An improved method for isolation of mouse primary hepatic stellate cells with high yield and purity

doi:10.3969/j.issn.2095-4344.0903

Chinese Journal of Tissue Engineering Research ›› 2018, Vol. 22 ›› Issue (21): 3376-3380.doi: 10.3969/j.issn.2095-4344.0903

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An improved method for isolation of mouse primary hepatic stellate cells with high yield and purity

Xu Ying¹, Zhang Ding-qi^{1, 2}, Mu Yong-ping¹, Chen Jia-mei¹, Wang Qing-lan², Ping Jian¹, Liu Wei¹, Liu Ping^{1, 2}

¹Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Key Laboratory of Liver and Kidney Disease (Ministry of Education), Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China; ²E-Institute of Traditional Chinese Internal Medicine, Shanghai Municipal Education Commission, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

Revised:2018-03-13 Online:2018-07-28 Published:2018-07-28
Contact: Liu Ping, M.D., Professor, Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Key Laboratory of Liver and Kidney Disease (Ministry of Education), Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China; E-Institute of Traditional Chinese Internal Medicine, Shanghai Municipal Education Commission, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China； Liu Wei, M.D., Assistant researcher, Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Key Laboratory of Liver and Kidney Disease (Ministry of Education), Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China
About author:Xu Ying, Doctorate candidate, Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Key Laboratory of Liver and Kidney Disease (Ministry of Education), Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China
Supported by:
the Key Project of National Natural Science Foundation of China, No. 81530101; National Natural Science Foundation of China, No. 81703681; Shanghai Sailing Program, No. 17YF1419800; China Postdoctoral Science Foundation, No. 2016M601643

Abstract

Abstract:

BACKGROUND: Due to the small liver and fine blood vessels in the mice, it is difficult to isolate primary hepatic stellate cells, resulting in a limited yield which cannot meet the needs of in vitro experiments.
OBJECTIVE: To explore the high-performance and stable method for isolation of mouse primary hepatic stellate cells, with high yield and purity, thereby laying an experimental foundation for studying the mechanism of hepatic fibrosis.
METHODS: Adult male C57BL/6 mice undertook liver infusion with prefusion solution with no calcium and magnesium ions via the portal vein, followed by digestion using 1 g/L pronase and 0.7 g/L collagenase. Then, the liver samples were centrifuged with Nycodenz density gradient solution to obtain primary hepatic stellate cells from the middle layer. Trypan blue staining method was used to calculate cell viability, and flow cytometry method was used to measure cell purity. Immunofluorescence method was used to detect the expression of Desmin in cells. Cell morphology was observed under inverted microscope at 3, 5, 7 days of primary culture, and the expression of α-smooth muscle actin was detected by PCR.
RESULTS AND CONCLUSION: The yield of cells per mouse was (5.5±0.4)×10⁶ cells. Trypan blue assay showed that the viability of isolated cells was over 95%, and the purity of isolated cells was 94% as measured by the flow cytometry. The cells highly expressed Desmin by immunofluorescence. With the prolongation of culture time in vitro, round cells were gradually changed into star-shaped cells, and the expression of α-smooth muscle actin and type I collagen gradually increased. This study successfully established the high-performance and stable method for isolation of mouse primary hepatic stellate cells, providing a technical scheme for obtaining high-purity and high-viability mouse primary hepatic stellate cells, and also providing a technical support for research on the mechanism of liver fibrosis.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Hepatocytes, Cell Separation, Tissue Engineering

CLC Number:

R394.2

Xu Ying, Zhang Ding-qi, Mu Yong-ping, Chen Jia-mei, Wang Qing-lan, Ping Jian, Liu Wei, Liu Ping. An improved method for isolation of mouse primary hepatic stellate cells with high yield and purity[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3376-3380.

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An improved method for isolation of mouse primary hepatic stellate cells with high yield and purity

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