Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Normal human endometrial cells in culture: Characterization and immortalization of epithelial and stromal cells by SV 40 large T antigen

Summary— Thirty endometrial biopsies were cultured in order to separate stromal and epithelial cells. Using epidermal growth factor (EGF), cortisol, cholera toxin, insulin with 5% horse serum for epithelial cells or a medium with 20% fetal calf serum for stromal cells, we could specifically enrich endometrial culture in epithelial or stromal cells and culture them for 1 or 2 months. These cultures retained the phenotypic characteristics of epithelial (cytokeratins, mucin HMFG 1) and stromal (vimentin, smooth muscle actin, desmin) lineage by immunostaining analysis. Epithelial and stromal cultures from one individual were respectively immortalized by the SV 40 large T antigen. The immortalized cell lines kept the phenotype of the normal cells from which they derived.     

Effects of reduced calcium on proliferation and cell viability in tumorigenic and nontumorigenic rat tracheal epithelial cell lines

We have compared the in vitro growth and viability of tumorigenic and nontumorigenic rat tracheal epithelial cell lines over a range of calcium concentrations from 0.003 to 0.85 mM. A greater dependence on calcium for proliferation was seen in the nontumorigenic line as compared to the tumorigenic line at both the colony formation level and in mass cultures. In the latter culture condition, a marked differential effect on cell survival was also demonstrated. These differences in calcium dependence were seen in media containing fetal bovine serum or low concentrations of newborn calf serum and in a serum-free medium developed for these cells. The effect was also independent of the method used for calcium removal i.e., either by chelex treatment or the inclusion of EGTA. Therefore, loss of calcium dependence may be associated with tumorigenicity in rat tracheal epithelial cells offering a selectable marker for neoplastic cells in carcinogen-exposed preneoplastic cell populations.     

Primary culture in chemically defined medium of human fetal mammary epithelial cells within reconstituted matrix]

A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal human fetal mammary epithelial cells within an extracellular matrix preparation. Human fetal mammary epithelial cells were isolated from the mammary glands of human female fetuses, 17 to 39 weeks-old. The "organoids" were embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm (EHS) sarcoma according to the method of Hahm and Ip. "Organoids" were grown in either serum-free medium or in medium with fetal calf serum (FCS). The "organoid" proliferated over a 2 to 3 weeks culture period and remained viable for 1 or 2 months within the basement membrane matrix in serum free medium. Several types of colonies were observed; including alveolar-like budding clusters obtained from cultures of mammary gland from fetuses of over 20 weeks age, units with ductule-like projections and stellate-type colonies. Cell proliferation was dependent on the culture medium (with FCS no proliferation was obtained) and on the substratum (without matrix, significantly less growth and development occurred). These types of colonies are obtained when a glandular differentiation of cells budding from the malpighian epithelium is observed. Light microscopic and transmission electron microscopic studies were undertaken at the time of culture. This unique system using normal fetal mammary epithelial cells thus provides a model in which the regulation of human mammary development can be investigated.     

Optimization of bovine satellite cell-derived myotube formation in vitro

Post-natal myogenic satellite cells, isolated from the sternomandibularis muscles of bovine at slaughter were used for primary culture studies. Isolated satellite cells tended to differentiate into multinucleated myotubes more efficiently if initially plated on to a fibronectin substratum. Bovine-derived satellite cells displayed greater fused cell numbers when exposed to Dulbecco's Modified Eagle's Medium (DMEM) supplemented with horse serum than similar supplementation with fetal calf serum (P less than 0.05) or sheep serum (P less than 0.05). In addition, differentiation appeared nearly complete after 4 days exposure to DMEM-1% horse serum as verified by beta-D-arabinofuranosyl-cytosine addition to cultures. Collectively, these data provide the first evidence that satellite cells can be isolated from a bovine skeletal muscle. Furthermore, these data indicate that bovine-derived satellite cells can be induced to undergo substantial morphological differentiation in vitro.     

Serum effects on the response of mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae.

The response of mammalian cells to Pseudomonas and diphtheria exotoxins was studied. A method was developed whereby the sensitivity of cells to these two toxins could be quantitated. The method is versatile and can be used to study the effects of toxins on many cellular metabolic or transport processes. The type of serum used in the culture medium significantly influenced the response of cells to the toxins. Calf, horse, and human sera protected cells while fetal calf serum did not. Precipitation with (NH4)2SO4 demonstrated the probable presence of toxin-specific antibody in the protective calf serum while none was detected in the nonprotective fetal calf serum. The level of antibody in calf serum, as titrated by hemagglutination, was sufficient to account for all the observed protection. It is suggested that fetal calf serum be used for all future cell culture studies of bacterial toxins.     

Primary culture and maintenance of steroid-secreting human placenatal monolayers.

