ISOLATION, CULTURE, AND IDENTIFICATION OF CANINE UMBILICAL VEIN VASCULAR ENDOTHELIAL CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HU Quan , CHAI Jiake , LIU Lingying , HOU Yusen , WANG Yihe , LI Bailing , YANG Hongming.

Burns Institute, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, 100048, P.R.China. Corresponding author: CHAI Jiake, E-mail: cjk304@126.com;

Corresponding author：

Keywords：

Vascular endothelial cells; Umbilical vein; Cell culture; Cell identification; Canine

DOI：

10.7507/1002-1892.20130105

Video：

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Objective To establish a simple and efficient method to isolate and culture the umbilical vein vascular endothelial cells in canine. Methods Twelve umbilical cords [(13.0 ± 1.5) cm in length] were taken from 12 newborn pups of Beagles. And then the vascular endothelial cells were isolated from these umbilical cords digested by 1% collagenase type I for 5, 7, and 10 minutes respectively (4 umbilical cords in each group). After cultured, the vascular endothelial cells were identified by morphology, immunofluorescence, and flow cytometry. And the growth curvature of umbilical vein vascular endothelial cells was detected by MTT assay. Results Few vascular endothelial cells were collected at 5 and 10 minutes after digestion; many vascular endothelial cells were seen at 7 minutes, and became cobblestone with culture time, with a large nucleus; after passage, cell morphology had no obvious change. Fluorescence microscope results showed that positive von Willebrand factor (vWF) and CD31 cells were observed in most of cells. The flow cytometry test displayed that the positive cell rates of vWF and CD31 were 99.0% ± 0.7% and 98.0% ± 1.2%, respectively. The above results indicated that cultured cells were vascular endothelial cells. MTT assay showed that vascular endothelial cells proliferation increased significantly with culture time. Conclusion Enzyme digestion is a convenient method to isolate vascular endothelial cells from canine umbilical vein, and a large number of cells and high purity of cells can be obtained by the method.

Citation： HU Quan,CHAI Jiake,LIU Lingying,HOU Yusen,WANG Yihe,LI Bailing,YANG Hongming.. ISOLATION, CULTURE, AND IDENTIFICATION OF CANINE UMBILICAL VEIN VASCULAR ENDOTHELIAL CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(4): 460-463. doi: 10.7507/1002-1892.20130105 Copy

1.	Shupp JW, Nasabzadeh TJ, Rosenthal DS, et al. A review of the local pathophysiologic bases of burn wound progression. J Burn Care Res, 2010, 31(6): 849-873.
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11.	黄起壬, 戴育成, 李剑, 等. 三种人脐静脉内皮细胞分离方法的比较. 江西医学院学报, 2005, 45(5): 8-14.
12.	Fehrenbach ML, Cao G, Williams JT, et al. Isolation of murine lung endothelial cells. Am J Physiol Lung Cell Mol Physiol, 2009, 296(6): L1096-1103.
13.	Jin Y, Liu Y, Antonyak M, et al. Isolation and characterization of vascular endothelial cells from murine heart and lung. Methods Mol Biol, 2012, 843: 147-154.
14.	张臣, 李彤, 侯跃龙, 等. 人脐静脉血管内皮细胞的高效分离与培养. 天津医药, 2010, 38(7): 605-607.
15.	Winiarski BK, Acheson N, Gutowski NJ. An improved and reliable method for isolation of microvascular endothelial cells from human omentum. Microcirculation, 2011, 18(8): 635-645.
16.	刘长乐, 郑心田, 李广平, 等. 体外人脐静脉内皮细胞的培养鉴定及抗原表达分析. 山东医药, 2011, 51(36): 25-26.
17.	Lip GY, Blann A. von Willebrand factor: a marker of endothelial dysfunction in vascular disorders? Cardiovasc Res, 1997, 34(2): 255-265.
18.	Horvath B, Hegedus D, Szapary L, et al. Measurement of von Willebrand factor as the marker of endothelial dysfunction in vascular diseases. Exp Clin Cardiol, 2004, 9(1): 31-34.
19.	Janeczek Portalska K, Leferink A, Groen N, et al. Endothelial differentiation of mesenchymal stromal cells. PLoS One, 2012, 7(10): e46842.
20.	Sobczak M, Dargatz J, Chrzanowska-Wodnicka M. Isolation and culture of pulmonary endothelial cells from neonatal mice. J Vis Exp, 2010, (46): doi: pii: 2316.

