A new approach to develop a biohybrid artificial liver using a tightly regulated human hepatocyte cell line

Currently patients with liver failure have been treated with a various liver support systems including a whole liver perfusion, a non-biological artificial liver, and a biohybrid artificial liver. In a hepatocyte-based bioreactor, porcine hepatocytes or transformed human liver tumor cells have been utilized because of the ease of preparation. According to the clinical data reported as of now, satisfactory results have not been obtained from the use of currently available liver support devices. One of the problems is limited availability of primary human liver cells for developing live support systems because of the shortage of human liver. To resolve this issue, human hepatocytes were immortalized with a retroviral vector SSR#69 which contained the genes of simian virus 40 large T antigen (SV40Tag) and herpes simplex virus-thymidine kinase (HSV-TK). One of the immortal cell lines, NKNT-3, showed the gene expression of differentiated liver functions, grew steadily in chemically defined serum-free CS-C medium, and doubled in number in about 48 hours. Essentially unlimited availability of NKNT-3 cells supports their clinical use for liver support devices. To realize the high density culture of NKNT-3 cells in a bioartificial liver device, we have developed cellulose microspheres (CMS) which contain cell adhesive GRGDS (Gly-Arg-Gly-Asp-Ser) peptides. Within 24 hours after starting a stirring suspension culture, GRGDS-CMS efficiently immobilized NKNT-3 cells. An electron microscopic examination demonstrated that NKNT-3 cells attached on GRGDS-CMS had well-developed mitochondria, rough reticulums, and villous extensions. In this article, we review the history of extracorporeal liver support systems and describe an attractive strategy for developing a novel extracorporeal liver assist device using NKNT-3 cells and GRGDS-coated cellulose microspheres.