Isolation and Culture of Equine Umbilical Vein Endothelial Cells and Their Functional Validation
[Objective]The aim of this study was to establish a simple and efficient method for isolation and culture of equine umbilical vein endothelial cells(eUVECs)in vitro and to provide a reliable cell model for the study of angiogenic functions in horses.[Method]Veins were isolated from equine umbilical cords under sterile conditions and rinsed thoroughly with PBS solution.The umbilical veins were digested using 0.2％type Ⅰ collagenase,and after digestion for 25-30 min,the isolated eUVECs were collected for culture.The growth of eUVECs was observed every 12 h using an inverted microscope.The D450 nm value of first-generation(P1)and P3 generation eUVECs was determined over a 7 d period using the CCK-8 method,and the growth curves were plotted.The expression of endothelial cell markers CD31 and CD34 was identified by immunofluorescence staining.eUVECs were induced using MSC adipogenic and osteogenic differentiation induction reagents to verify whether they had differentiation properties.After the endothelial cells were identified,eUVECs were stimulated by adding a gradient of increasing concentrations of tumour necrosis factor-α(TNF-α)(5,10,15,20 and 25 ng/mL)and cell activity was detected and morphological changes were observed using the CCK-8 assay.eUVECs were cultured using the Matrigel method,and ImageJ software was used to analyse the data and count the four indexes of neovascularisation:Junctions number,branching lenght,tube formation rate and meshes area.The optimal TNF-α stimulation concentration for in vitro angiogenic capacity was searched.[Result]The eUVECs isolated using 0.2％type Ⅰ collagenase digestion reached 90％confluence within 4-5 days.These cells were observed under an inverted microscope to be well-grown and arranged in a paving stone-like pattern.Positive expression of CD31 and CD34 in eUVECs was confirmed by immunofluorescence staining,and eUVECs showed weak adipogenic and osteogenic differentiation.Under the culture conditions of the Matrigel method,the indicators of angiogenic capacity,such as the junctions number,branching lenght,tube formation rate and meshes area,were highest in the 20 ng/mL TNF-α-treated group,and were extremely significantly higher than those in the control group(P＜0.01).However,under conventional culture conditions,with the increase of TNF-α concentration up to 15 ng/mL,the proliferation of eUVECs was significantly inhibited or even started to lead to apoptosis.[Conclusion]Type Ⅰ collagenase umbilical cord-filled digestion was able to successfully isolate and culture primary eUVECs.20 ng/mL TNF-αsignificantly enhanced the angiogenic ability of eUVECs in vitro,and eUVECs could be used as a cellular model to study angiogenesis in vitro.
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