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Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix
Author(s) -
Benali Rachid,
Dupuit Florence,
Jacquot Jacky,
Fuchey Claudette,
Hinnrasky Jocelyne,
Ploton Dominique,
Puchelle Edith
Publication year - 1989
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1111/j.1768-322x.1989.tb00843.x
Subject(s) - biology , hyaluronidase , basement membrane , fetal bovine serum , microbiology and biotechnology , centrifugation , extracellular matrix , subculture (biology) , trypsinization , collagenase , cell culture , matrix (chemical analysis) , differential centrifugation , trypsin , biochemistry , cell , enzyme , chemistry , genetics , chromatography
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°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 (×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 F 12 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 epithelloid 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.