Separation and long-term cultivation of rat hepatocytes / 吉林大学学报(医学版)

ABSTRACT

Objective:To study a simplified method of isolation of rat hepatocytes and to observe the pro-cess of cell morphology in long-term culture. Methods:Rat hepatocytes were isolated by a single two-stepperfusion method. The yield and viability were assessed by trypan blue exclusion. [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide] (MTT) was used to test the effect of serum concentration of newborn calf serum on the proliferation of hepatocytes. Hepatocytes were inoculated in the culture mediumconsisted of Williams' E supplemented with insulin,dexamethasone and 10％ new born calf serum. Themorphologic change of cultured hepatocytes was observed. Results:The average yield of hepatocytes was 2.26× 108 cells per rat, with an average viability of 95.6％. New born calf serum had strong biological activi-ty to stimulate the proliferation of hepatocytes and there was close-effect relationship followed by the in-crease of new born calf serum concentration. The rat hepatocytes can be cultured for 5～ 6 weeks withpreservation of normal morphologic appearance. Conclusion:The rat hepatocytes isolated by the abovemethod have high yields and viability and can be long-term cultured in vitro.