Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVEC)

Ambrose L. Cheung1

1 Dartmouth Medical School, Hanover, New Hampshire
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 4B
DOI:  10.1002/9780471729259.mca04bs4
Online Posting Date:  February, 2007
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Human‐derived endothelial cells can now be routinely harvested from human umbilical veins. Studies with human umbilical vein endothelial cells (HUVEC) have been conducted with cells from passage 2 to 5. It is now also possible to cryopreserve primary and early‐passaged HUVEC for future propagation and for forwarding to an end user by express courier. Stored HUVEC have been stably retrieved even after several years. These retrieval techniques have facilitated the deployment of HUVEC for many studies, including those for homeostasis, inflammatory disorders, atherosclerosis, cancer, and microbial adhesion and invasion. In this unit, we will delineate the procedure for harvesting, propagation, and storage of HUVEC.

Keywords: endothelial cell culture; HUVEC; propagation and storage; methods

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Table of Contents

  • Basic Protocol 1: Harvest and Culture of HUVEC
  • Support Protocol 1: Cryopreservation and Thawing of HUVEC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Harvest and Culture of HUVEC

  • Human umbilical cord (cords held for more than 3 hr should be discarded)
  • HEPES‐buffered saline (HBS; see recipe), 37°C
  • 1× (0.1% w/v) collagenase, type I (lyophilized; 150 to 200 U/mg; Invitrogen); store in 10‐ml aliquots at –20°C
  • M199 medium (Bio‐Whittaker) supplemented with 20% (v/v) FBS
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete HUVEC culture medium/20% FBS (see recipe)
  • 0.02% (w/v) EDTA/0.5% (w/v) bovine serum albumin
  • 400‐ml beakers
  • Gauze
  • Nylon tie, 2–0 size, 6‐in. (15‐cm) length
  • Cannulas: blunt, hubless 10‐G needles
  • Hemostat
  • 1‐, 30‐ and 50‐ml syringes
  • 10‐G needles, blunt and hubless
  • No. 10 surgical blade
  • Surgical gloves
  • Polyethylene tubing, 1/8 in. (0.3 cm) outer diameter
  • 50‐ml conical polypropylene centrifuge tubes (e.g., Falcon 2070)
  • Tabletop centrifuge
  • 25‐cm2 gelatin‐coated tissue culture flasks (see recipe) and other gelatin‐coated tissue culture vessels as needed for passaging
  • Additional reagents and equipment for phase‐contrast microscopy (unit 2.1) and cryopreservation of HUVEC ( protocol 2)
NOTE: Utilize aseptic technique in all manipulations of human umbilical cords.

Support Protocol 1: Cryopreservation and Thawing of HUVEC

  • Confluent primary HUVEC growing in 25‐cm2 tissue culture flasks ( protocol 1)
  • M199 medium (Bio‐Whittaker) supplemented with 20% (v/v) FBS
  • 92.5% (v/v) complete culture medium (containing 20% FBS; see recipe)/7.5% (v/v) DMSO
  • 100% ethanol
  • 50‐ml conical polypropylene centrifuge tubes
  • 1.22‐ml sterile cryogenic vials (Nalgene)
  • Cryogenic workstation (Corning)
  • Liquid nitrogen cryogenic storage system (e.g., Fisher)
  • 15‐ml conical polypropylene centrifuge tubes
  • 75‐cm2 tissue culture flasks (Corning), gelatin‐coated (see recipe)
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Literature Cited

Literature Cited
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