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| Isolation and culture of hepatocytes and sinusoidal endothelial cells and preparation of hepatocyte scaffolds |
| CHEN Shizuan, YU Fuxiang, CHEN Junyu, TANG Yinhe |
| Department of Hepatobiliary Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China |
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| Cite this article: |
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CHEN Shizuan,YU Fuxiang,CHEN Junyu, et al. Isolation and culture of hepatocytes and sinusoidal endothelial cells and preparation of hepatocyte scaffolds[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2019, 49(9): 630-633.
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Abstract Objective: To explore the methods of isolation and culture of rat hepatocytes and sinusoidal endothelial cells and preparation of hepatic acellular scaffolds. Methods: Rat hepatocytes isolated from liver tissues were digested by collagenase IV and purified in culture medium after washing, filtration and centrifugation. Cell immunohistochemistry was used to detect the cells after culture. Hepatic sinusoidal endothelial cells were isolated by Percoll density gradient centrifugation and purified in culture medium before they were detected by immunofluorescence. The scaffolds of rat hepatocyte acellular scaffolds were prepared by circulating perfusion and acellular treatment. The scaffolds were confirmed by scanning electron microscopy. Results: The isolated rat hepatocytes were round, full and clear under microscope. Immunohistochemical staining of cytokeratin 18 in hepatocytes revealed brown granules in the cytoplasm. Thehepatic sinusoidal endothelial cells were spindle-shaped and the nucleus was located in the center. Endothelin-1 immunofluorescence staining showed red signal on the cell surface. The scanning electron microscopy of the rat liver decellularized scaffold showed that the extracellular matrix was retained, and no nuclear and cytoplasmic components were found. Conclusion: Hepatocytes and sinusoidal endothelial cells are successfully isolated and hepatic acellular scaffolds were prepared.
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Received: 29 October 2018
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[1] KAJBAFZADEH A M, JAVAN-FARAZMAND N, MONAJEMZADEH M, et al. Determining the optimal decellularization and sterilization protocol for preparing a tissue scaffold of a human-sized liver tissue[J]. Tissue Eng Part C Methods, 2013, 19(8): 642-651.
[2] DUNNE L W, HUANG Z, MENG W, et al. Human decellularized adipose tissue scaffold as a model for breast cancer cell growth and drug treatments[J]. Biomaterials, 2014, 35(18): 4940-4949.
[3] JIANG W C, CHENG Y H, YEN M H, et al. Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering[J]. Biomaterials, 2014, 35(11): 3607-3617.
[4] 张晨曦, 卞勉励, 陈星燃, 等. 肝窦内皮细胞在肝脏疾病中的作用[J]. 中国药理学通报, 2017, 33(2): 149-152, 153.
[5] DING B S, NOLAN D J, BUTLER J M, et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration[J]. Nature, 2010, 468(7321): 310-315.
[6] XIE G, WANG X, WANG L, et al. Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats[J]. Gastroenterology, 2012, 142(4): 918-927.
[7] 吕文良, 徐晨光, 张莎莎, 等. 肝窦内皮细胞的分离培养及体外生长规律的观察[J]. 中华生物医学工程杂志, 2015, 21(1): 20-25.
[8] BREMER C M, BUNG C, KOTT N, et al. Hepatitis B virus infection is dependent on cholesterol in the viral envelope[J]. Cell Microbiol, 2009, 11(2): 249-260.
[9] CONSOLO F, FIORE G B, TRUSCELLO S, et al. A computational model for the optimization of transport phenomena in a rotating hollow-fiber bioreactor for artificial liver[J]. Tissue Eng Part C Methods, 2009, 15(1): 41-55.
[10] 牟卉卉, 胡康洪. 原代肝细胞分离培养技术现状及展望[J]. 世界华人消化杂志, 2009, 17(21): 2164-2170.
[11] 王红霞, 邓永键, 蒋强, 等. 裸鼠肝窦内皮细胞的分离、培养及鉴定[J]. 中国组织工程研究, 2012, 16(15): 2789-2792.
[12] FAULK D M, WILDEMANN J D, BADYLAK S F. Decellularization and cell seeding of whole liver biologic scaffolds composed of extracellular matrix[J]. J Clin Exp Hepatol, 2015, 5(1): 69-80.
[13] 黄俊杰, 王志斌, 陈纳, 等. 大网膜包埋移植肝脏去细胞生物支架血管化过程观察[J]. 温州医科大学学报, 2017, 47(10): 703-707.
[14] 蔡伟祥, 方建凤, 庄伟, 等. 分离、鉴定与培养大鼠肝细胞、
肝星状细胞和Kupffer细胞[J]. 温州医科大学学报, 2016, 46(9): 638-643, 648.
[15] 陈云志, 余华军, 孔鸿儒, 等. 一种新型大鼠胰腺去细胞化支架的制备研究[J]. 肝胆胰外科杂志, 2018, 30(2): 127-133. |
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