An Adapted Protocol for the Isolation and Culture of Human Umbilical Vein Endothelial Cells

Abstract

The umbilical cord is a valuable source of fetal stem cells, progenitor cells, and early-stage developmental cells, including human umbilical vein endothelial cells (HUVECs). HUVECs are widely used as a model for endothelial biology and are increasingly being investigated for their regenerative potential. Efficient isolation of these cells from the umbilical vein is a critical first step for both research and therapeutic applications. A key challenge in isolating HUVECs is the variability in success rates between laboratories. Most published protocols utilize Collagenase A for isolation. However, despite its widespread use, Collagenase A may not be the optimal enzyme due to batch-to-batch variability in enzymatic activity and potential negative effects on cell viability. In this study, we present an adapted HUVEC isolation protocol that employs dispase, demonstrating consistent and reproducible results across isolations. This approach also incorporates refined tissue and cell culture handling practices to further enhance reliability and cell health. We characterised the isolated cells using established HUVEC markers CD31 and CD146, and demonstrated in situ detachment of the cells from the vessel wall through immunofluorescence imaging. Our method achieved a success rate exceeding 95.6% across all umbilical cords processed. These findings highlight the protocols reproducibility and potential for broad applicability across research settings, using readily accessible reagents and equipment.

Keywords: Human umbilical vein endothelial cells, HUVECs, Umbilical cord, Vein, Endothelium, Endothelial cells, Cell isolation protocol