大量幼猪原代肝细胞的分离与培养

Large-Scale Isolation and Cultivation of Primary Porcine Hepatocytes

研究分离幼猪原代肝细胞培养过程中肝细胞的数量和功能活性。方法采用本地幼猪肝脏供体,应用体外胶原酶灌流法分离猪肝细胞,采用台盼蓝拒染法测定肝实质细胞活性,在质量浓度为10%FCS(灭活胎牛血清)的培养液中培养,在光镜及电镜下观察培养过程中肝细胞形态学变化,检测不同时期培养上清液中白蛋白和尿素氮BUN的浓度。结果每只猪肝脏平均肝细胞产量为1.55×10个,即时活性为90.2%±2.5%,24h后85%以上的肝细胞形成10肝细胞球,并可保持细胞功能活性达7天,其中前3天活性最高。结论本法分离获取的肝细胞产量高、功能活性好,可作为生物型人工肝的生物材料。

Objective To study the cell amount of young pig primary hepatocytes and their functional viability in isolation and culture. Methods The young pig primary hepatocytes were isolated by extracorporeal collagenase perfusion technique. Their viability was assessed by trypan blue exclusion. Hepatocytes were inoculated in the culture medium consisted of 10 % fetal calf serum. The morphological changes of cultured porcine hepatocytes was observed by optical microscope and electronic microscope .The concentrations of albumin and BUN in the supernatant in different culture period were determined. Results The average yield of porcine hepatocytes was 1.55×1010 cells per pig with an average viability of 90.2 %±2.5 %. Hepatocytes had a tendency to aggregate and 85 % of cells formed spheroids within 24 hours in culture and their viability and differentiating function during in vitro culture were valid for 7 days. The viability and differentiating function of hepatocytes were higher in the first three days during cultivation. Conclusion These data indicate that the young pig hepatocytes can be harvested with high yields and retain viability, thus it is an excellent hepatocyte source for bioartificial liver.