Characterization of epithelial cell cultures derived from human tracheal glands

Summary Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 μg/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 μg/ml), and bovine pituitary extract (25 μg/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide β-d-galactose-(l→ 3)N-acetyld-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl⁻ channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established. This research was supported by USPHS grants HL 41979 and HL 33142 from the National Heart, Lung and Blood Institutes.     

Differentiated cultures of primary hamster tracheal airway epithelial cells

Summary Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating tracheal epithelial cultures from Syrian golden hamsters and characterized the cultures for cell composition and function. Soon after initial plating, the epithelial cells reached a high transepithelial resistance and formed tight junctions. The cells differentiated into a heterogeneous, multicellular culture containing ciliated, secretory, and basal cells after culture at an air-liquid interface (ALI). The, secretory cell populations initially consisted of MUC5AC-positive goblet cells and MUC5AC/CCSP double-positive cells, but the makeup changed to predominantly Clara cell secretory protein (CCSP)-positive Clara cells after 14 d. The ciliated cell populations differentiated rapidly after ALI as judged by the appearance of β tubulin IV-positive cells. The cultures produced mucus, CCSP, and trypsin-like proteases and were capable of wound repair as judged by increased expression of matrilysin. Our method provides an efficient, high-yield protocol for producing differentiated hamster tracheal epithelial cells that can be used for a variety of in vitro studies including tracheal cell differentiation, airway disease mechanisms, and pathogen-host interactions.     

Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

Summary The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated [methyl-³H]-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 μm, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating [methyl-³H]thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration. The present study was financially supported by the Scientific Advisory Committee on Smoking and Health (Dutch Cigarette Industry Foundation) and the Ministry of Welfare, Health and Cutural Affairs.     

Secretion of rat tracheal epithelial cells induces mesenchymal stem cells to differentiate into epithelial cells

Bone marrow MSCs (mesenchymal stem cells) can differentiate into various tissue cells, including epithelial cells. This presents interesting possibilities for cellular therapy, but the differentiation efficiency of MSCs is very low. We have explored specific inducing factors to improve the epithelial differentiation efficiency of MSCs. Under inducing conditions, MSCs differentiated into epithelial cells and expressed several airway epithelial markers using RTE (rat tracheal epithelial) cell secretions. Rat cytokine antibody array was used to detect cytokines of the RTE secretion components, in which 32 kinds of protein were found. Seven proteins [TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), VEGF (vascular endothelial growth factor), BDNF (brain-derived neurotrophic factor), TGFβ1 (transforming growth factor β1), MMP-2 (metalloproteinases-2), OPN (osteopontin) and activin A in RTE secretions] were assayed using ELISA kits. The four growth factors (VEGF, BDNF, TGFβ1 and activin A) were involved in regulating stem cell growth and differentiation. We speculated that these four play a vital role in the differentiation of MSCs into epithelial cells by triggering appropriate signalling pathways. To induce epithelial differentiation, MSCs were cultured using VEGF, BDNF, TGFβ1 and activin A. Differentiated MSCs were characterized both morphologically and functionally by their capacity to express specific markers for epithelial cells. The data demonstrated that MSCs can differentiate into epithelial cells induced by these growth factors.     

Selective inhibition of fibroblasts by spermine in primary cultures of normal human skin epithelial cells

Summary Overgrowth with fibroblasts has been a major problem in the cultivation of normal human skin epithelium. In the present study it is shown that the addition of spermine to the culture medium in micromolar concentrations has a differential cytotoxic effect on fibroblasts allowing the cultivation of human skin epithelial cells in primary culture without fibroblastic overgrowth. Putrescine, another polyamine, is shown to be equally cytotoxic to fibroblasts and epithelial cells when added in millimolar concentrations; below this concentration range no cytotoxic effect could be demonstrated. This difference in cytotoxicity between spermine and putrescine is suggested to depend on the conversion of spermine, but not putrescine, and to highly cytotoxic products by an amine oxidase present in fetal bovine serum. This project was supported by the Novo foundation.     

Extracellular matrix-dependent differentiation of rabbit tracheal epithelial cells in primary culture

Summary The differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis. The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.     

