Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Summary

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Abstract

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Protocol

Procedure

1. Lay cord on clean pad and dab off excess blood. Make fresh cuts on both ends of the cord.
2. Insert 21 1/2 G needle with the plastic needle sheath ON, into the vein. (The vein is the largest opening; the 2 smaller ones are arteries)
3. Clamp the needle in place with a hemostat and attach the 20cc syringe of Hanks to the needle.
4. Push the Hanks through the vein with moderate pressure. Collect the waste in the beaker with bleach. (Holding the needle and cord with one hand while pushing would prevent the needle popping out of the vein.) If there is a lot of blood in vein, wash a se....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1% Collagenase			10 mL per cord, warmed to 37 °C
Tissue culture flasks			T75, one per cord.
Hanks medium
scissors			sterile
beaker			sterile
50 mL tubes