Hepatocyte attachment on biodegradable modified chitosan membranes: in vitro evaluation for the development of liver organoids.

Extracellular matrix structures including glycosaminoglycans play a critical role in cell attachment, differentiation, and morphogenesis. We evaluated chitosan ([1-->4] linked 2-amino-2-deoxy-beta-D-glucan) as a biomaterial for hepatocyte attachment because of its structural similarity to glycosaminoglycans. Freshly isolated rat and fetal porcine hepatocytes were seeded on chitosan membranes that had been previously blended with collagen, gelatin, or albumin to improve biocompatibility and surface roughness. The optimal cell density and attachment kinetics were quantified. The metabolic activity was investigated by measuring daily urea and total protein secretion by the cells for 2 weeks. While collagen blended-chitosan membranes provided a good attachment surface for rat hepatocytes, albumin and gelatin blended chitosan membranes were superior for fetal porcine hepatocyte attachment. The optimal attachment was maintained with membranes of medium molecular weight (Mr = 750,000 daltons) chitosan, at 3-4 x 10(4) cells/cm2 after 3 h of incubation. In vitro experiments demonstrated that fetal porcine hepatocytes survived at least 14 days when seeded on the chitosan-albumin matrix, demonstrating that this biomaterial can provide suitable cell attachment scaffolds for creating liver tissue organoids.