摘要
目的采用普通型中空纤维及编织型中空纤维两种人工肝生物反应器,比较其内培养的肝细胞生存时间及功能,为生物反应器的设计提供新的方法和思路。方法将新生中国实验小型猪的肝细胞与微载体共同培养,待肝细胞与微载体充分粘附形成微载体-球形聚集体后,将其注入生物反应器的外腔,在培养液内加入氯化氨、醋胺酚检测生物反应器的生物转化和代谢功能,行无血清培养检测肝细胞白蛋白的合成功能,并检测肝细胞酶的漏出量。锥虫蓝染色测定肝细胞的活力,透射电镜观察其超微结构。结果肝细胞与微载体相互聚集,形成了具有一定结构层次的肝细胞聚集体,重构了肝细胞之间的相互连接;接种入生物反应器后,在编织型生物反应器中肝细胞1周内保持较高的活力,至第7天肝细胞活力仍高达(75±6.3)%,对醋氨酚和氯化铵有良好的代谢、生物转化功能,白蛋白合成在第7天为(0.57±0.13)mg/106个肝细胞,酶的漏出量也少,各项指标均明显优于普通型生物反应器组(P<0.05)。结论编织型生物反应器为肝细胞的生长、增殖提供一个类似体内的三维立体环境,是一种性能良好的生物反应器。
Objective To provide some new ideas and means for designing bioreactor by comparing two types of artificial liver bioreactor. Methods Microcarrier culture of piglet hepatocytes was performed in the culture bottle, after formation of microcarrierspheroided aggregates the hepatocytes were seeded into extralumen of the bioreactor and cultured with a artificial capillary cell culture system. Then NH4 CL and Acetaminophen were added in the culture medium, the biological transforming function of the bioreactor was examined. The synthetic function of albumin of hepatocytes was tested, and the enzyme release of hepatocytes was examined. Viability of hepatocytes by both the trypan blue exclusion test and the ultrastructure of hepatocytes were determined. Results Hepatocytes which formed microcarrier-spheroided aggregates had some cell structures and reconstructed the cell-cell connections. Hepatocytes in the woven bioreactor maintained high viability within one week（viability of hepatocytes reached 75% ± 6.3% at 7 d）. The bio-transformating function both for NH4CL and Acetaminophen and synthetic function of albumin of hepatocytes were well maintained （0.57 ± 0.13 mg/10^6 hepatocytes at 7 d）, only a little of AST and LDH released from the cultured hepatocytes within one week. All these were much better than the common bioreactor（ P 〈 0.05 ）. Conclusion Hepatocyte-organoids having a tissue-like structure were equaly distributed into the extralumen of woven bioreactor under the support of woven hollow fiber which provide a three dimensions environment similar to that in vivo for growth and proliferation of hepatocytes. So it is a type of good performance bioreactor.
出处
《肝脏》
2006年第6期377-380,共4页
Chinese Hepatology
基金
国家自然科学基金资助项目(30027001)
全国优秀博士基金资助项目(199947)
关键词
人工肝
生物反应器
肝细胞
微载体
组织化培养
Artificial liver
Bioreactor
Hepatocytes
Microcarrier
Organoid culture
