Mai Gang, Nguyen Tuan Huy, Morel Philippe, Mei Jie, Andres Axel, Bosco Domenico, Baertschiger Reto, Toso Christian, Berney Thierry, Majno Pietro, Mentha Gilles, Trono Didier, Buhler Leo H
Surgical Research Unit, Department of Surgery, University Hospital Geneva, Switzerland.
Xenotransplantation. 2005 Nov;12(6):457-64. doi: 10.1111/j.1399-3089.2005.00248.x.
The aim of this study was to evaluate in vitro and in vivo functions of isolated hepatocytes after immortalization, cryopreservation, encapsulation and xenotransplantation into mice with fulminant liver failure (FLF).
Rat and human hepatocytes were isolated from normal liver tissue by collagenase digestion. Human hepatocytes were immortalized using lentiviral vectors coding for SV 40 large T antigen, Bmi-1 and telomerase. Rat and immortalized human hepatocytes (IHH) were encapsulated in 400 micron alginate-PLL-alginate membranes and cryopreserved using a computerized device. In vitro, encapsulated hepatocytes (cryopreserved or freshly isolated) were cultured in albumin-free medium and albumin production was measured by enzyme-linked immunosorbent assay (ELISA). In vivo, a model of FLF was established in C57/BL6 mice by acetaminophen administration (700 mg/kg i.p.) followed 15 h later by a 30% hepatectomy. Microencapsulated (cryopreserved or freshly isolated) hepatocytes were transplanted intraperitoneally to mice with FLF and the following experimental groups were performed: group 1 (n = 10) Tx of empty capsules; group 2 (n = 12) Tx of free primary rat hepatocytes; group 3 (n = 12) Tx of cryopreserved encapsulated rat hepatocytes; group 4 (n = 10) Tx of fresh encapsulated rat hepatocytes; group 5 (n = 9) Tx of cryopreserved encapsulated IHH; group 6 (n = 10) Tx of fresh encapsulated IHH. Animals were killed at regular intervals and histopathology of microcapsules and liver tissue was obtained.
In vitro, cryopreserved or fresh encapsulated rodent hepatocytes showed a progressively decreasing albumin secretion over 1 week in culture. In contrast, cryopreserved or fresh encapsulated IHH showed minimal, but stable albumin secretion. In vivo, FLF was achieved by combination of acetaminophen with 30% hepatectomy, resulting in a reproducible survival of 23% +/- 5%. In groups 1 and 2, survival rates were not improved significantly compared with untreated mice. In groups 3 and 4, Tx of cryopreserved or fresh encapsulated rat hepatocytes significantly increased survival rate to 66% and 80%, respectively (P < 0.01). In groups 5 and 6, Tx of cryopreserved or fresh encapsulated IHH improved survival to 50% and 55%, respectively (P < 0.05). Histopathology revealed that encapsulated hepatocytes were viable up to 2 weeks post-Tx.
Primary rodent hepatocytes maintained synthetic functions after encapsulation and cryopreservation short-term. IHH showed minimal albumin secretion in the absence of encapsulation and cryopreservation, suggesting that hepatocytes loose specific functions after immortalization. After induction of FLF in mice, intraperitoneal Tx of encapsulated (primary or immortalized, fresh or cryopreserved) xenogeneic hepatocytes significantly improved survival. These results indicate that naïve and genetically modified hepatocytes can successfully be encapsulated, stored using cryopreservation, and be transplanted into xenogeneic recipients with liver failure and sustain liver metabolic functions.
本研究旨在评估永生化、冷冻保存、封装及异种移植到暴发性肝衰竭（FLF）小鼠体内后分离的肝细胞的体外和体内功能。
通过胶原酶消化从正常肝组织中分离大鼠和人肝细胞。使用编码SV 40大T抗原、Bmi-1和端粒酶的慢病毒载体使人肝细胞永生化。将大鼠和永生化人肝细胞（IHH）封装在400微米的海藻酸盐-聚赖氨酸-海藻酸盐膜中，并使用计算机控制的设备进行冷冻保存。体外，将封装的肝细胞（冷冻保存的或新鲜分离的）在无白蛋白培养基中培养，并通过酶联免疫吸附测定（ELISA）测量白蛋白产生。体内，通过腹腔注射对乙酰氨基酚（700 mg/kg）建立C57/BL6小鼠FLF模型，15小时后进行30%肝切除术。将微封装的（冷冻保存的或新鲜分离的）肝细胞腹腔内移植到FLF小鼠体内，并进行以下实验组：第1组（n = 10）为空胶囊移植组；第2组（n = 12）为游离原代大鼠肝细胞移植组；第3组（n = 12）为冷冻保存的封装大鼠肝细胞移植组；第4组（n = 10）为新鲜封装的大鼠肝细胞移植组；第5组（n = 9）为冷冻保存的封装IHH移植组；第6组（n = 10）为新鲜封装的IHH移植组。定期处死动物，获取微胶囊和肝组织的组织病理学结果。
体外，冷冻保存的或新鲜封装的啮齿动物肝细胞在培养1周内白蛋白分泌逐渐减少。相比之下，冷冻保存的或新鲜封装的IHH白蛋白分泌极少，但稳定。体内，对乙酰氨基酚与30%肝切除术联合导致FLF，可重复性生存率为23%±5%。在第1组和第2组中，与未治疗的小鼠相比，生存率没有显著提高。在第3组和第4组中，冷冻保存的或新鲜封装的大鼠肝细胞移植分别使生存率显著提高到66%和80%（P < 0.01）。在第5组和第6组中，冷冻保存的或新鲜封装的IHH移植分别使生存率提高到50%和55%（P < 0.05）。组织病理学显示，封装的肝细胞在移植后2周内仍存活。
原代啮齿动物肝细胞在封装和短期冷冻保存后仍保持合成功能。IHH在未进行封装和冷冻保存时白蛋白分泌极少，表明肝细胞永生化后失去了特定功能。在小鼠诱导FLF后，腹腔内移植封装的（原代或永生化、新鲜或冷冻保存的）异种肝细胞显著提高了生存率。这些结果表明，未处理的和基因修饰的肝细胞能够成功地进行封装、冷冻保存，并移植到异种肝衰竭受体中，维持肝脏代谢功能。
