Aoki Takeshi, Koizumi Tomotake, Kobayashi Yasuna, Yasuda Daisuke, Izumida Yoshihiko, Jin Zhenghao, Nishino Nobukazu, Shimizu Yoshinori, Kato Hirohisa, Murai Noriyuki, Niiya Takashi, Enami Yuta, Mitamura Keitaro, Yamamoto Toshihiro, Kusano Mitsuo
Second Department of Surgery, School of Medicine, Showa University, Tokyo, Japan.
Cell Transplant. 2005;14(9):609-20. doi: 10.3727/000000005783982710.
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 1 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.
封装肝细胞移植是一种很有前景的细胞移植方法，无需免疫抑制，可作为全器官肝移植的替代方案。然而，肝细胞移植供体细胞短缺的问题尚未得到解决，在这一关键节点，建立一种肝细胞冷冻保存方法似乎很有必要，以便在不久的将来实现肝细胞移植的临床应用和生物人工肝系统的发展。在本研究中，我们证明了冷冻保存的微囊化大鼠和人肝细胞能够保留其肝功能，并且将冷冻保存的微囊化人肝细胞移植到大鼠脾脏中在无需免疫抑制的情况下仍能存活。大鼠和人肝细胞通过胶原酶消化法分离，并用聚-L-赖氨酸进行微囊化。将微囊化的大鼠肝细胞转移到培养基（含10%胎牛血清和10%二甲基亚砜的DMEM）中，立即在液氮中冷冻。用温水浴（37℃）解冻微囊化肝细胞。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。冷冻保存30天后评估人肝细胞的形态。将冷冻保存的人肝细胞移植到大鼠脾脏中以评估其形态。冷冻保存的微囊化肝细胞保持了活力，并且白蛋白、OAT2、CYP3A2和CYP3A9的表达呈强阳性。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至少30天保留其肝功能。此外，植入的冷冻保存的人肝细胞在移植后也在大鼠脾脏中存活并表达白蛋白。我们展示了一种通过封装技术对大鼠和人肝细胞进行长期冷冻保存的新方法，冷冻保存的微囊化人肝细胞移植到大鼠脾脏后保留了生物活性并具有良好的存活率。我们相信，这种肝细胞冷冻保存的新方法为封装细胞治疗提供了一个新方向，目标是在不久的将来实现临床应用。
