Differentiation and proliferation of primary rat hepatocytes cultured as spheroids.

We studied spheroid (multicellular aggregate) formation by hepatocytes and the expression of liver-specific functions such as albumin secretion when hepatocytes were cultured with various extracellular matrices. Hepatocytes cultured on Primaria(R) and poly-D-lysine coated dishes, and in the presence of a polymer, Eudragit, formed spheroids, and they also exhibited higher liver-specific functions and poor growth compared to monolayer cultures. The results indicated that the cell morphological change and cell-cell interaction caused by the spheroid formation were key factors promoting the expression of the liver-specific functions. To elucidate the mechanism underlying the poor growth in spheroids, we examined the HGF signaling pathway. Phosphorylation and down-regulation of the HGF receptor (c-Met proto-oncogene product) were observed for the cells from both monolayer and spheroid cultures, but Ras activation was partly blocked in spheroids. Furthermore, we found that CDK inhibitors, p21 and p27, were highly expressed in spheroids. These results suggested that the reduced Ras signaling and high expression of the CDK inhibitors might cause the lower growth in spheroids. We then examined the relationship between liver-enriched transcription factors (C/EBPalpha and beta) and liver-specific functions. The results revealed that the high expression of C/EBPalpha was maintained during cultures when hepatocytes formed spheroids. Antisense oligonucleotides of C/EBPalpha repressed albumin secretion and the expression of p21, suggesting that the transcription factor, C/EBPalpha, may play a crucial role in the growth and differentiation of hepatocytes in spheroids.