Abstract
We recently developed a collagen vitrigel membrane (CVM) chamber possessing a scaffold composed of high-density collagen fibrils. In this study, we first confirmed that the advantage of CVM chamber in comparison to the traditional culture chamber with porous polyethylene terephthalate membrane is to preserve a culture medium poured in its inside even though the under side is not a liquid phase but solid and gas phases. Subsequently, we designed three different culture systems to grow HepG2 cells in a culture medium (liquid phase) on the CVM which the under side is a culture medium, a plastic surface (solid phase) or 5 % CO2 in air (gas phase) and aimed to develop a brief culture method useful for activating the liver-specific functions and analyzing the pharmacokinetics of fluorescein diacetate. HepG2 cells cultured for 2 days on the liquid–solid interface and subsequently for 1 day on the liquid–gas interface represented excellent cell viability and morphology in comparison to the others, and remarkably improved albumin secretion and urea synthesis to almost the same level of freshly isolated human hepatocytes and CYP3A4 activity to about half the level of differentiated HepaRG cells. Also, the cells rapidly absorbed fluorescein diacetate, distributed it in cytosol, metabolized it into fluorescein, and speedily excreted fluorescein into both bile canaliculus-like networks and extracellular solution. These data suggest that hepatic structure and functions of monolayered HepG2 cells can be induced within a day after the oxygenation from beneath the CVM.
Highlights
It was previously reported that HepG2 cells, a human hepatoma cell line, inherently possess most biochemical and histological features of normal human parenchymal hepatocytes (Bouma et al 1989; Knowles et al 1980)
collagen vitrigel membrane (CVM) chambers pre-set on all conditions and the control Millicell chambers pre-set on the liquid culture medium surface preserved the culture medium for 2 h (Fig. 2a–c, e, h) and this phenomena continued more than 3 days
The focus of this study is to develop a novel culture method for activating the liver-specific functions of HepG2 cells and to investigate its utility for analyzing the ADME behavior of a model chemical, fluorescein diacetate (FD)
Summary
It was previously reported that HepG2 cells, a human hepatoma cell line, inherently possess most biochemical and histological features of normal human parenchymal hepatocytes (Bouma et al 1989; Knowles et al 1980). HepG2 cells that can sufficiently express liver-specific functions are considered as a valuable candidate of human hepatic cell source for drug development. The microenvironment of HepG2 cells in the culture system in vitro has been devised for activating their liver-specific functions. The continuous oxygenation culture system utilizing a gas-permeable PDMS (polydimethylsiloxane) was used for fabricating both thick multilayers and large multicellular spheroids with excellent viability, resulting in the improvement of albumin secretion of HepG2 cells (Anada et al 2012; Evenou et al 2010). The above culture systems are still insufficient for activating various liver-specific functions of HepG2 cells equivalent to human primary parenchymal hepatocytes in spite of the fact that they require complicate manipulations and a long period of more than 10 days
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