Serum-free culture of fractionated bovine bronchial epithelial cells

Abstract

Procedures for the serum-free culture of a density fractionated population of bovine bronchial epithelial cells have been established. Epithelial cells dispersed by protease digestion were fractionated by density equilibrium centrifugation, followed by plating of the small basal-like population on type I collagen-coated culture dishes. Two or three passages of 1:4 split enriched for a population of actively dividing cells, which could be stored in liquid nitrogen for subsequent use. Clonal growth assays revealed optimum proliferation using a 1:1 mixture of medium RPMI 1640 and LHC-9, a medium employed for human bronchial epithelial cells. Cellular growth rate, which was 0.6 to 1.3 doublings per day depending on the cell preparation, was conveniently decreased by supplementing LHC-9 medium with less than 50% RPMI. In contrast to airway epithelial cell cultures from other species, serum stimulated the growth of bovine bronchial epithelial cells in this system. Transforming growth factor beta 1, however, inhibited growth and induced differentiation into a squamous phenotype. Also in contrast with other systems, the bovine cells were resistant to growth inhibition by 100 nM tetradecanoyl phorbol acetate or 1 microM calcium ionophore A23187. Combination of phorbol ester with ionophore decreased mitotic activity, although induction of squamous morphology was not observed. Therefore, growth inhibition and squamous differentiation were not tightly coupled in this system. Finally, biologically synthesized matrix deposited by these cells stimulated growth rate. This culture system will therefore be useful in assessing the activities of both soluble and matrix-associated factors in the absence of serum.

RACK1, a PKC Targeting Protein, Is Exclusively Localized to Basal Airway Epithelial Cells

“…LHC9 media contains LHC basal media (Invitrogen; Carlsbad, CA), 0.5 mM phosphoethanolamine-ethanolamine (Sigma-Aldrich), 0.11 mM calcium (Fisher; Springfield, NJ), 50 U/ml penicillin and streptomycin (Gibco), 2 mg/ml fungizone (Gibco), trace elements, 5 mg/ml bovine insulin (Sigma), 5 ng/ml epidermal growth factor (Sigma), 10 mg/ml bovine transferrin (Sigma), 10 nM 3,3 ¶,5-triiodothyronine (Invitrogen), bovine pituitary extract (50 mg protein/ml; Pel Freeze, Rogers, AR), 0.2 mM hydrocortisone (Invitrogen), 0.5 mg/ml epinephrine (Sigma), and 0.1 mg/ml retinoic acid (Sigma) (Beckmann et al 1992). RPMI 1640 media was purchased from Gibco.…”

Endothelin-1 expression in airway epithelial cells

“…We agree that the action of dexamethasone may be influenced by exposure to FCS, since the serum consists of a variety of cytokines which can alter the responsiveness of epithelial cells [3]. Another possibility is again the condition of the cells; the decreased responsiveness to the exogenous stimuli might cause an apparent lack of the effect of dexamethasone on ET-1 release.…”

“…Although not explicit in the Materials and methods section, it appears that both primary and BEAS-2B cells analysed by TAKIZAWA et al [1] were exposed to serum-containing medium, otherwise ET-1 would have been barely detectable. This point may be clinically relevant in bronchial asthma where the epithelial layer, normally exposed to low amount of serum-derived proteins, is repeatedly flooded with massive extravasation from the mucosal blood vessels [3]. This mechanism might, indeed explain the increased ET-1 levels in bronchoalveolar lavage fluid from asthmatic patients, as measured by several authors, better than induction by pharmacological doses of pro-inflammatory cytokines.…”

Effects of dynamic compression on lentiviral transduction in an in vitro airway wall model

“…The utility of previously developed in vitro models for studying transepithelial transport-related phenomena is limited by the level of bronchial epithelial cell differentiation and ciliary function attainable (10 -12, 50, 51, 55). Culture conditions that were previously developed to promote epithelial differentiation towards a pseudostratified state include coculture with fibroblasts within a 3D matrix (1,11,50,51,63,64,75), the addition of specific factors to a serum-free media (15,27,55), and culture in ALI after epithelial cells have reached confluence (3,15,18,49,65). We further optimized the medium composition for ciliogenesis and cultured for 21 days at ALI.…”