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.     

Characterization of bovine pancreatic ductal cells isolated by a perfusion-digestion technique

Summary Bovine pancreatic ductal cells isolated by perfusing an enzyme solution into the lumen of the main duct were obtained as sheets of cells. Morphologic features of these cells were those of pancreatic ductal epithelial cells. These cells also contained alcian blue/periodic acid-Schiff positive material and bound lectins, and they stained for keratin in the same manner as intact ductal epithelium. In culture, the plating efficiency was high (13.6%) as determined by DNA content before and after 24 h plating, perhaps due to the gentle isolation technique and the isolation of sheets of cells rather than a single cell. Cell doubling time was 34.4 h in Eagle's minimal essential medium with 10% heat inactivated fetal bovine serum and antibodies, and over 95% of the cell incorporated [³H]thymidine during a 6 h labeling period after 4 d in primary culture. Isolated cells grew best in medium CMRL 1066 with 10% heat inactivated fetal bovine serum as determined by measuring DNA content. The paper is Publication 1100 from the Department of Pathology, University of Maryland School of Medicine. This study was supported in part by National Cancer Institute (Bethesda, MD) Contract NO1-CP-75947 and Grant CA-19197-06 through the National Pancreatic Cancer Project.     

Morphological,growth, and chromosomal changes in bovine pancreatic duct epithelial cells exposed toN-methyl-N′-nitro-N-nitrosoguanidine

Summary Epithelial cells cultured from bovine pancreatic ducts were given a single treatment ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Multinucleated cells and giant cells were observed more frequently in carcinogen-treated cultures than in controls. The MNNG-treated cultures also contained a sizeable population of small, dense cells that were not observed in control cultures. At the concentration of 1.0 μg/ml, MNNG caused an initial depression in the growth rate of the cells followed by growth stimulation for several weeks. The MNNG produced chromosomal damage in the cells as indicated by the observation that a substantial proportion of carcinogen-treated cells were heteroploid and contained a high frequency of metacentric and submetacentric chromosomes and a dicentric marker chromosome. The MNNG treated and control cultures did not acquire the ability to grow in soft agar or to produce tumors after transplantation into athymic, nude mice. This work was supported in part by Public Health Service Contract and Interagency Agreement Y01-CP60204 from the Division of Cancer Cause and Prevention, National Cancer Institute.     

Culture of adult rat lung cells: Benzo(a)pyrene metabolism and mutagenesis

Summary A method is described for obtaining and culturing large numbers of lung cells from normal adult male rats. The lungs were perfused in situ to remove blood cells and then perfused via the trachea with a trypsin-collagenase solution to initiate tissue digestion. The tissue was further digested in the enzyme solution and approximately 2×10⁸ viable lung cells were obtained per animal. Primary cultures contained a mixed cell population. Through eight subcultures about 70% of the cell population possessed an epithelial-like morphology, whereas the remaining 30% was fibroblast-like. Three clones of epithelial-like cells were isolated at the fourth subculture. The mass culture lung cells and the epithelial-like clone that was studied retained a normal karyotype and did not grow in soft agar. Both the mass culture cells and the epithelial clone metabolized the lung carcinogen benzo(a)pyrene (BP) to water-soluble products. Furthermore, the mass culture lung cells metabolized BP to intermediate(s) which mutated Chinese hamster V79 cells from ouabain sensitivity to ouabain resistance. These lung cell cultures have potential use in cell transformation, mutation and carcinogen metabolism studies. Visiting scientist from Hungary. This research was supported by Grant 5 R01 CA20022 and Public Health Service Contract N01 CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health.     

Isolation and characterization of epithelial cells from bovine pancreatic duct

Summary Epithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90 doublings of the cell population). The cells were grown in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 μg per ml) and insulin (1,5 and 10 μg per ml) had no effect on the growth of the cells. β-Retinyl acetate inhibited growth rate and cell yield at a concentration of 5 μg per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria, Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones are different as well as the period of time in which the clones can be propagated in vitro. This work was supported in part by Y01 CP 60204 and N01 CP 43237.     