Cellular retinoic acid-binding protein and its relationship to the biological activity of four synthetic retinoids in hamster tracheal organ culture

Summary The mechanism of action of retinoid in reversing keratinization in hamster trachea is yet unknown. The purpose of this study was to determine if cellular retinoic acid binding protein (CRABP) is present in tracheal epithelium following incubation in serum-free, vitamin A-deficient culture medium for 10 days, and if the effectiveness of a retinoid in reversing keratinization in organ culture is correlated with its ability to compete for CRABP sites. The cytosol prepared from tracheal cultures contained CRABP at a concentration of 2.61 pmoles per mg protein. Of the four retinoids with carboxyl end group selected for the study, two of the biological active retinoids competed for the CRABP sites. However, no correlation was observed between the biological activity of the inactive retinoids and their ability to associate with the CRABP sites. These results indicate that even though the action of retinoid may be mediated by retinoid binding protein, it cannot be used as a sole predicator of retinoid response in hamster trachea. This investigation was supported by Contract N01-CP-31012 and U. S. P. H. Grants CA30512 and CA32428, which were awarded by the Division of Cancer Etiology, National Cancer Institute, DHHS. Editor's Statement Tracheal organ cultures provide a useful model for the study of epithelial differentiation and carcinogenesis. Much attention has been given to the action of retinoids in this process. Mehta et al. demonstrate a lack of correlation between biological activity and specific cytosolic binding of members of this class of compounds, pointing out the need for a more complete biochemical understanding of the mechanism of action and active forms of retinoids in this and other systems in vivo and in vitro. David W. Barnes     

Critical variables controlling cell proliferation in primary cultures of rat tracheal epithelial cells

Summary The purpose of our experiments was to examine variables affecting early events in the establishment of rat tracheal epithelial (RTE) cultures as well as factors regulating long-term RTE cell growth. The experiments showed that when RTE cells were seeded into complete serum-free medium between 13 and 30% of the seeded cells attached. Of the seeded cells, only ∼2% entered into DNA synthesis and underwent repeated cell divisions to form colonies containing >20 cells. Coating the dishes with extracellular matrix components had little effect on cell attachment or colony forming efficiency (CFE). However, coating the dishes with fetal bovine serum markedly increased CFE. The media components bovine serum albumin and bovine pituitary extract were shown to be important in promoting cell attachment as well as CFE. Cholera toxin on the other hand had no effect on cell attachment but significantly increased CFE. These and other studies showed that cell attachment and cell proliferation are independently regulated. Studies on long-term culture growth indicated that the number of progeny produced per colony forming unit (CFU) is inversely proportional to the number of CFUs seeded. Inasmuch as the cultures did not become confluent under any of the culture conditions tested and media obtained from high density cultures were shown to be growth inhibitory, these findings suggest that a diffusible growth restraining factor is being produced by the cultures limiting clonal expansion. Experiments showing growth inhibitory effects of media conditioned by high cell density cultures support this interpretation. The putative factor reaches critical concentrations earlier in cultures seeded with high numbers of CFU than in cultures seeded with low numbers of CFU. Because the cultures are known to produce transforming growth factor-beta, this growth regulator probably plays a role in controlling RTE cell proliferation. However, it is likely than other events, such as depletion of growth factors from the media, also are significant in regulating the growth of the cultures.     

Differential effects of simvastatin on IL-13-induced cytokine gene expression in primary mouse tracheal epithelial cells

Background

Asthma causes significant morbidity worldwide in adults and children alike, and incurs large healthcare costs. The statin drugs, which treat hyperlipidemia and cardiovascular diseases, have pleiotropic effects beyond lowering cholesterol, including immunomodulatory, anti-inflammatory, and anti-fibrotic properties which may benefit lung health. Using an allergic mouse model of asthma, we previously demonstrated a benefit of statins in reducing peribronchiolar eosinophilic inflammation, airway hyperreactivity, goblet cell hyperplasia, and lung IL-4 and IL-13 production.

Objectives

In this study, we evaluated whether simvastatin inhibits IL-13-induced pro-inflammatory gene expression of asthma-related cytokines in well-differentiated primary mouse tracheal epithelial (MTE) cell cultures. We hypothesized that simvastatin reduces the expression of IL-13-inducible genes in MTE cells.

Methods

We harvested tracheal epithelial cells from naïve BALB/c mice, grew them under air-liquid interface (ALI) cell culture conditions, then assessed IL-13-induced gene expression in MTE cells using a quantitative real-time PCR mouse gene array kit.

Results

We found that simvastatin had differential effects on IL-13-mediated gene expression (inhibited eotaxin-1; MCP-1,-2,-3; and osteopontin (SPP1), while it induced caspase-1 and CCL20 (MIP-3α)) in MTE cells. For other asthma-relevant genes such as TNF, IL-4, IL-10, CCL12 (MCP-5), CCL5 (RANTES), and CCR3, there were no significant IL-13-inducible or statin effects on gene expression.

Conclusions

Simvastatin modulates the gene expression of selected IL-13-inducible pro-inflammatory cytokines and chemokines in primary mouse tracheal epithelial cells. The airway epithelium may be a viable target tissue for the statin drugs. Further research is needed to assess the mechanisms of how statins modulate epithelial gene expression.     