A simple method is described for primary culture and for maintenance of hormone-producing cells from normal human placenta. A consistent yield of cells was obtained and an average survival of 3 to 4 months in culture using 1 mm3 explants from the most vascular area of the placentas. These explants were placed in a variety of culture media in 30 ml flasks and incubated at 37 degrees C in an atmosphere of 5% CO2 and 95% air. The best yields in terms of cell growth were observed with Eagle's MEM (minimum essential medium) with supplements of horse serum and fetal calf serum or human cord serum. (Ham's F-10 with supplement of horse serum and fetal calf serum supports growth for the longest period and media containing human cord serum had the best yield of steroids.     

Obligatory Relationship Between the Sterol Biosynthetic Pathway and DNA Synthesis and Cellular Proliferation in Glial Primary Cultures

Primary cultures of newborn rat brain, which are composed predominantly of astroglia, were used to examine the relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation. Reduction of the fetal calf serum content of the culture medium from 10 to 0.1% (vol/vol) for an interval of 48 h between days 4 and 6 in culture resulted in a quiescent state characterized by inhibition of DNA synthesis and cellular proliferation. When 10% fetal calf serum was returned to the medium for these quiescent cells, within 24 h DNA synthesis increased markedly. Preceding the rise in DNA synthesis was an increase in sterol synthesis, which occurred within 12 h of the return of the quiescent cells to the 10% fetal calf serum. Exposure of the quiescent cells to mevinolin, a specific inhibitor of sterol synthesis at the 3-hydroxy-3-methylglutaryl-CoA reductase step, completely inhibited the increase in DNA synthesis that followed serum repletion. The increase in total protein synthesis that followed serum repletion was not similarly inhibited by mevinolin. When mevinolin was removed after causing the 24-h inhibition of DNA synthesis, the cultured cells underwent active DNA synthesis and proliferation. Thus, inhibition of the sterol biosynthetic pathway resulted in a specific and reversible inhibition of DNA synthesis and glial proliferation in developing glial cells. These findings establish a valuable system for the examination of glial proliferation, i.e., primary glial cultures subjected to serum depletion and subsequent repletion. Moreover, the data establish an obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation in developing glia.     

Cultivation of chicken proventricular epithelial cells and their potential for differentiation

Epithelial cells of chicken proventriculus (glandular stomach) differentiate into two types; luminal and glandular epithelial cells. The molecules regulating the differentiation of proventricular epithelial cells are not well understood. As the first step in screening the molecular determinants involved in the cell differentiation process, we tried to establish an in vitro culture system for isolated proventricular epithelial cells. Various basal media, growth factors and sera were tested. The medium that supports well the proliferation of epithelial cells was composed of Ham's F12 as the basal medium with epidermal growth factor (10 μg/mL), insulin (10 μg/mL), cholera toxin (1 μg/mL) and bovine pituitary extract (100 μg/mL). Fetal calf serum stimulated cell proliferation 1.7-fold, while horse serum was rather toxic. Proventricular epithelial cells proliferated for 3 days, but began to die out within 1 week of culture. Cultured epithelial cells never expressed embryonic chicken pepsinogen (ECPg), a marker gene of glandular epithelial cells, or maintained ECPg expression. The capacity for ECPg expression in cultured epithelial cells was analyzed by recombination with the proventricular mesenchyme and ECPg was detected in epithelial cells cultured up to 3 days. We concluded therefore, that epithelial cells keep the capacity for ECPg expression for 3 days of cultivation and proventricular mesenchymal cells are required for the actual expression of the ECPg gene.     

Effect of serum and 12-O-tetradecanoyl-phorbol-13-acetate on FSH-stimulated conversion of 4-androstene-3,17-dione to oestrogens in cell and organ cultures of suckling mouse ovaries

The effect of serum and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the FSH-stimulated oestrogen production was studied in both cell and organ cultures. Ovaries were removed from (WB X C57BL/6)F1 mice at 9-days of age, and the conversion of 4-androstene-3,17 -dione to oestrogens was stimulated by the addition of FSH in vitro. Either 10% serum (fetal calf, mouse, rat and horse) or 0.1 microM TPA markedly inhibited the FSH-stimulated oestrogen production by dispersed and cultured ovarian cells. In contrast, neither serum nor TPA influenced the oestrogen production in the organ culture. This suggests that the presence of tissue architecture may prevent the inhibitory effect of serum and TPA on the FSH-stimulated oestrogen production.     

Mitogenic action of substance P on the epithelial cells of the tongue

Substance P (10 and 100 ng/ml) stimulates the proliferation of basal cells from rat's tongue epithelium in primary cultures with 2.5% fetal calf serum. In serum-free medium substance P have no effect on the epithelial cell growth. This neuropeptide secreted by afferent nerve fibers of the tongue epithelium is suggested to have a neurotrophic influence on epithelial cells controlling their proliferation.     

