新型编织型生物反应器内大量乳猪肝细胞的组织化培养  

Organoid culture of mass porket hepatocytes in the novel bioreactor with woven hollow fibers

作　　者：何礼荣[1] 戴坤扬[1] 王英杰[2] 刘俊[2] 郭海涛[2] 刘鸿凌[2] 戎建杰[1] 

机构地区：[1]上海交通大学附属第六人民医院普外科,200233 [2]第三军医大学西南医院传染科

出　　处：《肝脏》2004年第1期24-27,共4页Chinese Hepatology

基　　金：国家自然科学基金资助项目 (30 0 2 70 0 1 );全国优秀博士基金资助项目 (1 99947)

摘　　要：目的　使反应器内培养的肝细胞能在较长时间保持肝细胞的特殊功能和活力 ,提高生物反应器的功效并为反应器内储存和运输肝细胞提供可能。方法　将新生实验型小猪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 ,将其置入新型编织型生物反应器的外腔 ,用培养液循环式人工毛细管培养系统进行培养 ,在培养液内加入氯化氨、醋胺酚检测生物反应器的转化功能 ,行无血清培养检测肝细胞的白蛋白的合成功能 ,观察肝细胞的酶漏出量。锥虫蓝染色观察肝细胞的活力 ,电镜观察其超微结构。结果　生物反应器内培养的肝细胞在 1周内能保持较高的活力 ,并保持着较高的氯化氨、醋胺酚的生物转化及白蛋白合成功能 ,酶的漏出量也少。肝细胞超微结构示内质网、线粒体等细胞器丰富 ,核内染色体分布均匀 ,肝细胞间的微绒毛形成胆小管样结构。结论　肝细胞与微载体及肝细胞之间的聚集 ,形成直径大小不等的肝细胞 微载体球形聚集体 ,这种由肝细胞重新结合而成的聚集体类似于体内的肝组织结构 ,在新型编织型生物反应器内的中空纤维网架的支持下 ,肝细胞聚集体均匀地分散其中 ,为肝细胞的生长提供了一个类似体内内环境的三维空间 ,因而在无氧合器供氧的情况下 ,肝细胞也能在 1周内保持较好的形态和功?Objective To maintain the liver-specific functions and viability of hepatocyte well for a long time in the bioreactor, so as to improve the functions of hollow fiber bioreactor and provide the possibility. Methods Microcarrier culture of porket hepatocyte was made in the culture bottle , after forming microcarrier-spheroided aggregates the hepatocytes was seeded into extralumen of the bioreactor and cultured with a artificial capillary cell culture system. After a period of time NH4CL and Acetaminophen were added in the culture medium, the transforming function of the bioreactor containing hepatocytes was examined. The synthetic function of albumin of hepatocytes was tested under the culture without serum. And the enzyme release of hepatocytes was examined. Both the viability of hepatocytes with trypan blue exclusion test and the ultrastructure of hepatocytes were observed. Results Within one week the hepatocytes in the bioreactor were maintained high viability. Both the bio-transformational function for NH4CL and Acetaminophen and synthetic function of albumin of hepatocytes were maintained very well , the enzyme release of porket hepatocytes was little within one week. The ultrastructures of hepatocytes within one week: the cell organelles were well preserved. endoplasmic reticulum and glycogen granules and mitochondria were present in normal quantity and distribution .The nuclear membrane and chromatin appeared normal. Neighboring cells reconstituted bile canaliculi-like structures with typical microvilli and tight junctions between the hepatocytes. Conclusion Hepatocyte organoids having a tissue-like structure assembled from hepatocytes and microcarriers were equably distributed into the extralumen of bioreactor under the support of hollow fiber and could express a high function and be maintained for two weeks without oxygenic supply via oxygenor.

关 键 词：编织型生物反应器 乳猪 肝细胞 组织化培养 微载体 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]