原代猪肝细胞培养体系及培养模式的初步探讨

Study on proximal culturing module and medium for primary porcine hepatocytes

目的 探索一高密度、高效率的原代猪肝细胞培养方法 ,为生物人工肝的生物材料提供一种有效的培养模式。方法 采用改良Seglen二步胶原酶灌注法分离肝细胞 ,分别将 5× 10 6 的肝细胞加入胎牛血清、猪门静脉血清的培养体系中进行方瓶静止培养及聚球培养。结果 肝细胞在接种后呈明显的Cytodex 3聚集趋势。门静脉血清培养体系中肝细胞的Albumin、Urea合成能力明显高于胎牛血清组。在培养后的前 3周门静脉血清培养体系中聚球细胞模式优于微载体静止培养模式。结论 与胎牛血清培养体系相比 ,猪门静脉血清培养体系更适合原代猪肝细胞高密度培养。聚球培养是一种较为理想的原代猪肝细胞培养模式。

Objective To explore a high-density and effective culturing method for primary porcine hepatocytes to obtain an effective culturing module for bioartificial liver. Methods Porcine hepatocytes were harvested by two-step perfusion collagenase method. Then they were respectively cultured at a concentration of 5×10 6 with 2 g/L cytodex-3 in DMEM medium supplemented with 100 ml/L fetal calf serum (FCS) and that with 100 ml/L porcine portal vein serum (PPVS). Results Most hepatocytes got together with microcarriers and some of them attached on microcarriers about 24 to 48 h after the culture and the multicellular aggregating spheroids were formed. The morphological characteristics and the synthetic functions of albumin and urea were maintained for 5 weeks in FCS culture system and 8 weeks in PPVS culture system. The mode of spheroids was better than that of microcarriers. Conclusion As compared with FCS culture system, PPVS culture system is more suitable to culturing primary hepatocytes in high density and the culturing mode of spheroid is the most promising module.