原代小型猪肝细胞微载体培养及其功能特性

Cultivation and identification of biologic activity of minitype porcine hepatocytes on microcarriers

目的 研究原代小型猪肝细胞微载体培养方法的建立及肝细胞功能特性。方法将3.20×107个的肝细胞及2g/L cytodexTM 3微载体连同40ml含100ml/L Hyclone小牛血清及其他辅助因子的RPMI 1640培养基加入250ml已硅化的方瓶中培养。对不同培养时间的肝细胞进行形态学观察。同时测定不同培养时期肝细胞生物合成及生物转化功能。结果肝细胞产量为6.8×109～8.1×109(7.58±0.57)×109/肝细胞;肝细胞活率为92.0%～99.0％(96.25％±3.10％);肝细胞具有正常肝细胞的超微结构特征;接种培养后肝细胞呈明显的朝CytodexTM 3聚集,二者易相粘贴,粘附在微载体上的肝细胞增殖生长旺盛;当肝细胞数为3.20×107个时,接种后24h肝细胞尿素及白蛋白合成量分别为1.22mmol/L及0.075g/L;随着时间的延长,利多卡因的转化率逐渐增加,在24h时即达到100％。结论 使用微载体培养的小型猪肝细胞具有较旺盛的增殖生长能力、良好的生物转化及生物合成功能,可作为体外生物人工肝较为理想的细胞来源。

Objective To explore a method for culture of minitype porcine hepatocytes on microcarriers and to investigate the functional features of the cultured hepatocytes. Methods Liver cells from minitype porcine liver were harvested by using a two - step extracorporeal perfusion apparatus with ethylene diamineteraacetic acid and collagenase soiutions. The isolated hepatocytes were inoculated in the fresh RPMI 1640 medium supplemented with 2 g/L cytodexTM 3, 100mL/ 1 calf serum and so on. The morphologic features of cultured hepatocytes were recorded. The biological function of biosynthesis and biotransformation of hepatocytes during different cultural intervals was evaluated. Results The harvested primary hepatocytes consisted of 99 % of the total isolated mass liver cells with the cellular viability as high as 96.25 % and gave an average yield of 7.58×109 cells each porcine liver, Primary hepatocytes in vitro cultured possessed the features of morphology of normal porcine primary hepatocytes; The primary hepatocytes attached to cytodexTM 3 after 3h of incubation, and began to actively proliferate after 20h of incubation in a mode of similar monolayer growth; The culture mass of primary hepatocytes has been identified to have the active synthetic and metabolic functions, e.g. synthesized urea and albumin being 1.22mmol/L and 0.075 g/L in 3.2×107 hepatocytes within 24 hours, respectively, and the transform rate of lidocanine into 3 - hydroxy - lidocanine being 100% in 3 .2×107 hepatocytes within 24 hours. Conclusions The primary hepatocytes from minitype porcine on microcarriers posses the features of active proliferation activity, morphology of normal porcine primary hepatocytes, active biosynthesis and biotransformation, which may be served as the better support model of extracorporeal bioartifical liver support system for patients with hepatic failure.