Development of an oxygenation culture method for activating the liver-specific functions of HepG2 cells utilizing a collagen vitrigel membrane chamber.

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.