DEVELOPMENT OF THE THREE-DIMENSIONAL ARTIFICIAL LIVER USING A LONG-ACTING VITAMIN C DERIVATIVE

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We established the procedure for isolation of hepatic stellate cells (vitamin A-storing cells) and parenchymal cells by perfusion of collagenase through the hepatic portal vein of rat. These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum or William's medium E.Isolated rat stellate cells and human hepatoma HepG2 cells contacted directly and formed a three-dimensional structure when co-cultured in the medium supplemented with a long-acting vitamin C derivative (L-ascorbic acid 2-phosphate ; Asc 2-P). Expression and three-dimensional distribution of phosphorylated proteins in HepG2 cells changed dramatically by contact with the stellate cells.Asc 2-P regulates cellular functions through promotion of the production of extracellular matrix. A kind and three-dimensional structure of extracellular matrix was found to regulate reversibly morphology, proliferation, and functions of the stellate cells. A peptide DGEA and integrin alpha2beta1 was essential for the direct adhesion of the stellate cells and type I collagen. Our present study demonstrated that signals from integrin stimulated phosphorylation of tyrosine and phosphatidylinositol, assembly of microtubules and fibrillar actin, and regulation of the morphology of the stellate cells. The native form of type I collagen which production is stimulated by Asc 2-P was important for the regulation of morphology and function of the hepatic stellate cells. The data obtained in our present study will promote the development of the three-dimensional artificial liver.