Three-dimensional culture of fetal mouse, rat, and porcine hepatocytes

Abstract

In this chapter, we introduce and summarize the results from our groups on typical macroporous and biodegradable poly-L-lactic acid (PLLA) scaffolds-based 3D shaking culture of fetal hepatocyte populations isolated from mice, rats, and pigs, and on some preliminary implantation of the cell-loaded scaffolds to mice and rats. In such 3D microenvironment, inoculated cells were organized into heterogenic 3D aggregates or multilayers, functional levels, and their in vitro stability was greatly enhanced when compared with those in 2D monolayer cultures. Although detoxification capacity in terms of EROD measurement did not seemed to be fully matured, other typical functions such as albumin production attained the adult level. This was enabled by the synergistic effects of 3D culture and soluble factors cocktails. Combination of nicotinamide (NA), dimethyl sulfoxide (DMSO), and oncostatin M (OSM) was very effective in fetal mice culture, but it does not support the growth and maturation of fetal rat hepatocytes, for which other cocktail composed of NA, HGF, FGF-1, FGF-4, OSM, and sodium butyrate was effective. In the case of fetal porcine hepatocytes, presumably because the obtained hepatocytes were in better matured stage than mice and rats, dependency on soluble factors was low, and 3D culture itself remarkably enhanced their spontaneous growth and maturation. The biggest problem in such 3D culture, cellular growth was limited only to the periphery of macropores of the scaffolds even with the thin disk shape of the scaffolds and with continuous shaking, resulted in about at most several times growth and one-tenth cellular density that in vivo. This indicated the insufficient mass transfer (primarily oxygen) between culture medium and inner spaces of the scaffolds. However, upon implantation to mesentery leaves of animals, almost all the remaining spaces in the scaffolds were finally filled with proliferated hepatocytes in mice and rats. These results clearly demonstrate that fetal cells that grow and mature in 3D culture with appropriate cocktails of soluble factors show promise in partly supporting the insufficient host liver functionality upon implantation.