[Isolation and separation of human adult hepatocytes from resected liver: cellular yield and purity using different...

Cultivated human hepatocytes (HC) for in vitro cell culture models are not frequently used yet. Besides, there are still several different methods of isolation, separation and cultivation of HC in use. We compared 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 method. The methods of disintegration having a stronger mechanical component (i.e. disintegration of tissue in a magnetic stirrer) yielded only few vital cells. To separate HC from non-parenchymal cells (NPC) after isolation we tested two methods. Best results could be reached by centrifugation 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 having viabilities ranging between 74 and 100%. Contamination with NPC was lower than 1% as assessed by immunocytochemistry. In contrast, only lower yield and purity resulted by application of a 3-times centrifugation at 28xg for 5 minutes. For cultivation of HC, both initial adherence and survival was superior when collagen type I coated culture plates 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 culture period of 6 weeks was reached.