Isolation and culture of primary equine tracheal epithelial cells

Culture of airway epithelial cells is a useful model to investigate physiology of airway epithelia and airway disease mechanisms. In vitro models of airway epithelial cells are established for various species. However, earlier published method for isolation and culture of equine tracheal epithelial cells requires significant improvements. In this report, the development of a procedure for efficient isolation, characterization, culture, and passage of primary equine tracheal epithelial cells are described. Epithelial cells were isolated from adult equine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue and smooth muscle. The tissue was minced and dissociated enzymatically using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution for 2 h at 37 degrees C. Cells were collected by sieving and centrifugation, and contaminating fibroblasts were removed by differential adhesion. This procedure resulted in a typical yield of 1 x 10(7) cytokeratin-positive epithelial cells per gram tracheal lining tissue. Viability was 95% by trypan blue exclusion and isolates contained approximately 94% cytokeratin-positive cells of epithelial origin. Cells seeded at a density of 6.9 x 10(4) cells/cm(2) in serum-free airway epithelial cell growth medium formed monolayers near confluency within a week. Confluent cells were dissociated using dispase II and first passages (P1) and second passages (P2) were successfully established in serum-free medium. Collagen coating of tissue culture flask was not required for cell adhesion, and cultures could be maintained at the level of P2 over 30 d. In the present study, we could establish a high-yield protocol for isolation and culture of equine tracheal epithelial cells that can serve for in vitro/ex vivo studies on the (patho-)physiology of equine airway disease as well as pharmacological and toxicological targets relevant to airway diseases.     

Growth and differentiation of primary tracheal epithelial cells in culture: regulation by extracellular calcium

Growth and differentiation of primary monkey tracheal epithelial (MTE) cells maintained on collagen gel substrata were studied in a defined serum-free culture medium containing 0.03 to 3.0 mM extracellular calcium. Cell attachment efficiency (40-60%) was not altered by different calcium levels. Growth of primary MTE cells on collagen gel substrata, which was vitamin A dependent, was enhanced 50% in the medium supplemented with high calcium (greater than 0.3 mM). High calcium medium also increased cell-cell interactions, formation of desmosomes, and multi-cell layering. The relative content of mucous cells, which were identified by a mucin-specific monoclonal antibody and the presence of mucus-secreting granules at the ultrastructural level, was greater in the high-calcium medium. Furthermore, the secretion of mucin into the medium, determined either by an ELISA or by the incorporation of 3H-glucosamine into mucous glycoprotein fractions, was also increased more than 5-fold in media containing high calcium content (greater than 0.6 mM). In contrast, MTE cells cultured in low calcium medium (less than 0.15 mM) were squamous-like with prominent tonofilaments, and their secretory product was mainly hyaluronate. These results demonstrate that media containing a high calcium content promote conducting airway epithelium to express mucous cell differentiation, while media with low calcium content promote squamous cell differentiation.     

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.     

Growth requirements and characterization of rat cervical epithelial cells in culture

The extended culture of rat cervical epithelial cells can be achieved in the absence of a fibroblast feeder layer by utilizing collagen gels and a complex growth medium. The medium contains a 1:1 mixture of RPMI-1640 and Ham's F12 supplemented with 7.5% porcine serum and epidermal growth factor, cholera toxin, transferrin, insulin, and hydrocortisone. Under these culture conditions the cells show rapid log-phase growth and high saturation densities while retaining the ultrastructural characteristics of immature squamous metaplastic cells of the rat uterine cervix even after extended passage. In a manner similar to epithelial cells from a variety of sources, rat cervical epithelial cells form hemicysts at confluence in vitro when cultured on impermeable substrates. The development of these methods for culturing cervical epithelial cells provides an experimental system for the study of factors important in regulating the growth and differentiation of metaplastic squamous epithelial cells.     

