Maintaining hepatocyte differentiation in vitro through co-Culture with hepatic stellate cells

Abstract

Primary hepatocytes lose their differentiatedfunctions rapidly when in culture. Our aim was to maintainthe differentiated status of hepatocytes in vitro by means of vital hepatic stellate cells (HSCs), their soluble and particulate factors and lipid extracts. Hepatocytes wereplaced into collagen-coated culture dishes in the presenceof HSCs at different ages of pre-culture, with or withoutdirect cell to cell contacts, at different cell ratios and in monoculture with cellular HSC components in place ofvital cells. Changes in morphology and enhancement ofphosphoenolpyruvate carboxykinase (PCK) activity byglucagon were used to determine the differentiated statusof hepatocytes in 2d-short-term culture. HSCs proved ableto maintain the differentiated function of hepatocytes in cocultureeither by direct cell contacts or via factors derivedfrom HSC-conditioned medium. In comparison, however,without cellular contact to hepatocytes five to ten times asmany HSCs were necessary to increase the PCK activity to the same degree as in the presence of intercellular contacts. Whereas stimulation in the presence of HSC/hepatocyte contacts was independent of HSC culture age only quiescent,resting HSCs (precultured for 1-2 d) were able to stimulate hepatocytes significantly via soluble factors. Culturing of epatocytes with a lipid extract or a particulate fraction fromHSCs clearly displayed a very strong beneficial effect onenzyme activity and morphology. HSCs maintain hepatocyte function and structure through preferentially cell-bound signalling and transfer of lipids. © The Author(s) 2009.