Construction of Biological Artificial Liver and Its Application in The Experimental Research_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 GUOEn-ling ,  KONGJing , LIUBin-bin , JIANGQing-quan

Department of Biliary Surgery, Affiliated Shengjing Hospital of China Medical University, Shenyang 110021, Liaoning Province, China;

Corresponding author：

KONGJing, Email: kongjing1998@163.com

Keywords：

Biological artificial liver; Hepatocyte; Isolation; Culture

DOI：

10.7507/1007-9424.20150296

Video：

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Objective To provide theoretical and technological support for further study of liver metabolism and disease by comparing the advantages and disadvantages of various artificial liver models (biological). Methods Literatures were searched and compared to summarize the requirement for liver donor, isolation, and culture of hepatocyte. Results In the separation method of hepatocyte, mechanical separation method had no requirement for liver donor, and was easy to acquire hepatocyte, while the acquired hepatocyte would be destructed severely, and the survival rate was low. On the other hand, the restriction of the digestion of the hepatocytes to the liver cell samples was unlimited, while the key of the enzyme digestion method was to regulate the balance between enzyme concentration and digestion time, which was limited to function researches of hepatocyte, and research about the responds of hepatocyte against outside, and other few researches. Perfusion digestion method had been widely applied for animal test. The Ca²⁺, collagenase and perfusion rate, pH value, buffer, and intubation method all play vital roles. During the cultivation, we needed to choose different methods according to several experiments, and add different additives in the appropriate medium. Different biological reactors had different advantages, disadvantages, and applicable conditions. Conclusions The donor selection is based on various experimental purposes to harvest hepatocytes from different sources. Whether on the separation process or on the cultivation process, according to the specific circumstances, such as the concentration, perfusion time, and the choice of different kinds of culture medium, we can choose different kinds of bioreactors, but all kinds of methods are still remained with multiple insufficiencies, which require more researchers to improve.

Citation： GUOEn-ling, KONGJing, LIUBin-bin, JIANGQing-quan. Construction of Biological Artificial Liver and Its Application in The Experimental Research. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2015, 22(9): 1137-1142. doi: 10.7507/1007-9424.20150296 Copy