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)     

小鼠原代呼吸道上皮细胞分离培养的新方法

现有的呼吸道上皮细胞系大多来源于肿瘤组织或成系时与肿瘤细胞融合,其生物学行为与正常呼吸道上皮细胞差异较大.为更准确反映呼吸道疾病条件下该类细胞的生物学效应,本文对小鼠呼吸道上皮细胞分离的新技术和培养方法进行了探索.利用链霉蛋白酶消化法分离获得小鼠呼吸道上皮细胞,利用特殊的完全培养基和Ⅰ型胶原包被的培养皿进行原代培养.镜...     

Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: Cellular electrophysiology

Summary We examined the effect of adrenergic agents on the cellular electrical properties of primary cultures of canine tracheal epithelium. Both isoproterenol and epinephrine stimulated Cl secretion, as evidenced by an increase in transepithelial voltage and a fall in transepithelial resistance. Moreover, both agents appear to increase the conductance of apical and basolateral membranes. However, the pattern of response was different. Isoproterenol initially depolarized apical voltage _a and decreased the fractional resistance of the apical membranef _R. These changes are consistent with an initial increase in apical Cl conductance. In contrast, epinephrine acutely hyperpolarized _a and increasedf _R, changes consistent with an initial increase in basolateral K conductance. Following the acute effect of epinephrine, _a depolarized andf _R decreased to values not significantly different from those observed with isoproterenol. The acute increase in basolateral K conductance produced by epinephrine appeared to result from stimulation of adrenergic receptors because it was reproduced by addition of the agonist phenylephrine, and blocked by the antagonist phentolamine. The ability of prazosin but not yohimbine to block the acute epinephrine-induced increase in K permeability indicates the presence of ₁ adrenergic receptors. The acute adrenergic-induced increase in basolateral K conductance may be mediated by an increase in cell Ca because the response was mimicked by addition of the Ca ionophore A23187. In contrast, the response to isoproterenol was similar to that observed with addition of 8-bromo-cAMP and theophylline. These results indicate that both and adrenergic agents mediate the ion transport processes in canine tracheal epithelium. adrenergic agents have their primary effect on the apical Cl conductance, probably via an increase in cAMP. adrenergic agents exert their primary effect on the basolateral K conductance, possibly via an increase in cell Ca.     

Explant organ culture of hamster alimentary tract epithelium

Adult Syrian Golden hamster alimentary tract maintained as explants in organ culture was studied using the model system for hamster pancreas described by Resau et al. (1983a). Explants of esophagus, stomach, duodenum and colon were maintained in organ culture on Gelfoam® sponge rafts in a high-oxygen atmosphere with serum-supplemented CMRL-1066 medium. All of the tissues were observed to show evidence of sublethal acute cell injury during the first several days of culture. Subsequently, the epithelial tissues recovered from this injury, repopulated the denuded areas of the explants and replicated within the sponge matrix. Explants were maintained in a differentiated state for 30+ days and sampled for morphology to examine the process of cell injury, repair, differentiation and replication which occurs in mucosal epithelia. The percentage of basement membrane covered by epithelia in the explants from various tissues was compared to the level of LDH in the media to reveal the relationship between viability determined by biochemical and by morphological methods. Restitution of the mucosal surface occurred in all of the explants. We conclude that adequate populations of replicating cells are maintained within the epithelium of the hamster alimentary tract tissues in vitro so that restitution can occur through migration and subsequent differentiation of the epithelial cells within the mucosa of the explants.Abbreviations 4F-1G 4% formaldehyde 1% glutaraldehyde fixative - LDH lactate dehydrogenase - OsO₄ osmium tetroxide - PAS/PLH periodic acid, periodic acid Shiff lead hematoxylin stain     

Neoplastic progression of rat tracheal epithelial cells is associated with a reduction in the number of growth factors required for clonal proliferation in culture

Summary Factors regulating the proliferation of normal, preneoplastic, and neoplastic rat tracheal epithelial (RTE) cells were investigated to identify changes taking place during the progression of RTE cells to neoplasia. Normal RTE cells exhibit clonal proliferation in a serum-free medium containing pituitary extract, serum albumin, cholera toxin, epidermal growth factor, hydrocortisone, and insulin. All combinations of these six factors were examined for their abilities to support clonal proliferation of normal, preneoplastic, and neoplastic RTE cells. In general, preneoplastic RTE cells required fewer factors for proliferation than normal RTE cells, and neoplastic cells required fewer factors than preneoplastic cells. A common pattern of reductions has been identified in the growth factors required for the clonal proliferation of preneoplastic vs. normal RTE cells and for neoplastic vs. preneoplastic and normal RTE cells. Normal RTE cells exhibit clonal proliferation in a serum-free medium supplemented with a minimum of six factors: bovine serum albumin, bovine pituitary extract, cholera toxin, epidermal growth factor, hydrocortisone, and insulin. Preneoplastic RTE cells exhibit clonal proliferation in a serum-free medium supplemented with four factors: bovine serum albumin, bovine pituitary extract, hydrocortisone, and insulin. Finally, neoplastic RTE cells exhibit clonal proliferation in a serum-free medium supplemented with two factors: bovine serum albumin and bovine pituitary extract. These results suggest that the progression of RTE cells to neoplasia is associated with a series of changes in regulatory pathways that control cell proliferation.     

