Introduction of a Model for Study of Polarized Cholesterol and Phospholipid Efflux in Gallbladder Epithelial Cell

Lee, Jin

Abstract

BACKGROUND/AIMS: The cholesterol absorption by gallbladder epithelial cell (GBEC) is well known. However, the metabolism of the absorbed cholesterol in GBEC is not established yet. Thus, in this study, I introduced an effective system to investigate polarized cholesterol and phospholipid efflux in GBEC.
METHODS
Dog GBEC-CL1 was cultured on Transwell system. After apical loading of cells with mixture of model bile (MB) and 14C-cholesterol or 3H-choline, cholesterol and phospholipid efflux were measured in apical and basolateral media. Cellular proliferation and MB toxicity were evaluated by the method using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide. Paracellular permeability was measured by 125I-apo A-I and 14C-cholesterol leakage tests. Additionally, immunofluorescence staining and immunoblotting were performed to demonstrate specific transporters associated with cholesterol and phospholipid efflux.
RESULTS
Cellular proliferation without collagen matrix on Transwell showed normal confluent monolayer growth, not allowing leakage between apical and basolateral compartments. MB did not show any toxicity to cells. Only GBEC loaded with MB showed basolateral predominant cholesterol and phospholipid efflux following treatment of apo A-I and cAMP. Immunofluorescence and immunoblotting studies demonstrated expression of ATP-binding cassette transporter A1 (ABCA1) or scavenger receptor class type I.
CONCLUSIONS
Dog GBEC-CL1 culture on Transwell using MB is an efficient system in studying polarized cholesterol and phospholipid efflux.