Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes
Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes
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DOI：
10.1016/j.transproceed.2005.01.017
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发表时间：
2005-01-01
影响因子：
0.9
通讯作者：
Buhler, LH
中科院分区：
文献类型：
--
作者：
Mai, G;Huy, NT;Buhler, LH
背景。本研究的目的是评估暴发性肝衰竭（FLF）小鼠永生化、冷冻保存、封装和异种移植后肝细胞的体外和体内功能。方法。通过胶原酶消化分离大鼠和人肝细胞。使用慢病毒载体使人肝细胞永生化。将大鼠和永生化人肝细胞封装在 400 μm 的藻酸盐-聚-L-赖氨酸（PLL；Sigma，Buchs，瑞士）-藻酸盐膜中，并使用计算机化设备冷冻保存。在体外培养封装的肝细胞（冷冻保存或非冷冻保存）；通过酶联免疫吸附测定法测量白蛋白分泌。将微囊（冷冻保存或非冷冻保存）肝细胞腹膜内移植到 FLF 小鼠体内：第 1 组（n = 10）移植空胶囊；第2组（n=12）游离原代大鼠肝细胞移植；第3组（n = 12）移植冷冻保存的封装大鼠肝细胞；第4组（n = 10）封装大鼠肝细胞移植；第 5 组 (n = 9) 移植冷冻保存的封装 IHH；第6组（n=10）移植封装结果。与游离原代肝细胞相比，冷冻或非冷冻封装的啮齿动物肝细胞在 1 周内表现出相似的连续体外白蛋白分泌水平。与游离的原代人肝细胞相比，冷冻保存或非冷冻保存的封装 IHH 显示出最少的白蛋白分泌。由对乙酰氨基酚和 30% 肝切除术联合产生的暴发性肝衰竭导致宿主存活率为 20% 至 30%。与未治疗的小鼠相比，第 1 组和第 2 组的存活率没有变化。对于第 3 组和第 4 组，移植冷冻或非冷冻保存的封装大鼠肝细胞可将存活率分别显着提高至 66% 和 80% (P < .01)。对于第 5 组和第 6 组，移植冷冻或非冷冻保存的封装 IHH 分别将宿主存活率提高至 50% 和 55% (P < .05)。结论。原代啮齿动物肝细胞在封装和冷冻保存后仍保持合成功能。在没有封装和冷冻保存的情况下，永生化人肝细胞显示出最少的白蛋白分泌，表明永生化后肝细胞失去了一些特定功能。在小鼠中诱导FLF后，腹膜内移植封装的（原代或永生化、冷冻保存或非冷冻保存）异种肝细胞显着提高了存活率。这些结果表明，天然肝细胞和转基因肝细胞可以成功封装、冷冻保存，并移植到具有FLF的异种受体中以维持肝脏代谢功能。
Background. The aim of this study was to evaluate the in vitro and in vivo function of hepatocytes after immortalization, cryopreservation, encapsulation, and xenotransplantation into mice with fulminant liver failure (FLF).Methods. Rat and human hepatocytes were isolated by collagenase digestion. Human hepatocytes were immortalized using lentiviral vectors. Rat and immortalized human hepatocytes were encapsulated in 400 mu m of alginate-poly-L-lysine (PLL; Sigma, Buchs, Switzerland)-alginate membranes and cryopreserved using a computerized device. In vitro, encapsulated hepatocytes (cryopreserved or noncryopreserved) were cultured; albumin secretion was measured by enzyme-linked immunosorbent assay. Microencapsulated (cryopreserved or noncryopreserved) hepatocytes were transplanted intraperitoneally to mice with FLF: group 1 (n = 10) transplantation of empty capsules; group 2 (n = 12) transplantation of free primary rat hepatocytes; group 3 (n = 12) transplantation of cryopreserved encapsulated rat hepatocytes; group 4 (n = 10) transplantation of encapsulated rat hepatocytes; group 5 (n = 9) transplantation of cryopreserved encapsulated IHH; group 6 (n = 10) transplantation of encapsulatedResults. Compared with free primary hepatocytes, cryopreserved or noncryopreserved encapsulated rodent hepatocytes showed similar levels of continuous in vitro albumin secretion over 1 week. Cryopreserved or noncryopreserved encapsulated IHH showed minimal albumin secretion compared with free primary human hepatocytes. Fulminant liver failure, produced by a combination of acetaminophen and 30% hepatectomy, resulted in a 20% to 30% host survival. In groups 1 and 2, survival was unmodified, compared with untreated mice. For groups 3 and 4, transplantation of cryopreserved or noncryopreserved encapsulated rat hepatocytes significantly increased survival rates to 66% and 80%, respectively (P < .01). For groups 5 and 6, transplantation of cryopreserved or noncryopreserved encapsulated IHH improved host survival to 50% and 55%, respectively (P < .05).Conclusions. Primary rodent hepatocytes maintained synthetic functions after encapsulation and cryopreservation. Immortalized human hepatocytes showed minimal albumin secretion in the absence of encapsulation and cryopreservation, suggesting that hepatocytes lose some specific functions after immortalization. After induction of FLF in mice, intraperitoneal transplantation of encapsulated (primary or immortalized, cryopreserved or noncryopreserved) xenogeneic hepatocytes significantly improved survival. These results indicate that naive and genetically modified hepatocytes can be successfully encapsulated, stored by cryopreservation, and transplanted into xenogeneic recipients with FLF to sustain liver metabolic functions.