Isolation and primary cultures of human intrahepatic bile ductular epithelium

Summary A technique for the isolation of human intrahepatic bile ductular epithelium, and the establishment of primary cultures using a serum- and growth-factor-supplemented medium combined with a connective tissue substrata is described. Initial cell isolates and monolayer cultures display phenotypic characteristics of biliary epithelial cells (low molecular weight prekeratin positive; albumin, alphafetoprotein, and Factor VIII-related antigen negative). Ultrastructural features of the cultured cells show cell polarization with surface microvilli, numerous interepithelial junctional complexes and cytoplasmic intermediate prekeratin filaments.     

Regulation of differentiation and keratin protein expression by vitamin A in primary cultures of hamster tracheal epithelial cells.

Hamster tracheal epithelial (HTE) cells maintained in primary culture show the induction of specific keratin species under vitamin A-deficient conditions. A comparison was made between the morphology and the expression of keratins in HTE cells in vivo and in primary culture with and without vitamin A. HTE cells cultured in serum-free, vitamin A-supplemented medium formed a simple cuboidal, ciliated monolayer and produced four simple epithelial keratins (7, 8, 18, and 19). In contrast, vitamin A-deficient HTE cells, which were squamous-like and stratified in culture, produced a more complex keratin pattern, with the induction of four additional keratin species (5, 6, 14, and 17). A keratin pair whose expression serves as a marker of stratified epithelia was induced, as well as a single keratin species unique to lesions of squamous metaplasia in vitamin A-deficient hamster tracheal organ cultures. Thus it appears that HTe cells retain the ability to respond to a deficiency in vitamin A through squamous differentiation and increased keratin production when removed from the intact organ and maintained in primary culture in a chemically defined medium. This system may be useful for the study of mechanisms underlying the squamous differentiation of respiratory epithelial cells in the development of bronchogenic tumors.     

奶山羊乳腺上皮细胞的分离、培养及鉴定

应用组织块培养法高密度培养、连续传代法建立西农萨能奶山羊乳腺上皮细胞体外培养体系,通过生长曲线绘制、核型分析、免疫荧光染色 (角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白)、油红染色及β-酪蛋白基因的RT-PCR分析进行培养细胞鉴定。实验结果表明细胞生长曲线为典型的S型,染色体数目众数为60,细胞角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白表达均呈阳性,油红染色后可见细胞质内的脂滴,且细胞表达酪蛋白mRNA。说明运用本方法培养的细胞为正常的乳腺上皮细胞,并具有一定的泌乳功能。     

Defined medium for primary culture de novo of adult rat alveolar epithelial cells

Summary Isolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic bioelectric properties, and change morphologically with time in culture to resemble type I cells. Concurrently, the cells express type I cell surface epitopes, making this a potentially useful in vitro model with which to study regulation of alveolar epithelial cell function and differentiation. To define specific soluble growth factors and matrix substances that may regulate these processes, it would be preferable to culture isolated pneumocytes de novo under completely defined, serum-free conditions. In this study, we developed a completely defined serum-free medium that is capable of supporting alveolar epithelial cells in primary culture, allowing the formation of monolayers with characteristic bioelectric and phenotypic properties. Freshly isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters. Plating efficiency, bioelectric properties, morphology, and binding of a type I cell-specific monoclonal antibody were determined as functions of time. Plating efficiency plateaus at about 14% by Day 3 in culture. Transepithelial resistance rises to high levels, peaking at 1.76±0.14 KΩ-cm² by Day 5 in culture. Short-circuit current peaks on Day 3 in culture at 2.71±0.35 μA/cm². With time, the cells gradually become flattened with protuberant nuclei and long cytoplasmic extensions, more closely resembling type I cells, and begin to express a type I cell surface epitope. These observations indicate that it is feasible to culture alveolar epithelial cell monolayers under completely defined serum-free conditions de novo. This culture system should prove useful for identifying soluble growth factors and matrix substances that modulate alveolar epithelial cell biological properties.     

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.