1.	Naik S, Santangini HA, Trenkler DM, et al. Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems. Cell Transplant, 1997, 6(5):447-454.
2.	Donato MT, Castell JV, Gómez-Lechón MJ. Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices:comparison with other hepatic cellular models. J Hepatol, 1999, 31(3):542-549.
3.	崔杨. 生物人工肝的几种细胞来源. 透析与人工器官, 2005, 16(4):32-36.
4.	Hillis WD, Bang FB. The cultivation of human embryonic liver cells. Exp Cell Res, 1962, 26:9-36.
5.	陈慧梅, 廖红, 高静. 肝细胞培养方法研究进展. 细胞生物学杂志, 2002, 24(3):163-166.
6.	龚建平, 韩本立. 肝脏细胞的分离、培养和鉴定技术. 世界华人消化杂志, 1999, 7(5):417-419.
7.	Howard RB, Christensen AK, Gibbs FA, et al. The enzymatic preparation of isolated intact parenchymal cells from rat liver. J Cell Biol, 1967, 35(3):675-684.
8.	Berry MN, Friend DS. High-yield preparation of isolated rat liver parenchymal cells:a biochemical and fine structural study. J Cell Biol, 1969, 43(3):506-520.
9.	Berry MN, Grivell AR, Grivell MB, et al. Isolated hepatocytes-past, present and future. Cell Biol Toxicol, 1997, 13(4-5):223-233.
10.	Sudhakaran PR, Stamatoglou SC, Hughes RC. Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes. Exp Cell Res, 1986, 167(2):505-516.
11.	Shen L, Hillebrand A, Wang DQ, et al. Isolation and primary culture of rat hepatic cells. J Vis Exp, 2012, 29(64):3917.
12.	Edwards M, Houseman L, Phillips IR, et al. Isolation of mouse hepatocytes. Methods Mol Biol, 2013, 987:283-293.
13.	Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol, 1976, 13(1):29-83.
14.	Smedsrød B, Pertoft H, Eggertsen G, et al. Functional and morpho-logical characterization of cultures of Kupffer cells and liver endothe-lial cells prepared by means of density separation in Percoll, and selective substrate adherence. Cell Tissue Res, 1985, 241(3):639-649.
15.	Ishii M, Vroman B, LaRusso NF. Isolation and morphologic charac-terization of bile duct epithelial cells from normal rat liver. Gastroen-terology, 1989, 97(5):1236-1247.
16.	Ouji Y, Yoshikawa M, Moriya K, et al. Isolation and characterization of murine hepatocytes following collagenase infusion into left ventricle of heart. J Biosci Bioeng, 2010, 110(4):487-490.
17.	余招焱, 白燕南, 张涛, 等. 经心脏逆行置管灌注法在小鼠原代肝细胞分离中的应用. 四川大学学报:医学版, 2014, 45(1):138-141.
18.	Marion TL, Perry CH, St Claire RL 3rd, et al. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes. Toxicol Appl Pharmacol, 2012, 261(1):1-9.
19.	周星辉, 王丙力, 谭焕然. 大鼠肝实质细胞原代培养模型的研究及其功能鉴定. 中国临床药理学与治疗学, 2005, 10(7):743-746.
20.	Ismail T, Howl J, Wheatley M, et al. Growth of normal human hepatocytes in primary culture:effect of hormones and growth factors on DNA synthesis. Hepatology, 1991, 14(6):1076-1082.
21.	Farivar M, Wands JR, Isselbacher KJ, et al. Effect of insulin and glucagon on fulminant murine hepatitis. N Engl J Med, 1976, 295(27):1517-1519.
