一种改良大鼠肝细胞分离和原代培养技术

A modified method of isolation and primary culture of rat hepatocytes

目的探讨并改进大鼠肝细胞分离和培养技术。方法Ⅲ型胶原预铺培养板，改良原位胶原酶两步灌流法分离肝细胞，适宜密度接种，含10％胎牛血清的RPMI 1640培养基培养；观察细胞产量、活率、贴壁和生长情况评价方法的可行性。结果肝细胞分离时间明显缩短；胶原酶用量减少1／3；每鼠可获得（1．81±0．65）×10^8个肝细胞；活率为（87．46±6．90）％；细胞接种4h即可贴壁，可存活2～3周。结论该法操作简便、经济，细胞产量和存活率高，贴壁和生长良好，可满足各种实验需求。

Objective To explore and modify the method of isolation and primary culture of rat hepatocytes. Methods Six-well plates were pre-coated with collagen m about 24 h before use. Rat hepatocytes were isolated by a modified two-step collagenase perfusion technique in situ, seeded in eligible density, and cultured in RPMI 1640 medium containing 10% fetal bovine serum. The yield, viability, attachment and growth of hepatocytes were observed to estimate the feasibility. Results The isolation time of rat hepatocytes was shortened obviously. One-third of collagenaseⅣ was saved. About （ 1.81 ±0.65 ） × 10^8 hepatocytes were obtained from every rat, and the viability was （ 87.46 ± 6.90 ） %. The cells attached to the plates about 4 h after seeded, grew well and had a two-to three-week survival. Conclusion The modi- fied method is economical, and less labor and time for isolation and culture of primary rat hepatocytes. The yield, viability and attachment rate are high enough to be used for various experiments.