1. Shupp JW, Nasabzadeh TJ, Rosenthal DS, et al. A review of the local pathophysiologic bases of burn wound progression. J Burn Care Res, 2010, 31(6): 849-873.
2. Pries AR, Kuebler WM. Normal endothelium. Handb Exp Pharmacol, 2006, (176 Pt 1): 1-40.
3. 胡静, 仲照东, 邹萍. 血管内皮细胞的生物学特性. 国际输血及血液学杂志, 2010, 33(1): 76-79.
4. Aird WC. Endothelium in health and disease. Pharmacol Rep, 2008, 60(1): 139-143.
5. Marin V, Kaplanski G, Grès S, et al. Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. J Immunol Methods, 2001, 254(1-2): 183-190.
6. Siow RC. Culture of human endothelial cells from umbilical veins. Methods Mol Biol, 2012, 806: 265-274.
7. Nachman RL, Jaffe EA. Endothelial cell culture: beginnings of modern vascular biology. J Clin Invest, 2004, 114(8): 1037-1040.
8. Baudin B, Bruneel A, Bosselut N, et al. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc, 2007, 2(3): 481-485.
9. Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest, 1973, 52(11): 2745-2756.
10. Geerts WJ, Vocking K, Schoonen N, et al. Cobblestone HUVECs: a human model system for studying primary ciliogenesis. J Struct Biol, 2011, 176(3): 350-359.
11. 黄起壬, 戴育成, 李剑, 等. 三种人脐静脉内皮细胞分离方法的比较. 江西医学院学报, 2005, 45(5): 8-14.
12. Fehrenbach ML, Cao G, Williams JT, et al. Isolation of murine lung endothelial cells. Am J Physiol Lung Cell Mol Physiol, 2009, 296(6): L1096-1103.
13. Jin Y, Liu Y, Antonyak M, et al. Isolation and characterization of vascular endothelial cells from murine heart and lung. Methods Mol Biol, 2012, 843: 147-154.
14. 张臣, 李彤, 侯跃龙, 等. 人脐静脉血管内皮细胞的高效分离与培养. 天津医药, 2010, 38(7): 605-607.
15. Winiarski BK, Acheson N, Gutowski NJ. An improved and reliable method for isolation of microvascular endothelial cells from human omentum. Microcirculation, 2011, 18(8): 635-645.
16. 刘长乐, 郑心田, 李广平, 等. 体外人脐静脉内皮细胞的培养鉴定及抗原表达分析. 山东医药, 2011, 51(36): 25-26.
17. Lip GY, Blann A. von Willebrand factor: a marker of endothelial dysfunction in vascular disorders? Cardiovasc Res, 1997, 34(2): 255-265.
18. Horvath B, Hegedus D, Szapary L, et al. Measurement of von Willebrand factor as the marker of endothelial dysfunction in vascular diseases. Exp Clin Cardiol, 2004, 9(1): 31-34.
19. Janeczek Portalska K, Leferink A, Groen N, et al. Endothelial differentiation of mesenchymal stromal cells. PLoS One, 2012, 7(10): e46842.
20. Sobczak M, Dargatz J, Chrzanowska-Wodnicka M. Isolation and culture of pulmonary endothelial cells from neonatal mice. J Vis Exp, 2010, (46): doi: pii: 2316.

Chinese Journal of Reparative and Reconstructive Surgery

ISOLATION, CULTURE, AND IDENTIFICATION OF CANINE UMBILICAL VEIN VASCULAR ENDOTHELIAL CELLS

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