22.	王贵民, 王忠毅, 谭毓铨. 地塞米松对单层培养的大鼠肝细胞分泌机能的影响. 中华肝胆外科杂志, 2003, 9(8):50-53.
23.	殷海涛, 滕皋军, 刘宝瑞. 夹层胶原"三明治"法短期体外培养肝细胞. 江苏医药, 2005, 31(1):36-39.
24.	Liu ML, Mars WM, Zarnegar R, et al. Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver. Hepatology, 1994, 19(6):1521-1527.
25.	Lang R, Stern MM, Smith L, et al. Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix. Biomaterials, 2011, 32(29):7042-7052.
26.	Reid LM, Jefferson DM. Culturing hepatocytes and other differen-tiated cells. Hepatology, 1984, 4(3):548-559.
27.	Koike M, Matsushita M, Taguchi K, et al. Function of culturing monolayer hepatocytes by collagen gel coating and coculture with nonparenchymal cells. Artif Organs, 1996, 20(2):186-192.
28.	Dabeva MD, Petkov PM, Sandhu J, et al. Proliferation and differen-tiation of fetal liver epithelial progenitor cells after transplantation into adult rat liver. Am J Pathol, 2000, 156(6):2017-2031.
29.	Choi HJ, Choi D. Successful mouse hepatocyte culture with sandwich collagen gel formation. J Korean Surg Soc, 2013, 84(4):202-208.
30.	Taguchi K, Matsushita M, Takahashi M, et al. Development of a bioartificial liver with sandwiched-cultured hepatocytes between two collagen gel layers. Artif Organs, 1996, 20(2):178-185.
31.	张先杰, 孙家邦, 李铎, 等. 三明治构型培养肝细胞——一种潜在的生物人工肝单位. 中华肝胆外科杂志, 2001, 7(5):292-294.
32.	Komoroski BJ, Zhang S, Cai H, et al. Induction and inhibition of cytochromes P450 by the St. John,s wort constituent hyperforin in human hepatocyte cultures. Drug Metab Dispos, 2004, 32(5):512-518.
33.	Cai Y, Gong LK, Qi XM, et al. Apoptosis initiated by carbon tetra-chloride in mitochondria of rat primary cultured hepatocytes. Acta Pharmacol Sin, 2005, 26(8):969-975.
34.	张瑞, 韩宝三, 吴薇, 等. 微载体及其在肝细胞培养中的作用与应用. 中国组织工程研究与临床康复, 2008, 12(32):6331-6334.
35.	Clark JM, Hirtenstein MD. Optimizing culture conditions for the production of animal cells in microcarrier culture. Ann N Y Acad Sci, 1981, 369:33-46.
36.	匡洁, 彭承宏, 韩宝三, 等. 微囊包载体肝细胞培养方式对肝细胞功能及活性的影响. 外科理论与实践, 2010, 15(5):531-535.
37.	Yalpani M, Hall LD. Some chemical and analytical aspects of poly-saccharide modifications. Ⅲ. Formation of branched-chain, soluble chitosan derivatives. Macromolecules, 1984, 17(3):272-281.
38.	Nam JH, Ermonval M, Sharfstein ST. Cell attachment to microcar-riers affects growth, metabolic activity, and culture productivity in bioreactor culture. Biotechnol Prog, 2007, 23(3):652-660.
39.	Seo SJ, Akaike T, Choi YJ, et al. Alginate microcapsules prepared with xyloglucan as a synthetic extracellular matrix for hepatocyte attachment. Biomaterials, 2005, 26(17):3607-3615.
40.	陈杰, 彭承宏, 沈柏用, 等. 肝细胞体外培养技术的研究与进展. 中国组织工程研究与临床康复, 2008, 12(53):10539-10542.
41.	李兰娟. 人工肝脏. 第2版. 杭州:浙江大学出版社, 2012:379-392.
42.	丛庆伟, 朱英. 人工肝支持系统治疗肝衰竭的研究进展. 医学与哲学, 2014, 35(8B):70-73.

