Culture of proliferating and differentiating fat-storing cells in 3T3-conditioned medium

There is growing evidence suggesting that hepatic fat-storing cells (FSC) or Ito cells have an important function in vitamin A storage and metabolism and in the synthesis of connective tissue components in normal liver and during fibrogenesis. The purified FSC acquire a fibroblastic morphology and their vitamin A content decreases in culture. We cultivated cells under in vitro conditions that allowed the expression of FSC morphological and functional characteristics for 3-4 weeks of primary culture. Cells were isolated from rat liver by the collagenase-perfusion method without further purification and cultured with 3T3-conditioned medium, which seemed to stimulate the selective proliferation of the FSC. After 8-10 days, round and stellate cells grew actively from a few precursor cells in the primary culture and were not subcultivated; the stellate cells had the ability to become round and vice versa and were highly motile. The cells had intracytoplasmic lipid droplets, a well developed rough endoplasmic reticulum, Golgi complex, numerous vesicles filled with electron-dense material, and extracellular matrix (ECM) components on their surface. Both stellate and round cells showed the presence of desmin by immunofluorescence and vitamin A autofluorescence, but lacked peroxidase activity. The culture conditions we describe allowed the selective proliferation of cells with morphological and functional characteristics of the FSC in the normal liver, raising the possibility of studying FSC proliferation and differentiation.