Publications (2)2.09 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Isolated human hepatocytes are of great value in investigating cell transplantation, liver physiology, pathology, and drug metabolism. Though hepatocytes possess a tremendous proliferative capacity in vivo, their ability to grow in culture is severely limited. We postulated that repeated medium change, common to most in vitro systems, may prevent long-term maintenance of hepato-specific functions and growth capacity. To verify our hypotheses we compared the DNA synthesis and differentiation status of isolated human hepatocytes, cultured in medium which was renewed every day or was not changed for 3 weeks ('autocrine' setting). Daily medium change led to rapid hepatocellular de-differentiation without any signs of DNA replication. In contrast, the autocrine setting allowed hepatocytes to become highly differentiated, demonstrated by an elevated ASGPr expression level, and increased albumin and fibrinogen synthesis and release. Cytokeratin 18 filaments were stably expressed, whereas cytokeratin 19 remained undetectable. Hepatocytes growing in an autocrine fashion were activated in the presence of hepatocyte growth factor (HGF), evidenced by c-Met phosphorylation. However, HGF response was not achieved when the culture medium was renewed daily. Furthermore, the autocrine setting evoked a late but strong interleukin 6 release into the culture supernatant, reaching maximum values after a 10-day cultivation period, and intense BrdU incorporation after a further 5-day period. Our data suggest that preservation of the same medium creates environmental conditions which allow hepatocytes to control their differentiation status and DNA synthesis in an autocrine fashion. Further studies are necessary to identify the key mediators involved in autocrine communication and to design the optimal culture configuration for clinical application.
- [Show abstract] [Hide abstract] ABSTRACT: Transplantation of isolated hepatocytes might be an alternative to orthotopic liver transplantation. However, a cell culture system has to be established which requires a sufficient amount of hepatocytes with stable liver-specific function. In the present study, an in vitro cell culture system has been optimized which allows the induction of cell mitosis of highly differentiated hepatocytes. Hepatocytes were derived from human liver tissue with a high pressure 2 step isolation method and grown on a two-dimensional collagen matrix. Expression and activity of growth receptors HGF-r and EGF-r and amount of CK18, CK19, albumin, factor VIII and fibrinogen were investigated by Facs or Western blot. BrdU-incorporation was evaluated fluorometrically. Interestingly, cultivation of human hepatocytes without medium renewal induced growth of cell islands, consisting of small heoatocytes (single nucleus, more granulated than adult human hepatocytes). Furthermore, albumin, factor VIII and fibrinogen were expressed at a high level. Maximal DNA-synthesis was measured on day 5 (15% of cells were Brdu positive). HGF-r and EGFr showed maximal peaks on days 5 and 9. In parallel, CK18-expression and de novo synthesis of CK19 were enhanced. We postulate that a high pressure isolation technique necessary to obtain hepatocytes with high growth potential. In addition, enrichment of soluble mediators in the cell culture supernatant might provide specific triggering factors responsible for stabilizing cell differentiation.
Goethe-Universität Frankfurt am Main
Frankfurt, Hesse, Germany
- Klinik für Urologie und Kinderurologie