Free amino groups of chitosan, a substance which has previously been shown to be a good scaffold for hepatocyte attachment, were covalently modified with fructose. The modification significantly increased the number of cells that could be attached on the surface of chitosan gel. Rat hepatocytes cultivated on fructose-chitosan behaved similarly to those on unmodified chitosan, i.e., they retained the spherical shape they have in vivo, and released much less lactate dehydrogenase than cells attached on a collagen-coated surface. The modification with fructose did not alter the important characteristics of chitosan for hepatocyte culture : liver-specific functions such as urea synthesis and drug metabolism were stably maintained for 5 d in the hepatocytes cultured on fructose-chitosan. In sharp contrast, hepatocytes attached on a collagen-coated surface underwent a severe morphological change, from spherical to flat, and lost almost all their lidocaine-removal activity within 5 d. A very thin fructose-chitosan layer was also applied onto the collagen-coated surfaces of polystyrene plates and a dextran microcarrier by crosslinking free amino groups in the chitosan and collagen with glutaraldehyde to fix the thin layer. Hepatocytes on the fructose-chitosan-coated surface retained their spherical shape, masking the cell-flattening effect of the collagen layer. Perfusion culture was then carried out using a hollow-fiber cartridge in which hepatocytes attached on fructose-chitosan-coated microcarriers were suspended in the extracapillary space : the liver-specific functions were stably maintained during 4 d of the culture. A fructose-chitosan-coated surface thus appears to be a very promising scaffold for hepatocyte attachment which can be used in cellular biological studies of liver functions, especially in relation to cytochrome P450, as well as in bioartificial liver support systems.
The Pharmaceutical Society of Japan