Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix

Abstract

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.

Electrophysiological and ultrastructural events evoked by methacholine and intracellular photolysis of caged compounds in cultured ovine trachea submucosal gland cells

“…In particular Ro 31-6930 (2-(6-cyano-2,2-dimethyl-2H-1-benzopyran-4-yl)-pyridine 1-oxide), levcromakalim (BRL38227) and pinacidil attenuate secretagogue-induced lysozyme secretion from serous cells in an in vitro air-filled ferret trachea preparation (Griffin, 1995). The development of cell culture techniques has enabled access to submucosal gland cells with serous cell properties (Benali, Dupuit, Jacquot, Fuchey, Hinnrasky, Ploton & Puchelle, 1989;Sommerhoff & Finkbeiner, 1990;Tournier, Merten, Meckler, Hinnrasky, Fuchey & Puchelle, 1990). Cultured submucosal gland cells express voltage-activated K+ channels and secrete lysozyme in response to methacholine, phenylephrine and substance P. Such lysozyme release induced by secretagogues is indicative of serous cell function.…”

Production of lipocortin‐like proteins by cultured human tracheal submucosal gland cells

“…Procedures for indirect immunofluorescence of cultured human tracheal submucosal gland cells were as previously described [28]. We applied the biotin-streptavidin fluorescein technique using rabbit polyclonal antiserum to lipocortin I (dilution 1/25), fluorescein streptavidin and biotinylated goat anti-rabbit IgG (dilution 1/25, Amersham).…”

In vitro effects of some differentiation inductors in metaplastic epithelium of the human nasal cavity

“…Several studies, both in animals (Steele et al 1978;Benali et al 1989;Sanderson and Dirksen 1989) and in humans (Zahm et al 1990;Chevillard et al 1991), demonstrate that explants and isolated cell cultures are a suitable and valid model system for studying the behaviour of respiratory epithelia. The present study aims at evaluating whether it is possible to restore ciliogenesis in vitro in metaplastic epithelium of the nasal cavity of patients affected by chronic nasopharyngitis.…”