Journal of Hebei Medical University ›› 2021, Vol. 42 ›› Issue (1): 7-11.doi: 10.3969/j.issn.1007-3205.2021.01.002

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Experimental study on isolation and culture of canine vascular endothelial cells by collagenase digestion scrape and repeat digestion techniques

  

  1. 1.Department of Thoracic and Cardiac Surgery, the Affiliated Hospital of Jiangsu University, Jiangsu 
    Province, Zhenjiang 212001, China; 2.Department of Thoracic and Cardiac Surgery, the Affiliated 
    Hospital of Jiangnan University, Jiangsu Province, Wuxi 214062, China; 3.Department of 
    Thoracic and Cardiac Surgery, Shanghai Ninth People′s Hospital of Shanghai 
    Jiaotong University, School of Medicine, Shanghai 200011,China
  • Online:2021-01-25 Published:2021-02-04

Abstract: Objective  To explore a novel culture method of vascular endothelial cells(ECs) in vitro from canine saphenous vein. 
Methods  Vascular inner membrane was inverted, and both ends of the vein were ligated, followed by digestion with 0.1% collagenase and mechanical scrape. Endothelial cells in canine saphenous vein were obtained by collagenase redigestion, and then cultured with low glucose DMEM medium. Appropriate amount of endothelial cell growth factor was then added for culture. Cells were cultured using 0.25% trypsin and then subcultured. The cultured cell lines were identified by immunofluorescence, immunohistochemistry, light and scanning electron microscopy. 
Results  The monolayer cells were formed at 14 days after culture in vitro. The ECs arrayed like pitching stone under light microscopy. By transmission electron microscopy, ECs were found spindle shaped and there were many microvilli on the cell surface; Ve-Cadherin in the ECs showed positive reaction by immunofluorescence and immunohistochemistry. Compared with the traditional Veronica Tisato separation method, the cell survival rate using this method was significantly improved [(75.39±12.61)% vs. (47.28±9.59)%, P<0.01]. 
Conclusion  Aplication of collagenase digestion, mechanical scrape and collagenase redigestion can reliably obtain purified ECs of canine saphenous vein, which could be proliferated and successively passaged in vitro.

Key words: great saphenous vein, endothelial cells, collagenase, Ve-Cadherin antigen