Nutrition and fetal brain maturation : I. Responses in vitro and in vivo

The glycolytic enzyme enolase increases during the perinatal period of brain development and was utilized as a marker for examining the effect of culture environment on differentiation of cells from 20-day fetal rat brain. Enolase activity in cell cultures increased from 0.91 +/- 0.03 (Day 0) to 2.11 +/- 0.10 mumol/min/mg protein (Day 6). Comparable levels were not reached in vivo until neonatal pups were 15 days old. The in vitro increase was inhibited by both cycloheximide and actinomycin D. Enolase activity in the cells responded to alterations in both incubation media and homologous serum. After 6 days in culture, cells incubated in rat serum (10%) added to MEM or RPMI produced twice as much enolase activity as cells incubated similarly in Ham's medium, i.e., 1.96 +/- 0.09 and 1.85 +/- 0.21 vs 1.02 +/- 0.09, P less than 0.001. Results of a comparable magnitude were obtained when fetal calf serum replaced adult rat serum, but enolase production was somewhat lower when newborn calf serum replaced adult rat or fetal calf serum. When cells were incubated for 6 days with graded concentrations of adult rat serum (2.5-15%), enolase activity increased progressively. The pattern of enolase response suggests that the fetal rat brain cell model described herein will provide a sensitive probe with which to gain insight into nutrition and fetal brain development.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

Cell culture of mammalian thymic epithelial cells: growth, structural, and antigenic properties

The thymus plays an important role in the maturation and differentiation of T lymphocytes. Many of its functions have been attributed to its epithelial component. Past in vitro studies of putative thymic epithelial cells have been hampered by the inability to produce well-characterized cultures of these cells. Using lethally irradiated 3T3 cells as a feeder layer, we have succeeded in growing virtually pure cultures of thymic epithelial (TE) cells from rabbits, mice, and humans. Antikeratin staining provides an unambiguous criterion for positive identification of the epithelial cells. These cells were found to lack Ia-like or theta-like antigens. The ability to culture large quantities of mammalian TE cells should allow for their detailed functional characterization.     

Enhancement of osteoblast proliferative capacity by growth factor-like molecules in bear serum

The use of animal serum in cell culture is vital for providing the nutrient factors required to promote proliferation and function. Fetal calf serum has become the preferred choice because of its abundance, reasonable cost, and ability to sustain human cells in vitro. Although a wide variety of serum sources have been tested and used, little is known about the ability of serum obtained from the American black bear (Ursus americanus) to support human cell growth in culture. The American black bear, an animal comparable in size to humans, is unique in that it hibernates for mo at a time but does not experience extensive bone loss normally associated with extended immobility. The aim of this study was to analyze the effect of bear serum on human osteoblast cultures. We discovered that three of the eight bear serum samples induced significantly higher proliferation rates in osteoblasts than did fetal calf serum over a 24-h period. Osteoblasts incubated in bear serum displayed higher messenger ribonucleic acid levels for phenotype markers osteocalcin and type I collagen than did those incubated in fetal calf serum. The mitogenic activity of the bear serum was reduced when heated at 56 degrees C for 30 min before use in culture. The molecular weight of the mitogenic factors was found to be primarily greater than 50 kDa. The present work demonstrates the capability of serum from American black bears to support human osteoblast proliferation in vitro.     

Heavy-metal toxicity in an insect cell line. Effects of cadmium chloride, mercuric chloride and methylmercuric chloride on cell viability and proliferation in Aedes albopictus cells

We evaluated the toxicity of CdCl2, HgCl2, and MeHgCl on the C6/36 cell line of Aedes albopictus. This cell line proved to be a suitable tool for studying heavy-metal toxicity in insect cells. Since data on heavy-metal toxicity in invertebrate cell cultures are almost nonexistent, our results are discussed in relation to in vivo invertebrate and in vitro vertebrate studies. Viability and proliferation were assessed by dye exclusion and DNA quantification, respectively. Viability tests were carried out with and without 5% fetal calf serum in the medium. The three metal species decreased viability to different extents (MeHgCl>HgCl2>CdCl2), and fetal calf serum had a protective effect. In serum-deprived cultures, LD50 values were 140.20, 2.51, and 2.08 µmol/L for CdCl2, HgCl2, and MeHgCl, respectively. For cultures with fetal calf serum, LD50 values were 149.71, 12.01, and 5.47 µmol/L, respectively. The viability curve for CdCl2 under serum-free conditions suggests the induction of a cell defense system. The three metal species also inhibited cell proliferation (MeHgCl> CdCl2> HgCl2). The IC50 values were 1.75, 18.36, and 0.96 µmol/L for CdCl2, HgCl2, and MeHgCl, respectively. In summary, low MeHgCl concentrations caused both cell death and inhibition of cell proliferation; HgCl2 primarily disrupted the plasma membrane, whereas CdCl2 primarily inhibited cell proliferation.     

