Departments of Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Liver Transpl. 2011 Feb;17(2):104-14. doi: 10.1002/lt.22200.
Liver transplantation is an established treatment for acute and chronic liver disease. However, because of the shortage of donor organs, it does not fulfill the needs of all patients. Hepatocyte transplantation is promising as an alternative method for the treatment of end-stage liver disease and as bridging therapy until liver transplantation. Our group has been working on the optimization of matrix-based hepatocyte transplantation. In order to increase cell survival after transplantation, freshly isolated human hepatocytes were seeded onto biodegradable poly(l-lactic acid) (PLLA) polymer scaffolds and were cultured in a flow bioreactor. PLLA discs were seeded with human hepatocytes and exposed to a recirculated medium flow for 6 days. Human hepatocytes formed spheroidal aggregates with a liver-like morphology and active metabolic function. Phase contrast microscopy showed increasing numbers of spheroids of increasing diameter during the culture period. Hematoxylin and eosin histology showed viable and intact hepatocytes inside the spheroids. Immunohistochemistry confirmed sustained hepatocyte function and a preserved hepatocyte-specific cytoskeleton. Albumin, alpha-1-antitrypsin, and urea assays showed continued production during the culture period. Northern blot analysis demonstrated increasing albumin signals. Scanning electron micrographs showed hepatocyte spheroids with relatively smooth undulating surfaces and numerous microvilli. Transmission electron micrographs revealed intact hepatocytes and junctional complexes with coated pits and vesicles inside the spheroids. Therefore, we conclude that primary human hepatocytes, precultured in a flow bioreactor on a PLLA scaffold, reorganize to form morphologically intact liver neotissue, and this might offer an optimized method for hepatocyte transplantation because of the expected reduction of the initial cell loss, the high regenerative potential in vivo, and the preformed functional integrity.
肝移植是治疗急性和慢性肝病的一种成熟方法。然而，由于供体器官短缺，它不能满足所有患者的需求。肝细胞移植作为治疗终末期肝病的替代方法和肝移植前的桥接治疗具有广阔的前景。我们的研究小组一直在致力于优化基于基质的肝细胞移植。为了提高移植后的细胞存活率，我们将新鲜分离的人原代肝细胞接种到可生物降解的聚（L-丙交酯）（PLLA）聚合物支架上，并在流控生物反应器中进行培养。PLLA 载玻片上接种人原代肝细胞，并暴露于循环培养液流中 6 天。人原代肝细胞形成具有类似肝脏形态和活跃代谢功能的球形聚集体。相差显微镜显示，在培养期间，聚集体的数量和直径都在增加。苏木精和伊红组织学显示聚集体内有存活且完整的肝细胞。免疫组织化学证实了持续的肝细胞功能和保留的肝细胞特异性细胞骨架。白蛋白、α-1-抗胰蛋白酶和尿素测定显示在培养期间持续产生。Northern blot 分析显示白蛋白信号增加。扫描电子显微镜显示肝细胞聚集体具有相对平滑的起伏表面和大量微绒毛。透射电子显微镜显示了完整的肝细胞和带有有被小泡和陷窝的连接复合体。因此，我们得出结论，经过预培养的人原代肝细胞在 PLLA 支架上流控生物反应器中可以重新组织形成形态完整的肝组织，这可能提供了一种优化的肝细胞移植方法，因为预计会减少初始细胞丢失、体内高再生潜能和预先形成的功能完整性。
