用于生物人工肝的猪肝细胞三维立体培养

Three-dimensional culture of porcine hepatocytes for bioartificial liver

目的 研究猪肝细胞在三维立体培养系统 (中空纤维生物反应器 )中的生长、增殖及功能表达情况 ,探讨肝细胞的生物活性与培养时间之间的关系 ,找出在该培养系统中生物人工肝的有效肝功能支持时段。方法 用体重 5～ 8kg健康杂种小猪作为肝细胞供体。用胶原酶原位灌注法分离肝细胞 ,将分离计数好的肝细胞悬液 ( 5× 1 0 7～ 1 0× 1 0 7·mL- 1 )注入中空纤维管内间隙进行培养。定期对肝细胞的活性、形态、生长情况及功能表达 (白蛋白合成 )情况进行观察和检测。结果 猪肝细胞在中空纤维管培养系统内生长、增殖及功能表达良好 ,能够在长达 35d的时间内保持活力 ,并呈现一定的生长规律。结论 该系统培养的猪肝细胞在第 5～ 2 5天的范围内能够达到生物人工肝支持治疗的要求 。

Objective To study the growth, multiplication and expression of porcine hepatocytes in the three dimensional culture system (hollow fiber bioreactor, HFB) so as to find out the relationship of the vitality and culture time of hepatocytes and find out how long the bioartificial liver (BAL) in this culture system can offer an effective hepatic function support. Methods Hepatocytes were isolated from hybrid pigs weighing 5～8?kg by means of colagenase. After isolating and counting, we put the suspending hepatocytes (5×10 7～10×10 7·mL -1 ) into the inside space of the HFB. In the beginning, we circuited the culture medium through outside space of the HFB. When the hepatocytes were glued on the side of the tube that had been stuck with little gel matrix, we changed the circuiting culture medium through inside space of the HFB. At regular intervals we observed and checked the vitality, conformation, growth and expression of the cultured hepatocytes. Results Porcine hepatocytes not only grew and reprocessed well in HFB but also expressed well in function. They could remain vigorous for about 35 days and take on an usual survival pattern to some extent. Conclusion The porcine hepatocytes cultured in this system can meet the requirements of the BAL support treatment from 5 to 25 days. During this period the porcine hepatocyte hollow fiber BAL can be applied experiment and clinical research.[HQ姜?[WTHZ]