Growth and characterisation of primary bovine colon epithelial cells in vitro

Epithelial crypts from the bovine colon were obtained by using a combined mechanical and enzymatic isolation method, followed by differential D-sorbitol gradient centrifugation. By using this isolation technique, a pure fraction of epithelial crypts with minimal mesenchymal contamination was obtained. The crypts were seeded in collagen-coated plastic flasks. The attached epithelial cells proliferated and formed a confluent monolayer after 6 days in culture. Under low-serum culture conditions (1% fetal calf serum), the cells had a population doubling time of 21-22 hours. During the culture period, the colonocytes were characterised morphologically and enzymatically. The morphology of the cultured cells was confirmed by scanning electron microscopy and transmission electron microscopy. The presence of microvilli, tight junctions and desmosomes demonstrated the ability of the cultured cells to restore an epithelial-like cell monolayer. The epithelial origin of the cells was demonstrated by labelling the cells with antibodies against epithelial-specific cytokeratins 7 and 13. The functional integrity of the cells was evaluated by measuring various marker enzymes (gamma-glutamyltranspeptidase, acid phosphatase, alkaline phosphatase, NADH-dehydrogenase) and membrane-associated Na+-K+-ATPase activity. Membrane integrity was determined by measuring the leakage of lactate dehydrogenase into the culture medium. This new culture system for bovine colon epithelial cells could be used as an in vitro model of the colon epithelium in physiological and toxicological studies.     

Plating and maintenance of epithelial tumor cells in primary culture: Interacting roles of serum and insulin

We have attempted to define the best conditions for primary culture of epithelial cells from mouse mammary tumors. Both fetal calf serum and insulin increase the rate of cell attachment to the culture substratum in dose-dependent ways. Under suboptimal conditions, epithelial cells begin to detach again from culture dishes after 2 days; this allows us to calculate the maintenance of cultures by comparing the amount of DNA in the monolayer at an early and a late time (‘maintenance ratio’). The cells are better maintained when more serum is used, up to about 4%. At low serum doses, maintenance is also responsive to the amount of insulin in the medium; insulin has little effect when more than 3% serum is present. However, the maintenance of the cells is responsive to the serum dose, regardless of the insulin dose. Furthermore, the optimum insulin concentration decreases as more serum is added to the medium. We conclude: that epithelial cells of mammary tumors require insulin to function properly in culture; that as little as 4% serum supplies an adequate dose of insulin-like activity for cell maintenance; and that fetal calf serum also supplies additional compounds which the cells need to survive.     

Primary culture and maintenance of steroid-secreting human placental monolayers

Summary A simple method is described for primary culture and for maintenance of hormone-producing cells from normal human placenta. A consistent yield of cells was obtained and an average survival of 3 to 4 months in culture using 1 mm³ explants from the most vascular area of the placentas. These explants were placed in a variety of culture media in 30 ml flasks and incubated at 37°C in an atmosphere of 5% CO₂ and 95% air. The best yields in terms of cell growth were observed with Eagle’s MEM (minimum essential medium) with supplements of horse serum and fetal calf serum or human cord serum. (Ham’s F-10 with supplement of horse serum and fetal calf serum supports growth for the longest period and media containing human cord serum had the best yield of steroids.) Presented in part at the 25th meeting of the Tissue Culture Association, June 5, 1974, Miami Beach, Florida. Supported by a contract from the Agency for International Development (2491/csd) and a grant from the National Institutes of Health (CA-12455).     

The lack of an inhibitory effect of hyaluronate on chondrogenesis in chick limb-bud mesoderm cells grown in culture

The effect of hyaluronate on chondrogenesis in cultures of chick limb-bud mesoderm cells, derived from stage 20–21, 23–24 and 26 embryos grown at different cell densities and in 3 different culture media, was studied. The results show that hyaluronate at a concentration of 500 μg/ml, does not consistently produce an inhibition of chondrogenesis in cultures of stage 20–21, 23–24 or 26 limb-bud mesoderm cells in contrast to what has been reported by Toole et al. (1972). It was demonstrated that under optimal conditions, stage 26 cells grown in the absence of hyaluronate do not form as many cartilage colonies in culture as do cells from stage 20–21 or 23–24 embryos. It was determined that culture medium composed of Eagle's MEM supplemented with 7% horse serum, 3% fetal calf serum and 5% 10-day chick embryo extract supported chondrogenesis significantly better than Ham's F-12 supplemented with 10% fetal calf serum. Our results suggest that the inhibition of chondrogenesis by hyaluronate reported earlier is most likely due to the sub-optimal conditions of growth medium, cell density and embryonic stage than to the hyaluronate treatment.