肝细胞大规模培养的一种简便方法

Spinner culture:a simple method for large scale culture of hepatocytes

目的 探讨肝细胞大规模培养的简便方法。方法 采用改良Seglen原位两步胶原酶灌注法分离乳猪肝细胞 ,将肝细胞悬液接种到预先用 1%多聚赖氨酸被覆的培养瓶中 ,置于 37℃普通培养箱低速旋转培养 ,定期测定其活率、葡萄糖合成功能、G 6 Pase活性及上清中白蛋白、LDH浓度 ,电镜观察培养细胞的超微结构。结果 旋转培养肝细胞保持较高的活率 ,葡萄糖合成功能和G 6 Pase活性明显增高 ;电镜下肝细胞形成紧密连接和胆小管样结构。结论 采用普通培养箱旋转培养能保持良好的肝细胞功能 。

Objective To explore a simple method for large scale culture of hepatocytes.Methods Suckling pig hepatocytes were isolated by modified two step in situ collagenase perfusion method described by Seglen.The suspension of hepatocytes was inoculated into spinner flasks precoated by 1% polylysine,then the flasks were stirred at lower speed in a 37℃ incubator.The viability,glucose syntheses,G 6 Pase activity,concentration of albumin and LDH in the supernatant were determined dynamically and the ultrastructure of the cultural hepatocytes was observed.Results The hepatocytes in spinner group showed enhanced viability,glucose synthesis capacity and G 6 Pase activity when compared to those in control group.Tight junctions and bile canaliculus like channels between cells within aggregates were observed under TEM.Conclusion The function of hepatocytes cultured by spinner method in an incubator was well maintained.This study provides a simple and better culture method for a large scale culture of hepatocytes and bioartificial liver.