成人脂肪肝整肝肝细胞分离及大量冻存的实验研究

肝脏 ›› 2017, Vol. 22 ›› Issue (5): 410-413.

成人脂肪肝整肝肝细胞分离及大量冻存的实验研究

李涛,佟辉,申川,祝哲诚

200025 上海交通大学附属瑞金医院肝移植中心

出版日期:2017-05-30 发布日期:2017-05-30
通讯作者: 祝哲诚,Email:transplant@126.com

Isolation and cryopreservation of human hepatocytes from adult whole liver with steatosis

LI Tao, TONG Hui, SHEN Chuan, ZHU Zhe-cheng

Liver Transplantation Center, Ruijin Hospital affiliated to Medical College of Shanghai Jiaotong University, Shanghai 200025, China

Online:2017-05-30 Published:2017-05-30
Contact: ZHU Zhe-cheng, Email: transplant@126.com

摘要/Abstract

摘要： 目的建立成人脂肪肝整肝肝细胞的分离以及人肝细胞大量冻存技术,为生物人工肝提供稳定的人肝细胞来源。方法采用胶原酶经肝静脉逆行灌注的方法分离成人重度脂肪肝的整肝肝细胞,并比较常规冻存和程序冻存后肝细胞在细胞活性、贴壁率、LDH漏出量及白蛋白合成能力的差异。结果采用添加N-乙酰半胱氨酸(NAC)的胶原酶灌注液分离肝细胞的产量为(7.4±0.5)×10⁶ cells/g肝组织,活性为(81.4±3.4)%,而未添加NAC组的肝细胞产量为(5.6±0.8)×10⁶ cells/g肝组织和活性为(67.3±5.0)%,差异均有统计学意义(P<0.05)。分离的人肝细胞采用程序冻存后在细胞活性、贴壁率及白蛋白合成能力方面均相应高于常规冻存组(P<0.05),LDH漏出量低于常规冻存组(P<0.05)。结论应用添加NAC的胶原酶灌注液经肝静脉逆行灌注可提高脂肪肝整肝肝细胞分离的活性及产量,采用程序冻存的方法可以提高冻存人肝细胞的活性,满足生物人工肝对肝细胞的需要。

关键词: 人肝细胞, 分离, 培养, 冻存, 生物人工肝

Abstract: Objective To isolate and cryopreserve human hepatocytes from adult whole liver with heavy steatosis to provide human hepatocytes source for bio-artificial liver. Methods Primary human hepatocytes were isolated using a modified two-step collagenase retrograde perfusion via hepatic vein from adult whole livers with heavy steatosis. The isolated hepatocytes were under programmed and standard cryopreservation, respectively. The viability, adhesion rate, lactate dehydrogenase (LDH) releasing level and albumin synthesis ability were compared between the thawed hepatocytes with the 2 different cryopreservation methods.Results The average yield and viability of hepatocytes from livers perfused with N-acetylcysteine (NAC) was significantly higher than that without NAC [(7.4±0.5)×10⁶ cells/g vs. (5.6±0.8)×10⁶ cells/g and (81.4±3.4)%vs. (67.3±5.0)%, both P<0.05], respectively. Compared with hepatocytes under standard cryopreservation, hepatocytes under programmed cryopreservation showed significantly higher in the viability, adhesion rate, LDH release level and albumin synthesis ability (all P<0.05), respectively.Conclusion A modified two-step retrograde perfusion using collagenase with the NAC via hepatic vein could improve the yield and viability of isolated hepatocytes from adult whole livers with heavy steatosis. Besides, the programmed cryopreservation program could improve the quality of the isolated hepatocytes to meet the grossing need of hepatocyte for the bio-artificial liver.

Key words: Human hepatocytes, Isolation, Cryopreservation, Bioartificial liver

李涛,佟辉,申川,祝哲诚. 成人脂肪肝整肝肝细胞分离及大量冻存的实验研究[J]. 肝脏, 2017, 22(5): 410-413.

LI Tao, TONG Hui, SHEN Chuan, ZHU Zhe-cheng. Isolation and cryopreservation of human hepatocytes from adult whole liver with steatosis[J]. Chinese Hepatolgy, 2017, 22(5): 410-413.

参考文献 14

[1]	van Wenum M, Adam AA, Hakvoort TB, et al. Selecting cells for bioartificial liver devices and the importance of a 3D culture environment: a functional comparision between the HepaRG and C3A cell lines. Int J Biol Sci, 2016,12: 964-978.
[2]	Carpentier B, Gautier A, Legallais C. Artificial and bioartificial liver devices: present and future. Gut, 2009, 58: 1690-1702.
[3]	Darr TB, Hubel A. Postthaw viability of precultured hepatocytes. Cryobiology, 2001, 41: 11-20.
[4]	Alexandrova K, Griesel C, Barthold M, et al. Large-scale isolation of human hepatocytes for therapeutic application. Cell Transplant, 2005, 14: 845-853.
[5]	Ibars EP, Cortes M, Tolosa L, et al. Hepatocytes transplantation program: lessons and learned and future strategies. World J Gastroenterol, 2016, 22: 874-886.
[6]	Sagias FG, Mitry RR, Hughes RD, et al. N-acetylcysteine improves the viability of human hepatocytes isolated from severely steatotic donor liver tissue. Cell Transplant, 2010, 19:1487-1492.
[7]	李涛,彭志海,孙星,等. 经肝静脉与门静脉离体灌注分离猪肝细胞的比较研究. 肝脏,2005,10:22-23.
[8]	李涛,祝哲诚,谢俊杰,等. 用于生物人工肝的大量猪肝细胞低温冻存技术. 肝脏,2013,12: 812-814.
[9]	Bhogal RH, Hodson J, Bartlett DC, et al. Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience. Plos One, 2011, 6: e18222.
[10]	Alexandre E, Cahn M, Abadie-Viollon C, et al. Influence of pre-, intra- and post-operative parameters of donor liver on the outcome of isolated human hepatocytes. Cell Tissue Bank, 2002, 3:223-233.
[11]	Terry C, Mitry RR, Lehec SC, et al. The effects of cryopreservation on human hepatocytes obtained from different sources of liver tissue. Cell Transplant, 2005, 14: 585-594.
[12]	Terry C, Dhawan A, Mitry RR, et al. Cryopreservation of isolated human hepatocytes for transplantation: state of the art. Cryobiology, 2006, 53: 149-159. Hewitt NJ, Li AP. Cryopreservation of hepatocytes. Methods Mol Biol, 2015, 1250: 13-26.
[13]	Terry C, Dhawan A, Mitry RR, et al. Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation. Liver Transpl, 2010, 16: 229-237.
[14]	Stéphenne X, Najimi M, Sokal EM. Hepatocyte cryopreservation: is it time to change the strategy World J Gastroenterol, 2010, 16: 1-14.