Three-dimensional co-culture of rat hepatocyte spheroids and NIH/3T3 fibroblasts enhances hepatocyte functional maintenance.

Functional maintenance of primary hepatocytes in culture can be improved by several distinct approaches involving optimization of the extracellular matrix microenvironment, media composition and cell-cell interactions, both homotypic and heterotypic. Using a galactose-decorated surface, we have developed a method to combine these two approaches by co-culturing rat primary hepatocyte spheroids with NIH/3T3 mouse fibroblast cells. Spheroids were performed by culturing hepatocytes for 3 days on galactosylated poly(vinylidene difluoride) membrane; NIH/3T3 cells were subsequently seeded and co-cultured with the spheroids. Results showed that although NIH/3T3 cells alone responded poorly to the galactosylated PVDF surface and displayed limited attachment, NIH/3T3 fibroblasts attached to the periphery of the hepatocyte spheroids and proliferated around them. Co-cultured hepatocyte spheroids exhibited significantly higher liver-specific functions as compared to spheroids cultured alone. Albumin secretion level in this co-culture system peaked on day 11, which was 1.8- and 2.9-times higher than the peak expression level in spheroid homo-culture control in serum-free (day 3) and serum-containing media (day 4), respectively. The albumin secretion function was maintained for at least two weeks; it was 5.1 (in serum-free medium) and 17.8 (in serum-containing medium) times higher than spheroid homo-culture on day 13. Similarly, the co-culture system also expressed approximately 5.5- and 3.1-times higher 3-methylcholanthrene-induced cytochrome P450 enzymatic activity on day 14 as compared to the homo-culture control in serum-free and serum-containing medium, respectively. In conclusion, this unique co-culture system demonstrated the synergistic roles of homotypic cell-cell interaction, heterotypic cell-cell interaction, cell-substrate interaction and soluble stimuli in hepatocyte functional maintenance.