期刊
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
卷 281, 期 3, 页码 F557-F570出版社
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajprenal.2001.281.3.F557
关键词
model system; epithelia; anion transport; pH regulation; cystic fibrosis; congenital bilateral absence of the vas deferens
A collagenase-based dissociation technique has been developed to routinely establish monolayer cultures of freshly isolated porcine vas deferens epithelium. Cells isolated from each tissue are transferred to 25-cm(2) tissue culture flasks and grown in a standard cell culture medium. Flasks reach confluency in 3-4 days, and cells are subsequently seeded onto permeable supports. Cultured cells display a monolayer cobblestone appearance and are immunoreactive to anti-ZO-1 and anti-cytokeratin antibodies. Electron microscopy is employed to demonstrate the presence of junctional complexes and microvilli. When evaluated in modified Ussing chambers, cultured monolayers exhibit a basal lumen negative potential difference, high electrical resistance (>1,000 Omega .cm(2)), and respond to norepinephrine, vasopressin, ATP, adenosine, and histamine, with changes in short-circuit current indicative of anion secretion. Responses are significantly attenuated in Cl(-) and/or HCO(3)(-)-free solutions. Attempts to further optimize culture conditions have shown that chronic exposure to insulin increases proliferation rates. Thus the culture method described will reliably produce viable neurotransmitter-responsive cell monolayers that will allow for the characterization of vas deferens epithelial function and associated control mechanisms.
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