Differentiated structure and function of cultures from human tracheal epithelium

Abstract

Here we describe the conditions which allow cultured human tracheal epithelial cells to retain the ion transport properties and ultrastructure of the original tissue. The order of potency of growth supports and media additives in elevating baseline short-circuit current (Isc) and responses to mediators were vitrogen gel (VIT) greater than extracellular matrix from bovine corneal endothelial cells (ECM) greater than human placental collagen (HPC), and 2% Ultroser G serum substitute (USG) greater than 5% fetal calf serum (FCS) greater than defined growth factors (GF). For all combinations of medium and growth supports, an air interface (AIR) gave better electrical properties than immersion feeding (IMM). As opposed to our earlier conditions (HPC/FCS/IMM), the best new combination (VIT/USG/AIR) produced higher baseline Isc (58.0 +/- 10.6 vs. 5.1 +/- 1.0 microA/cm2) and increased Isc responses to isoproterenol (6.1 +/- 1.5 vs. 0.8 +/- 0.3 microA/cm2) and bradykinin (9.6 +/- 2.0 vs. 1.0 +/- 0.2 microA/cm2), while retaining high transepithelial resistance (227 +/- 5 omega.cm2). VIT/USG/AIR led to the appearance of cilia, an increase in the depth of the cell sheets (50 vs. 10 microns), longer and more frequent apical microvilli, and increased interdigitations of the basolateral membrane. Protein and DNA content were also significantly increased. Secretory granules were present which stained with antibody to goblet cells, but not to serous or mucous gland cells. CF cells grown in VIT/USG/AIR showed high baseline Isc (69 +/- 18 microA/cm2) and a proportionately larger inhibition of Isc by amiloride (70 +/- 10 vs. 34 +/- 3%). Isc did not respond to isoproterenol, and the response to bradykinin was 22% normal.

Effect of Corticosteroids and Neuropeptides on the Expression of Defensins in Bovine Tracheal Epithelial Cells

“…To obtain a pseudostratified epithelium cultured at an air-liquid interface, methods previously described (35,53) were followed with minimal modification. Briefly, freshly isolated tracheal epithelial cells were seeded onto collagen type I-coated semipermeable membranes (0.6 cm 2 , Millicell-PCF; Millipore, Bedford, MA) at a density of 5 ϫ 10 5 cells/ml in culture medium.…”

Lectin binding and endocytosis at the apical surface of human airway epithelia

“…The cells for culture were isolated from trachea and bronchi by enzyme digestion as previously described. [20][21][22] Freshly isolated cells were seeded at a density of 5 × 10 5 cells/cm 2 on to collagen-coated, 0.6-cm 2 area Millicell polycarbonate filters (Millipore, Bedford, MA, USA). The cells were maintained at 37°C in a humidified atmosphere of 5% CO 2 and air.…”

“…Twenty-four hours after plating, the mucosal medium was removed and the cells were allowed to grow at the air-liquid interface. [20][21][22] The culture medium consisted of a 1:1 mix of DMEM:Ham's F12, 5% Ultraser G (Biosepra, Cergy-Saint-Christophe, France), 100 U/ml penicillin, 100 g/ml streptomycin, 1% nonessential amino acids, and 0.12 U/ml insulin. Airway epithelia were allowed to reach confluence and develop a transepithelial electrical resistance, indicating the development of tight-junctions and an intact barrier.…”

Rac1 and Cdc42 Differentially Modulate Cigarette Smoke–Induced Airway Cell Migration through p120-Catenin–Dependent and –Independent Pathways

“…Primary cultures were established using previously published methods. 27 Briefly, human tracheal tissue strips were washed in PBS. The bronchial epithelium was separated from underlying stroma using enzymatic digestion followed by vigorous agitation to dislodge the epithelial sheets.…”

“…The cells seeded on Transwell membrane were grown in defined air-liquid interface medium for 2 to 3 weeks to produce differentiated, polarized HBE cultures resembling natural airway epithelium as described elsewhere. 27 The cells seeded on cell culture plates were grown in bronchial epithelial cell growth medium (BEGM BulletKit; Lonza, Basel, Switzerland) according to the manufacturer's instructions.…”