Growth in primary culture of mouse submandibular epithelial cells embedded in collagen gels

Summary Mouse submandibular glands were dissociated and the epithelial cells embedded in a collagen gel matrix. A characteristic and reproducible pattern of growth was seen resulting in three-dimensional outgrowths with ductlike structures projecting into the matrix. A sustained cell growth leading to a 5 to 10-fold increase in cell number was observed in less than 2 wk. The extent of this growth was found to be dependent on serum concentration. Of the three sera tested, swine serum was found to promote greater growth compared to fetal bovine serum or horse serum. Swine serum dose response studies have shown that a concentration of 2 to 5% in the medium elicited only a modest increase, if any, in cell number compared to the initial value within a period of 2 wk. Various hormones and growth factors were then added to this “maintenance” medium. Insulin was found to stimulate growth consistently and reproducibly in a dose-dependent manner. Ultrastructurally, the resulting outgrowths were comprised of polarized cells joined by apical tight junctions and desmosomes. These outgrowths produced epidermal growth factor in response to dihydrotestosterone, triiodothyronine, and cortisol. The present system provides a method for sustaining growth and functional differentiation in primary culture of mouse submandibular gland epithelial cells. This investigation was supported by PHS Grants CA05388 and CA09041, awarded by the National Cancer Institute, Department of Health and Human Services.     

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture

Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm²) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to platic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium. Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared in Clin. Res. 30: 93A; 1982.     

Biochemical characterization of mucous glycoproteins synthesized and secreted by hamster tracheal epithelial cells in primary culture

Hamster tracheal epithelial cells growing on type I collagen gel synthesize and secrete high molecular weight glycoconjugates which elute in the void volume upon Sepharose CL-4B column chromatography. The presence of any proteoglycans in this void volume material was ruled out based on both enzymatic analysis and behavior on DEAE-ion exchange chromatography. Based on the incorporation of radioactive precursors, followed by strong acid hydrolysis or neuraminidase digestion, the material was shown to contain sialic acid, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sulfate. Complete susceptibility to papain digestion and reductive beta-elimination suggests that the material consists of O-linked glycoproteins. The identification of N-acetylgalactosaminitol in the beta-eliminated oligosaccharides confirms this notion. The molecular weight of the oligosaccharides following beta-elimination ranges from 4,000 to 15,000. We conclude that the high molecular weight glyconjugates produced by hamster tracheal epithelial cells in primary culture are mucous glycoproteins based on size, sensitivity to alkaline borohydride treatment, and monosaccharide composition. Further characterization of these mucous glycoproteins showed both size and charge microheterogeneity among molecules. Detailed structural analysis of oligosaccharides of these mucous glycoproteins is currently under way.     

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.     

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

Hamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham's F12 medium with 10(-8) M retinoic acid (RA+), or without retinoic acid (RA-). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA- cultures, but the large cells became flat in RA- medium on day 2. On days 3-5, thymidine incorporation and total labelling indices were less in RA- than RA+ cultures, and on days 4-6, cell numbers were decreased in RA- cultures. On day 3, the large cells of the RA- colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA- colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA- colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA- colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA+ cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA- cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA- cultures on days 5 and 6, respectively. The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a "basal cell hyperplasia".(ABSTRACT TRUNCATED AT 400 WORDS)     

Establishment and comparison of air-liquid interface culture systems for primary and immortalized swine tracheal epithelial cells

Background

Air-liquid interface (Ali) systems allow the establishment of a culture environment more representative of that in vivo than other culture systems. They are useful for performing mechanistic studies of respiratory epithelial cells as drug permeation barriers and can be used to study the interactions between hosts and respiratory pathogens. However, there have been few studies concerning Ali cultures of primary swine tracheal epithelial cells (STECs) and an immortalized STEC line, and the differences between these two systems remain poorly defined.

Results

In this study, we established Ali culture systems for primary STECs and for immortalized STEC line, and we systematically compared the differentiation capacities and immunological functions of these systems for the first time. Under Ali culture conditions, immortalized STEC line and primary STECs could survive for at least forty days, formed tight junctions and differentiated into stratified cells. They both possessed complete abilities to produce mucin and inflammatory cytokines and develop cilia. However, in contrast to primary STECs, which had a heterogeneous morphology, Ali-cultured immortalized STEC line appeared to be a homogenous population. The formation of tight junctions in Ali-cultured primary STECs was superior to that in immortalized STEC line. In addition, cilia in Ali-cultured immortalized STEC line were more pronounced, but their duration of expression was shorter than in primary STECs.

Conclusions

Ali-cultured primary STECs and immortalized STEC line systems possessing complete abilities to undergo ciliary differentiation and inflammatory cytokine production were established for the first time in this study, and several differences in morphology and the formation of tight junctions and cilia were observed between these two systems. These two systems will be important tools for drug screening studies, as well as for detailed analyses of the interactions between hosts and respiratory pathogens.

