原代猪肝细胞无血清培养

Serum-free medium for the culture of primary porcine hepatocytes

目的研究原代猪肝细胞无血清培养及肝细胞的功能。方法采用EGTA和胶原酶P两步法经肝静脉逆行灌注分离乳猪肝细胞,在无血清培养基中培养,并对不同培养时间的肝细胞生化合成及生物转化功能进行检测。结果肝细胞产量为(1.5±0.1)×1010/肝,活率为(90.3±1.5)%,接种培养后肝细胞增生旺盛,LDH漏出第2天达到高峰,然后逐渐下降并稳定于一定水平。随着时间的延长及肝细胞数量的增加,白蛋白合成及利多卡因转化率逐渐增加,呈时间及肝细胞数量的依赖关系。结论采用本法分离获取的猪肝细胞产率高、活性强、具有良好的生物合成及生物转化功能,可作为生物人工肝较理想的肝细胞来源。

Objective To investigate the effects of serum-free medium for culture of isolated primary hepatocytes from suckling pig and detect their metabolic activity. Methods Suckling pig hepatocytes were isolated by retrograde twostep collagenase perfusion method via portal vein and cultured at 2 × 10^8/L serum-free medium. Morphologic features of cultured hepatocytes were observed and LDH release, function of biosynthesis and biotransformation of hepatocytes during different cultural intervals were also evaluated. Results The yield of primary hepatoeytes was（1.5 ± 0.1 ） × 10^10 cells each liver with the cellular viability as high as（90.3 ± 1.5）%. The primary hepatocytes attached flask wall within the first 24h incubation, and began to actively proliferate after 48 h of incubation in a mode of monotayer growth. LDH release was at the highest level on the second day. The albumin synthesis and the transform rate of lidocanine into 3- hydroxy lidocanine increased with the time of culture and the quantity of hepatocyte during 7 days. Conclusion This method allows the high yield of primary porcine hepatocytes with high viability and normal metabolic activity in vitro serum-free culture system, which can be served as the better cell resource of bioartifieial liver devices.