In vitro cytodifferentiation of perinatal rat islet cells within a tridimensional matrix of collagen

Summary Cell suspensions prepared by collagenase digestion of pancreases obtained from rat fetuses (21.5 d old) and newborns (2.5 d old) were mixed with a collagen solution and inoculated on a collagen base layer. At the onset of the culture, most acinar cells became necrotic, whereas other epithelial cells proliferated. Most of the cell clusters arranged themselves into simple polarozed structures composed of epithelial cells forming hollow spheres, and from these budded neoformed endocrine islets. Scarce fibroblasts were located close to these structures. Immunocytochemical localization of insulin and glucagon, as well as ultrastructural characteristics of the cell types revealed an intrainsular distribution similar to the in vivo localization. Tridimensional matrix of collagen offers, to perinatal pancreatic cells in culture, an environment close to the in vivo conditions: cells reorganize themselves in tissuelike structures and cell interactions concerned in the cytodifferentiation of pancreatic islets occur. This system allows for the study of undifferentiated epithelial cells—the presumed stem cells—differentiating and differentiated endocrine cells in the same preparation. B.A. is supported by a doctoral scholarship from the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture, Brussels. This work was supported by grants from the Fonds National de la Recherche Scientifique, Brussels, and from Petrofina S.A., Brussels.     

Simulated microgravity culture system for a 3-D carcinoma tissue model

An in vitro organotypic culture model is needed to understand the complexities of carcinoma tissue consisting of carcinoma cells, stromal cells, and extracellular matrices. We developed a new in vitro model of carcinoma tissue using a rotary cell culture system with four disposable vessels (RCCS-4D) that provides a simulated microgravity condition. Solid collagen gels containing human pancreatic carcinoma NOR-P1 cells and fibroblasts or minced human pancreatic carcinoma tissue were cultured under a simulated microgravity condition or a static Ig condition for seven days. NOR-P1 cultures subjected to the simulated microgravity condition showed greater numbers of mitotic, cycling (Ki-67-positive), nuclear factor-kappa B-activating cells, and a lower number of apoptotic cells than were shown by cultures subjected to the static Ig condition. In addition, human pancreatic carcinoma specimens cultured under the simulated microgravity condition maintained the heterogeneous composition and cellular activity (determined by the cycling cell ratio and mitotic index) of the original carcinoma tissue better than static culture conditions. This new 3-D rotary cell culture system with four disposal vessels may be useful for in vitro studies of complex pancreatic carcinoma tissue.     

A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype

The development of in vitro models able to support the long-term viability and function of acinar cells is critical for exploring pancreatic pathophysiology. Despite considerable efforts, no long-term culture models for non-transformed pancreatic acini exist. Our aim was to develop and validate culture conditions for this purpose. An explant outgrowth culture design was established in which mouse pancreatic explants were cultured at the gas-liquid interphase. An enriched culture medium, pH 7.8, was employed to promote the selective outgrowth of acinar cells and to support their differentiated phenotype. After 7 days, the outgrown primary acinar cells were subcultured and maintained up to an additional 7 days as secondary monolayers on tissue culture plastic. Measurements of basal and caerulein-induced amylase secretion, phase-contrast microscopy and immunohistochemical analyses were used to characterize the cultures. Explants retained their pancreatic cytoarchitecture for 2 days in vitro. A triphasic dose response to caerulein was detected in 7-day primary cultures. The maximal rate of secretion was 1.2-fold versus basal (p=0.009) and 1.7-fold versus 1 pM caerulein (p=0.014). In secondary cultures the response was biphasic with maximal rates of secretion being 1.9-fold in 3- to 4-day cultures at 0.01 nM (p=0.049) and 2-fold in 6- to 7-day cultures at 0.1 nM (p=0.003). The present culture model provides a means to obtain functionally competent normal mouse acinar cells for long-term in vitro experimentation.     

