A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation
A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation
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DOI：
10.3727/000000005783982710
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发表时间：
2005-01-01
影响因子：
3.3
通讯作者：
Kusano, M
中科院分区：
文献类型：
--
作者：
Aoki, T;Koizumi, T;Kusano, M
微囊化肝细胞移植是一种无免疫抑制的细胞移植方法，可替代全器官肝移植。然而，肝细胞移植供体细胞的短缺问题尚未得到解决，在这个关键时刻，似乎有必要建立一种肝细胞冷冻保存的方法，使肝细胞移植的临床应用和生物人工肝系统的发展在不久的将来。在这项研究中，我们证明了冷冻保存的微囊化大鼠和人肝细胞可以保留其肝功能，冷冻保存的微囊化人肝细胞移植到大鼠脾脏中仍然是可行的，没有免疫抑制。采用胶原酶消化法分离大鼠和人肝细胞，并将其用聚-L-赖氨酸微囊化。将微囊化的大鼠肝细胞转移至培养基（含10%FBS和10%DMSO的DMEM）中，并立即在液氮中冷冻。使用温水浴（37 ℃）解冻微囊化肝细胞。在冷冻保存90天后评估肝功能、药物代谢和细胞形态。冷冻保存1周后，将微囊化肝细胞培养长达2周，以评估其肝功能和形态。在冷冻保存30天后评估人肝细胞的形态。将冻存的人肝细胞移植到大鼠脾脏中以评估其形态学。冷冻保存的微囊化肝细胞保持其活力，并且白蛋白、OAT 2、CYP 3A 2和CYP 3A 9表达呈强阳性。培养两周后，冷冻保存的微囊化大鼠肝细胞保留了其肝功能（尿素合成）。冷冻保存的微囊化人肝细胞在冷冻保存后也主要存活并保留其肝功能至少30天。此外，包埋冻存的人肝细胞移植后在大鼠脾脏中也存活并表达白蛋白。我们证明了一种新的方法，长期冷冻保存大鼠和人肝细胞，通过使用封装技术，与冷冻保存的微囊化人肝细胞移植到大鼠脾脏的生物活性和良好的生存保留。我们相信，这种新的方法来冻存肝细胞提供了一个新的方向，在包囊细胞治疗的临床应用的目标在不久的将来。
Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After I week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.