Chen Zhong, Ding Yitao, Zhang Heyun
Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital, Medical School, Nanjing, PR China.
Dig Surg. 2002;19(3):184-91. doi: 10.1159/000064211.
In bioartificial liver preparation, serum-contained medium is ordinarily replaced by serum-free medium and hepatocytes are generally cultured at high density. This study was to undertaken to evaluated the dynamic changes in morphology, viability and functions of porcine hepatocytes in serum-free medium at high density.
Hepatocytes were isolated from suckling pigs by modified two-step in situ collagenase perfusion method and cultured in serum-free medium at high density. Morphology, viability, protein and glucose syntheses, G-6-Pase activity, diazepam transformation of hepatocytes and release of LDH in supernatant during 7 days of culture were evaluated. These measurements were also determined on both groups of hepatocytes cultured at low-density in serum-free medium and serum-contained medium, which served as control groups.
Morphology and protein synthesis of hepatocytes cultured in serum-free medium at high density were stable over the course of 7 days. High viability (>90%) was obtained though it declined with time. Diazepam transformation of cells was higher on days 2 and 3. Glucose synthesis of cells declined from day 3 to day 7. G-6-Pase activity of the hepatocytes declined apparently after 1 day of culture and it was maintained at a low level from day 1 to day 7. Release of LDH in supernatant was higher on days 1, 2 and 3. There were no significant differences in viability and functions of hepatocytes except for G-6-Pase activity at low-density culture between the serum-free medium group and the serum-contained medium group. The functions of hepatocytes cultured at high density were lower than at low-density culture.
The results showed that the morphology, viability, protein synthesis and diazepam transformation of hepatocytes cultured in serum-free medium at high density were maintained during 7 days of culture. The serum-free medium provided indices of cell viability and functions that were comparable to serum-contained medium. The functions of hepatocyte cultured at high density (1 x 10(7) cells/ml) were lower than at low density (5 x 10(5) cells/ml).
在生物人工肝制备过程中，通常用无血清培养基替代含血清培养基，并且肝细胞一般以高密度进行培养。本研究旨在评估高密度下无血清培养基中猪肝细胞的形态、活力及功能的动态变化。
采用改良的两步原位胶原酶灌注法从乳猪中分离肝细胞，并在无血清培养基中进行高密度培养。评估培养7天期间肝细胞的形态、活力、蛋白质和葡萄糖合成、葡萄糖-6-磷酸酶（G-6-Pase）活性、地西泮转化以及上清液中乳酸脱氢酶（LDH）的释放。还对在无血清培养基和含血清培养基中低密度培养的两组肝细胞进行了这些测量，作为对照组。
在无血清培养基中高密度培养的肝细胞，其形态和蛋白质合成在7天的培养过程中保持稳定。尽管活力随时间下降，但仍获得了较高的活力（>90%）。细胞的地西泮转化在第2天和第3天较高。细胞的葡萄糖合成从第3天到第7天下降。肝细胞的G-6-Pase活性在培养1天后明显下降，并从第1天到第7天维持在低水平。上清液中LDH的释放在第1天、第2天和第3天较高。除了无血清培养基组和含血清培养基组在低密度培养时G-6-Pase活性外，肝细胞的活力和功能没有显著差异。高密度培养的肝细胞功能低于低密度培养。
结果表明，在无血清培养基中高密度培养的肝细胞，其形态、活力、蛋白质合成和地西泮转化在7天培养期间得以维持。无血清培养基提供的细胞活力和功能指标与含血清培养基相当。高密度（1×10⁷细胞/ml）培养的肝细胞功能低于低密度（5×10⁵细胞/ml）培养。
