Objective: To find out an optimal condition for isolation and primary culture of hepatocytes.
Method: Rat hepatocytes were isolated by a two-step collagenase perfusion, and cultured in hepatozyme-SFM. The reduction of MTT to formazan salt was examined. Supernatant medium was collected for analysis of alanine amino transferase (ALT) and ureagenesis.
Results: The two-step collagenase perfusion yielded 39+/-12x10(6) cells/g liver tissue with a viability of 88%+/-2%. Fine morphology and stable urea synthesis for one week could be achieved when hepatocytes were cultured in hepatozyme-SFM.
Conclusion: High yield of hepatocytes can be isolated with two-step collagenese perfusion. Hepatozyme-SFM is suitable for sustained growth of hepatocytes.