Heterocellular cultures of pulmonary alveolar epithelial cells grown on laminin-5 supplemented matrix

The pulmonary alveolar epithelium consists of alveolar type I (AT1) and alveolar type II (AT2) cells. Interactions between these two cell types are necessary for alveolar homeostasis and remodeling. These interactions have been difficult to study in vitro because current cell culture models of the alveolar epithelium do not provide a heterocellular population of AT1 and AT2 cells for an extended period of time in culture. In this study, a new method for obtaining heterocellular cultures of AT1- and AT2-like alveolar epithelial cells maintained for 7 d on a rat tail collagen-fibronectin matrix supplemented with laminin-5 is described. These cultures contain cells that appear by their morphology to be either AT1 cells (larger flattened cells without lamellar bodies) or AT2 cells (smaller cuboidal cells with lamellar bodies). AT1-like cells stain for the type I cell marker aquaporin-5, whereas AT2-like cells stain for the type II cell markers surfactant protein C or prosurfactant protein C. AT1/AT2 cell ratios, cell morphology, and cell phenotype-specific staining patterns seen in 7-d-old heterocellular cultures are similar to those seen in alveoli in situ. This culture system, in which a mixed population of phenotypically distinct alveolar epithelial cells are maintained, may facilitate in vitro studies that are more representative of AT1-AT2 cell interactions that occur in vivo.     

Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal 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, γ-glutamyltranspeptidase, peroxidase, and β-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. Epithelial 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. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

Requirements for maintaining the embryonic state of avian tendon cells in culture

Summary Primary avian tendon cells (PAT) maintain their embryonic state when cultured in medium F-12 with very low serum (0.2%) and ascorbate (50 μg per ml); that is, they retain the potential for devoting 20–30% of their total protein synthesis to collagen. However, if the cells are left at a confluent cell density or are derived from confluent cultures, this potential is irreversibly decreased. This effect, along with poor medium formulations, probably accounts for the “dedifferentiation” process that occurs when fibroblasts are cultured. In contrast, PAT cells kept at subconfluent cell densities retain the ability to synthesize high levels of collagen. The one limitation in obtaining long-term cultures of high collagen-producing tendon cells in the inability of serum at low concentrations to remain a potent mitogen after a few subcultures. The quantitative loss of function has long been considered to be a cell culture artifact; however, we propose that this drop in collagen synthesis is a reflection of the developmental programing of these cells. In separate series of experiments using organ cultures, we show that tendon tissue from the embryo makes over 30% collagen, whereas, “young” tendons make 18% and “older” tendons from the adult make less than 1%. Therefore, a quantitative drop in collagen synthesis would be expected if normal development were to occur in culture. Our data are consistent with the idea that cultures of embryonic tendon cells are triggered to mature by a mechanism that correlates with high cell density. This investigation was supported in part by National Science Foundation Grant PCM 77-14982; in part by the Division of Biomedical and Environmental Research of the Department of Energy under contract W-7405-ENG-48; and by a National Institutes of Health Fellowship IF32 CA 05807-01, from the National Cancer Institute to R. I. S.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Exogenous activin increases the proportion of insulin cells in the developing chick pancreas in culture

As activin is believed to be a key signalling factor during early pancreatic development, its influence on the proliferation and/or determination of insulin cells in the developing chick dorsal pancreatic bud was investigated. Dorsal pancreatic buds of 5-day-old chick embryos were explanted on to Matrigel and cultured in serum-free medium (Ham's F12.ITS), to which 1 or 10ng/ml activin was added. After 7 days in culture, the explants were processed for immunocytochemistry and the insulin-positive cells were scored and expressed as a proportion of the sum of insulin and glucagon cells. When compared to the control cultures (Hams F12.ITS alone), activin treatment resulted in respective increases in the proportion of insulin cells of 1.6 and 1.9 fold. It is suggested that activin treatment favours differentiation of the insulin cell pathway relative to glucagon cells.     

Nutritional requirements of papiliomavirus-transformed mouse cells and an unifected parent line in serum-free culture

Summary A serum-free culture system was used to compare the nutritional requirements of mouse mammary cells transformed by bovine papillomavirus type 1 (ID13 cells) and the uninfected parent line (C127 cells). The serum-free, chemically defined medium used for this study was an MCDB 151-based medium (MCDB 151+S+I), supplemented with epidermal growth factor, transferrin, hydrocortisone, ethanolamine, phosphoethanolamine, retinoic acid, trace metals, and insulin. Proliferation of either cell type in serum-free culture required the addition of 250 μg/ml of insulin. ID13 cells have a doubling time of greater than 96 h in MCDB 151+S+I, whereas C127 cells have a doubling time of 60 h. This is in sharp contrast to the growth characteristics of the two cell types in 10% fetal bovine serum, where doubling times for the ID13 and C127 cells are 24 and 30 h, respectively. Culture of the cells in a serum-free medium has therefore revealed that the papillomavirus-transformed cells have more stringent growth requirements than the uninfected parent line. This work was supported in part by grant #1-P01 NS19214 from the National Institutes of Health, Bethesda, MD, NSF grant #R11-8217798 from the National Science Foundation, Washington, DC, and by a grant from the Otolaryngology Foundation.     