     

Possible requirement of collagen gel substratum for production of mucin-like glycoproteins by primary rabbit tracheal epithelial cells in culture

Summary Primary rabbit tracheal epithelial cells growing on either plastic surface or collagen gel produce high molecular weight glycoconjugates. Biochemical characterization of these materials show they are exclusively hyaluronic acid when cells are grown on plastic surface, but a mixture of hyaluronic acid and mucin-like glycoproteins when grown on collagen gel. This research suggests that the substratum plays an important role in the maintenance or differentiation or both of mucous cells in culture.     

Metabolism of all-trans-retinol and all-trans-retinoic acid in rabbit tracheal epithelial cells in culture

As reported previously squamous cell differentiation of rabbit tracheal epithelial (RTE) cells in culture is a multi-step process. This program of differentiation is inhibited by retinoic acid and retinol; retinoic acid is about 100 times more effective than retinol. To examine the metabolism of these agents in this in vitro model system, RTE cells were grown in the presence of all-trans-[3H]retinol or all-trans-[3H]retinoic acid and their metabolites analyzed by high-pressure liquid chromatography. RTE cells converted most of the retinol to retinyl esters, predominantly retinyl palmitate. A small fraction was metabolized to polar compounds, one of which coeluted with retinoic acid. After methylation this compound eluted as 13-cis-methyl retinoate and as all-trans-methyl retinoate. Conversion to 13-cis-retinol was also observed. All-trans-retinoic acid was rapidly taken up by RTE cells and converted to more polar (peak 1) and less polar (peak 3) metabolites. A proportion of all-trans-[3H]retinoic acid was metabolized to 13-cis-[3H]retinoic acid. These metabolic reactions appeared to be constitutive and were not induced by pretreatment with retinoic acid. The peak 1 metabolites were rapidly secreted into the medium whereas the peak 3 metabolites were retained by the cells and were not detected in the medium. Alkaline hydrolysis of the metabolites in peak 3 yielded retinoic acid, indicating the formation of retinoyl derivatives. Our results establish that RTE cells can convert all-trans-retinol to 13-cis-retinol and retinoic acid. RTE can metabolize all-trans-retinoic acid to 13-cis-retinoic acid and to an unidentified ester of retinoic acid.     

Induction in the expression of an unusual proline-rich protein by pig tracheal surface epithelial cells maintained in primary culture.

Primary cell culture is a valuable tool for studying the regulation of gene expression since many differentiated traits are conserved. Cells cultured from the epithelial lining of pig trachea were selected as a model system for mucin synthesis. RNAs were isolated from pig trachea epithelial linings and from pig trachea surface epithelial cells cultured in serum-free media. Cell free translations showed an unusually high incorporation of [3H]proline into a relatively small protein (about 20 kDa), but only with RNA from the cells in culture. RNA prepared from pig trachea cells immediately before placing the cells in culture (day 0) did not contain mRNA encoding this unusual proline-rich protein. However, the expression of this protein was dramatically induced within 2 days of maintaining the cells in culture.     

Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells

Summary Studies on the regulation of differentiation in airway epithelial cells have been hampered by the lack of cell culture systems that differentiate in vitro. One such system that does exhibit differentiation is hamster tracheal epithelial cells (HTE). A major problem with this system, however, is that at the time cells differentiate, they lyze the collagen gel upon which they grow, resulting in termination of the culture. Here we report that by growing the HTE cells at 32° instead of 37°C we can totally prevent lysis of the collagen gel. Cells grown at this lower temperature maintain their differentiated phenotype as evidenced by abundant mucus granules and the secretion of authentic mucus glycoproteins into the culture media. We have also developed a method for subculturing the primary cells which allows growth and differentiation in secondary culture. The HTE cells were capable of being passaged at least three times and did not become transformed as judged by their inability to grow in soft agar and to produce tumors in syngeneic animals. This improved HTE cell culture system will allow detailed studies on the mechanisms which regulate growth, differentiation, and mucus secretion in surface airway epithelial cells. This work was supported in part by grants HL-19717 and HL-36854 from the National Institutes of Health, Bethesda, MD.