Isolation of Human Umbilical Vein Endothelial Cells (HUVECs) from Umbilical Cord and their characterization of HUVECs in terms of cell surface marker via immunofluroscence and flow cytometry
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Endothelial cells , the lining cells of blood vessels , are involved in many physiological and pathophysiological process, including haemostasis, vasoregulation, inflammation, angiogenesis, and the extravasation of fluids, macromolecules, hormones and leucocytes. This work was carried out to investigate the isolation, culture and characterization of human umbilical vein endothelial cells (HUVECs) in vitro by the help of Flow cytometry and Immunocytochemistry. Moreover, protocol was standardized to improve the cell yield and growth of these cells in culture. The endothelial cells were isolated by collagenase digestion and were seeded in tissue culture flasks using Dulbeccos modified Eagles medium (DMEM) –LG(Low Glucose) and M-200 media specific for ECGM (Endothelial Cell Growth Media ) containing LSGS( Low Serum Growth Supplement) and growth factor containing the (VEGF) and (bFGF) This study showed that HUVECS attached to to the gelatin coated tissue culture plates and become confluent after 3 days in vitro .HUVECs displayed cobblestone morphology at confluence with granular cytoplasm. HUVECs were positive for CD31- 3 % positivity, VE-Cadherin - 18.8% and vWF- 30.3%. Immunocytochemistry helped to anatomically a visualize the localization of endothelial cells marker. The cells produced from this technique grew well for extend periods and retained the normal characteristics of primary HUVECs, making them invaluable tools for researchers. Keywords: HUVECs., Flow Cytometry, Immunocytochemistry,VE-Cadherin,Vwf
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