Culture of human main pancreatic duct epithelial cells

Summary Attempts to grow human pancreatic duct epithelial cells in long-term culture have proven difficult. We have developed a system of growing these cells for several passages by adapting methods used to culture dog pancreatic duct cells. Epithelial cells were enzymatically dissociated from the main pancreatic duct and plated onto collagen-coated culture inserts suspended above a human fibroblast feeder layer. After primary culture, the cells were either passaged onto new inserts or plastic tissue culture plates in the absence of collagen. Cells grown on the latter plates were maintained in a serum-free medium. Primary pancreatic duct epithelial cells grow steadily to confluence as a monolayer in the feeder layer system. After primary culture, cells passaged onto new inserts with fresh feeder layer or plastic plates and fed with serum-free medium continued to develop into confluent monolayers for up to four passages. The cells were columnar with prominent apical microvilli, sub-apical secretory vesicles, and lateral intercellular junctions resembling the morphology of normal in vivo epithelial cells. These cells were also positive for cytokeratin 19, 7, and 8 and carbonic anhydrase II, as measured by immunohistochemistry. Metabolically, these cells synthesized and secreted mucin, as measured by incorporation of tritiated N-acetyl-d-glucosamine. In conclusion, we demonstrated that human pancreatic epithelial cells from the main duct can be successfully grown in culture and repeatedly passaged using a feeder layer system, with serum-free medium, and in organotypic cultures.     

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.     

Use of pancreatic beta cells in culture to identify diabetogenic N-nitroso compounds

Epidemiological observations suggest that environmental factors play a role in the pathogenesis of insulin dependent diabetes mellitus (1). Several chemicals have been identified as specific beta cell toxins (2-4). We report here studies to determine the feasibility of using monolayer cultures of pancreatic beta cells from neonatal rat to screen potential diabetogenic chemicals. Cytotoxicity was monitored both by phase microscopy and the release of insulin into the culture medium. In comparative studies, cellular protein and release of 51chromium (51Cr) were measured after addition of test compounds to cultures of fibroblasts derived from pancreatic tissue. The nitrosoamides 1 methyl-l-nitrosourea (MNU), 1,3 bis (2-choroethyl) nitrosourea (BCNU), chlorozotocin (CLZ), and the beta cell toxin, streptozotocin (SZ), were examined. CLZ and SZ were more toxic to pancreatic beta cells than to fibroblasts. In contrast, MNU and BCNU damaged both beta cells and fibroblasts at identical concentrations. These results suggest that in vitro techniques can be used to identify chemicals that selectively injure beta cells. Although SZ-induced toxicity was ameliorated with addition of nicotinamide to cultures of beta cells, nicotinamide did not prevent damage caused by CLZ. This observation indicates different mechanisms of drug-induced cytotoxicity.     

Protein-free medium for C-1300 mouse neuroblastoma cells

Summary An optimized medium, designated MCDB 411, has been developed for mouse neuroblastoma cells. At cell densities of 1×10⁴ cells/cm² or greater, several different clones of C1300 mouse neuroblastoma cells can be cultured serially in Medium MCDB 411 with no serum or other undefined supplementation and with a doubling time of about 24 h. At clonal densities it is necessary to supplement the medium with 1.0 μg/ml insulin. Alternately, good clonal growth can be obtained without direct supplementation by coating the culture dishes with insulin and rinsing off all that is not tightly bound. Primary cultures of cells from serially transplanted C1300 tumors that have never been cultured previously in vitro can be established directly in unsupplemented Medium MCBD 411 with rapid initiation of multiplication and no apparent crisis or selection for minority cell types. The availability of a synthetic medium that supports growth of neuroblastoma cells without supplementation should facilitate the use of these cells as model systems for the study of neuronal function and differentiation. This work was supported by Grant CA-15305 from the National Cancer Institute.