1. Naik S, Santangini HA, Trenkler DM, et al. Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems. Cell Transplant, 1997, 6(5):447-454.
2. Donato MT, Castell JV, Gómez-Lechón MJ. Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices:comparison with other hepatic cellular models. J Hepatol, 1999, 31(3):542-549.
3. 崔杨. 生物人工肝的几种细胞来源. 透析与人工器官, 2005, 16(4):32-36.
4. Hillis WD, Bang FB. The cultivation of human embryonic liver cells. Exp Cell Res, 1962, 26:9-36.
5. 陈慧梅, 廖红, 高静. 肝细胞培养方法研究进展. 细胞生物学杂志, 2002, 24(3):163-166.
6. 龚建平, 韩本立. 肝脏细胞的分离、培养和鉴定技术. 世界华人消化杂志, 1999, 7(5):417-419.
7. Howard RB, Christensen AK, Gibbs FA, et al. The enzymatic preparation of isolated intact parenchymal cells from rat liver. J Cell Biol, 1967, 35(3):675-684.
8. Berry MN, Friend DS. High-yield preparation of isolated rat liver parenchymal cells:a biochemical and fine structural study. J Cell Biol, 1969, 43(3):506-520.
9. Berry MN, Grivell AR, Grivell MB, et al. Isolated hepatocytes-past, present and future. Cell Biol Toxicol, 1997, 13(4-5):223-233.
10. Sudhakaran PR, Stamatoglou SC, Hughes RC. Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes. Exp Cell Res, 1986, 167(2):505-516.
11. Shen L, Hillebrand A, Wang DQ, et al. Isolation and primary culture of rat hepatic cells. J Vis Exp, 2012, 29(64):3917.
12. Edwards M, Houseman L, Phillips IR, et al. Isolation of mouse hepatocytes. Methods Mol Biol, 2013, 987:283-293.
13. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol, 1976, 13(1):29-83.
14. Smedsrød B, Pertoft H, Eggertsen G, et al. Functional and morpho-logical characterization of cultures of Kupffer cells and liver endothe-lial cells prepared by means of density separation in Percoll, and selective substrate adherence. Cell Tissue Res, 1985, 241(3):639-649.
15. Ishii M, Vroman B, LaRusso NF. Isolation and morphologic charac-terization of bile duct epithelial cells from normal rat liver. Gastroen-terology, 1989, 97(5):1236-1247.
16. Ouji Y, Yoshikawa M, Moriya K, et al. Isolation and characterization of murine hepatocytes following collagenase infusion into left ventricle of heart. J Biosci Bioeng, 2010, 110(4):487-490.
17. 余招焱, 白燕南, 张涛, 等. 经心脏逆行置管灌注法在小鼠原代肝细胞分离中的应用. 四川大学学报:医学版, 2014, 45(1):138-141.
18. Marion TL, Perry CH, St Claire RL 3rd, et al. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes. Toxicol Appl Pharmacol, 2012, 261(1):1-9.
19. 周星辉, 王丙力, 谭焕然. 大鼠肝实质细胞原代培养模型的研究及其功能鉴定. 中国临床药理学与治疗学, 2005, 10(7):743-746.
20. Ismail T, Howl J, Wheatley M, et al. Growth of normal human hepatocytes in primary culture:effect of hormones and growth factors on DNA synthesis. Hepatology, 1991, 14(6):1076-1082.
21. Farivar M, Wands JR, Isselbacher KJ, et al. Effect of insulin and glucagon on fulminant murine hepatitis. N Engl J Med, 1976, 295(27):1517-1519.
22. 王贵民, 王忠毅, 谭毓铨. 地塞米松对单层培养的大鼠肝细胞分泌机能的影响. 中华肝胆外科杂志, 2003, 9(8):50-53.
23. 殷海涛, 滕皋军, 刘宝瑞. 夹层胶原"三明治"法短期体外培养肝细胞. 江苏医药, 2005, 31(1):36-39.
24. Liu ML, Mars WM, Zarnegar R, et al. Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver. Hepatology, 1994, 19(6):1521-1527.
25. Lang R, Stern MM, Smith L, et al. Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix. Biomaterials, 2011, 32(29):7042-7052.
26. Reid LM, Jefferson DM. Culturing hepatocytes and other differen-tiated cells. Hepatology, 1984, 4(3):548-559.
27. Koike M, Matsushita M, Taguchi K, et al. Function of culturing monolayer hepatocytes by collagen gel coating and coculture with nonparenchymal cells. Artif Organs, 1996, 20(2):186-192.
28. Dabeva MD, Petkov PM, Sandhu J, et al. Proliferation and differen-tiation of fetal liver epithelial progenitor cells after transplantation into adult rat liver. Am J Pathol, 2000, 156(6):2017-2031.
29. Choi HJ, Choi D. Successful mouse hepatocyte culture with sandwich collagen gel formation. J Korean Surg Soc, 2013, 84(4):202-208.
30. Taguchi K, Matsushita M, Takahashi M, et al. Development of a bioartificial liver with sandwiched-cultured hepatocytes between two collagen gel layers. Artif Organs, 1996, 20(2):178-185.
31. 张先杰, 孙家邦, 李铎, 等. 三明治构型培养肝细胞——一种潜在的生物人工肝单位. 中华肝胆外科杂志, 2001, 7(5):292-294.
32. Komoroski BJ, Zhang S, Cai H, et al. Induction and inhibition of cytochromes P450 by the St. John,s wort constituent hyperforin in human hepatocyte cultures. Drug Metab Dispos, 2004, 32(5):512-518.
33. Cai Y, Gong LK, Qi XM, et al. Apoptosis initiated by carbon tetra-chloride in mitochondria of rat primary cultured hepatocytes. Acta Pharmacol Sin, 2005, 26(8):969-975.
34. 张瑞, 韩宝三, 吴薇, 等. 微载体及其在肝细胞培养中的作用与应用. 中国组织工程研究与临床康复, 2008, 12(32):6331-6334.
35. Clark JM, Hirtenstein MD. Optimizing culture conditions for the production of animal cells in microcarrier culture. Ann N Y Acad Sci, 1981, 369:33-46.
36. 匡洁, 彭承宏, 韩宝三, 等. 微囊包载体肝细胞培养方式对肝细胞功能及活性的影响. 外科理论与实践, 2010, 15(5):531-535.
37. Yalpani M, Hall LD. Some chemical and analytical aspects of poly-saccharide modifications. Ⅲ. Formation of branched-chain, soluble chitosan derivatives. Macromolecules, 1984, 17(3):272-281.
38. Nam JH, Ermonval M, Sharfstein ST. Cell attachment to microcar-riers affects growth, metabolic activity, and culture productivity in bioreactor culture. Biotechnol Prog, 2007, 23(3):652-660.
39. Seo SJ, Akaike T, Choi YJ, et al. Alginate microcapsules prepared with xyloglucan as a synthetic extracellular matrix for hepatocyte attachment. Biomaterials, 2005, 26(17):3607-3615.
40. 陈杰, 彭承宏, 沈柏用, 等. 肝细胞体外培养技术的研究与进展. 中国组织工程研究与临床康复, 2008, 12(53):10539-10542.
41. 李兰娟. 人工肝脏. 第2版. 杭州:浙江大学出版社, 2012:379-392.
42. 丛庆伟, 朱英. 人工肝支持系统治疗肝衰竭的研究进展. 医学与哲学, 2014, 35(8B):70-73.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction of Biological Artificial Liver and Its Application in The Experimental Research

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