ISOLATION AND SEPARATION OF HUMAN ADULT H EPATOCYTES - YIELD AND PURITY BY USING DIFFERENT METHODS

Cultivated human hepatocytes (HC) for in vitro cell culture models are not frequently used yet. Besides, there are still several different m ethods of isolation, separation and cultivation of HC in use. We compa red various methods to obtain isolated HC. Cell yield as well as high purity of the HC cultures were the main objectives of this study. For isolation, best results were made with a modified 2-step perfusion met hod. The methods of disintegration having a stronger mechanical compon ent (i.e. disintegration of tissue in a magnetic stirrer) yielded only few vital cells. To separate HC from non-parenchymal cells (NPC) afte r isolation we tested two methods. Best results could be reached by ce ntrifugation in Percoll adjusted to a density of 1.065 g/ml for 10 min . at 50xg. Thus, we were able to obtain nearly pure HC suspensions hav ing viabilities ranging between 74 and 100%. Contamination with NPC wa s lower than 1% as assessed by immunocytochemistry. In contrast, only lower yield and purity resulted by application of a 3-times centrifuga tion at 28xg for 5 minutes. For cultivation of HC, both initial adhere nce and survival was superior when collagen type I coated culture plat es were used instead of uncoated plates. Up to now, a maximum yield of 2.14 x 10(7) viable HC per gram processed tissue and a maximum cultur e period of 6 weeks was reached.