments or isolated endometrial glands. Endometrial adenocarcinoma cells (HEC-1, HEG-50), both in monolayers and suspension, also responded to medium renewal by increasing ODC activity about 10-fold after 4 h, with subsequent reduction to control levels after 7 h. These effects were blocked by actinomycin D and cycloheximide. Endometrial stromal cells exhibited highly variable ODC activities at different passages. Difluoromethylornithine (DFMO) and sodium molybdate had marked antiproliferative effects in HEC-50 cultures, reducing cell numbers to 10 to 20% of control values 11 d after plating and inhibiting ODC activity by approximately 80% on Day 7. The antiproliferative effect of DFMO, but not that of molybdate, was reversed by 10 μM putrescine, the product of ODC activity. In contrast to DFMO, molybdate had no effect on ODC activity of cell homogenates. Molybdate did not elicit antizyme formation in HEC-50 cells under conditions in which putrescine did. These results indicate that ODC activity, present in both epithelial and stromal cells, as shown analytically and also by autoradiography after labeling with [³H]DFMO, may be related to cell proliferation in vivo and that proliferation of human endometrial cancer cells in culture can be arrested by DFMO and by molybdate. This investigation was supported by PHS grant HD 07197, awarded by the National Institute of Child Health and Human Development and PHS grant CA 15648, awarded by the National Cancer Institute.     

Biological characteristics and viral susceptibility of a stable dog kidney cell line

Summary A cell line derived in 1956 from normal dog kidney is described. The cells are epithelial, contact-inhibited, and can be maintained in the same culture vessels for periods of more than 2.5 yr. Karyologically, the cells are hypodiploid with a modal number of 72 as opposed to the diploid number of 78. The karyotype indicates male origin of the cells and clonal derivation of extant cultures due to the presence of two marker chormosomes in all metaphases observed. At the 159th passage the dog kidney (DK) cells did not produce tumors in athymic rats. At least 13 viruses of various types produced transmissible cytopathogenic effects in the DK cells, including all of the human influenza viruses investigated. The project was supported in part by an Institutional Research and Development grant from SRI International and Contract Y01 CP8-0500, National Cancer Institute, National Institutes of Health, Bethesda, MD.     

A simplified technique for the short-term tissue culture of rabbit corneal cells

Summary A technique for the short-term culture of pure populations of rabbit corneal endothelial and epithelial cells has been developed. Rabbit corneas were placed on concave agarose surfaces, treated briefly with a solution of trypsin and ethylenediamine tetracetic acid, and transferred, either epithelial cell surface or endothelial cell surface down, to microscope slide culture chambers. Within 6 to 12 h the epithelial cells or endothelial cells attached to the slide chamber surface and the cornea was removed, leaving behind a pure population of cells which spread out and grew to fill the surface of the slide chamber. This technique provides a simple and economic means for the reproducible initiation of primary cultures of rabbit corneal epithelial and endothelial cells for us in a variety of experiments. This study was supported in part by Public Health Service grants EY03150, EY02580, and EY02377 from the National Eye Institute, National Institutes of Health, Bethesda, MD, and a Foreign Fellowship (Dr. Xie) from Research to Prevent Blindness, Inc., New York, NY.     

Cytoplasmic suppression of malignancy

Summary Using both normal and transformed rat liver epithelial cells to prepare cytoplasmic hybrids (cybrids) we have found evidence to support the theory that the cytoplasm from a normal cell can suppress tumorigenicity. A unique aspect of this study is that all of the cells utilized, both normal and malignantly transformed, were derived from an original cloned cell. We found that fusing cytoplasts from normal cells to malignantly transformed whole cells resulted in cybrid clones which, when injected into newborn rat pups, isogenic with those from which the cell culture was initiated, yielted tumors in 51% of the animals injected compared to 92% of the animals injected with the tumorigenic parent. Those animals that did develop tumors from the cybrid cells survived longer than those injected with cells from the tumorigenic parent. Thus, the cybrid, formed of cytoplasm from both parents, was less tumorigenic than the malignantly transformed parent cell. When reconstituted cells were prepared by fusing cytoplasts from normal cells with karyoplasts from malignantly transformed cells, a situation in which essentially all of the cytoplasm of the reconstituted cell is derived from normal cells, the tumorigenic phenotype was extinguished. This work was supported in part by United States Public Health Service grant CA12056, and grant CA09100 from the National Cancer Institute, Bethesda, MD. This work is partial fulfillment for the degree of Doctor of Philosophy for B.A.I.     

Establishment of duck cell line derived from experimental tumor induced by 20-methylcholanthrene

Summary Experimental tumors developed in white Pekin ducks after intramuscular implantation of 20-methylcholanthrene. Cells derived from the primary tumor were adapted successfully to grow in vitro and have growth characteristics similar to that of established cell lines of mammalian origin. The cell density rises rapidly and the doubling time is approximately 19 hr. The duck cells have been cultured succesfully for at least 80 passages in vitro. The continuously cultured cells have the characteristic chromosome pattern of duck, and the DNA of the duck cell line hybridized with duck liver DNA. We believe we have established a continuous cell line of avian origin. Electron-microscopic examinations of the tumor cells and RNA-directed DNA polymerase of the cell-free supernate show no evidence of endogenous virus production. This study was supported by Public Health Service Research Grants CA 16479 and CA 20012 from the National Cancer Institute and RR 00890 from the National Institutes of Health.     

CA 125 is an excretory product of human endometrial glands

The present investigation was undertaken to study the cellular localization and kinetics of synthesis of CA 125 in the endometrium. CA 125 was localized by immunohistochemistry to the infranuclear region of epithelial cells during the proliferative phase and to the apical luminal border during the secretory phase. In gestational endometrium, both the cytoplasm and the apical luminal border of epithelial cells were intensely positive. No staining was seen in endometrial stromal cells during the normal cycle or in decidualized endometria. Results obtained from in vitro cultures of separated glandular and stromal cells were similar to those obtained by immunohistochemistry. That is, epithelial cells released between 5 and 25 times more CA 125 into the culture medium than did stromal cells. The release of CA 125 was highest in epithelial and stromal cells obtained during the early secretory phase. CA 125 concentrations were markedly elevated in endometrial aspirations obtained during the secretory phase or in endometria with crumbling stroma compared to plasma levels. Plasma levels of CA 125 were slightly elevated during menses. These results suggest that CA 125 is an exocrine product of endometrial epithelial cells. Plasma levels of CA 125 may be of endometrial origin only when the membrane barriers, which normally prevent its entry into the circulation, are damaged.     

Maintenance of adult hamster pancreas cells on fibroblastic cells

Summary The maintenance of primary cultures of adult hamster pancreatic cells on layers of irradiated C3H/10T1/2 cells was studied. Various types of pancreatic cells, acinar, islet and ductular cells could be identified in the cultures by light and electron microscopy. Morphologically the various pancreatic cells retained many differentiated characteristics of their respective in vivo cell types. Insulin production was maintained at near Day 1 levels for the 16 d in culture for which it was measured. Colonies of epithelial cells continued to grow during a 20 d culture period. It is believed that this procedure for maintaining functional and growing pancreas cells in culture may be a useful in vitro model for studying the initiation of pancreatic carcinogenesis. Supported by Grant R01 CA 20022 and Contract N01 CP33278 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland.