Preparation of Endothelial Cells

Hynda K. Kleinman1, Maria C. Cid2

1 National Institute of Dental Research/NIH, Bethesda, Maryland, 2 Hospital Clinic i Provincia, Barcelona, Spain
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 2.3
DOI:  10.1002/0471143030.cb0203s00
Online Posting Date:  May, 2001
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Abstract

Endothelial cells, which line blood vessels, can be prepared from a variety of tissues. They are frequently prepared from the umbilical vein, which is relatively easy to obtain. The procedure is clearly described and provides a large population of highly purified endothelial cells. There are also methods for obtaining endothelial cells from other tissues such as fat, skin, and mucosa. These methods require special care and generate smaller populations of cells.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Endothelial Cells from Human Umbilical Vein
  • Alternate Protocol 1: Isolation of Endothelial Cells from Retroperitoneal Adipose Tissue or Nasal Mucosa
  • Alternate Protocol 2: Isolation of Microvascular Endothelial Cells from Dermis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Endothelial Cells from Human Umbilical Vein

  Materials
  • Fresh human umbilical cord
  • RPMI 1640 medium, 4° and 37°C
  • Phosphate‐buffered saline (PBS; Life Technologies)
  • 5 mg/ml collagenase CLSII (Worthington) in PBS (filter sterilized), 37°C
  • HUVEC medium (see recipe), 37°C
  • 250‐ml wide‐mouthed tissue culture flasks
  • Sterile pads and gauze
  • Sterile scissors, razors, hemostats, and non‐toothed forceps
  • Syringes and yellow 200‐µl pipet tips
  • 50‐ml conical centrifuge tubes
  • Tabletop centrifuge
  • Nunc T‐75 tissue culture flasks or 75‐cm2gelatin‐coated flasks (see recipe)
  • 150‐mm‐diameter Nunc tissue culture dishes
  • Additional reagents and equipment for trypsinization of cells (unit 1.1)

Alternate Protocol 1: Isolation of Endothelial Cells from Retroperitoneal Adipose Tissue or Nasal Mucosa

  • Retroperitoneal adipose tissue or nasal mucosa
  • 0.02% (w/v) collagenase A (Boehringer Mannheim) in PBS (filter sterilized)
  • Cell scraper
  • Laminin‐coated 6‐well tissue culture plates (see recipe)
  • Additional reagents and equipment for trypsinization of cells (unit 1.1)

Alternate Protocol 2: Isolation of Microvascular Endothelial Cells from Dermis

  • Dermal tissue (e.g., neonatal foreskins)
  • Discontinuous Percoll gradient (see recipe)
  • 5 mM tetrasodium EDTA in PBS (also available as Versene from Life Technologies), filter‐sterilized using 0.22‐µm Nalgene filter, prewarmed to 37°C
  • PBS containing 5% FBS, room temperature to 37°C (store up to 1 month at 4°C)
  • Laminin‐coated 6‐well tissue culture plates (see recipe)
  • Anti‐CD31‐coated petri dishes (see recipe)
  • Gelatin‐ or fibronectin‐coated 6‐well tissue culture plates (see reciperecipes)
  • Additional reagents and equipment for trypsinization of cells (unit 1.1)
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Figures

Videos

Literature Cited

Literature Cited
   Folkman, J., Haudenshild, C.C., and Zetter, B.R. 1979. Long term culture of capillary endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 76:5217‐5221.
   Fukuda, K., Imamura, Y., Koshihara, Y., Ooyama, T., Hanamure, Y. and Ohyama, M. 1989. Establishment of human mucosal microvascular endothelial cells from inferior turbinate culture. Am. J. Otolaryngol. 10:85‐91.
   Gimbrone, M. 1979. Culture of vascular endothelium. Prog. Hemostasis Thromb 3:1‐28.
   Gordon, P.B., Sussman, I.I., and Hatcher, V.B. 1983. Long term culture of human endothelial cells. In Vitro 19:661‐671.
   Jaffe, E.A. 1980. Culture of human endothelial cells. Transplant. Proc 12:49‐53.
   Kibbey, M.C., Grant, D.S., and Kleinman, H.K. 1992.Role of the SIKVAV site of laminin in promotion of angiogenesis and tumor growth: An in vivo Matrigel model. J. Natl. Cancer Inst. 84:1633‐1638.
   Kubota, Y., Kleinman, H.K., Martin, G.R., and Lawley, T.J. 1988. Role of laminin and basement membrane in morphological differentiation of human endothelial cells into capillary‐like structures. J. Cell Biol. 107:1589‐1598.
   Voest, E.E., Kenyon, B.M., O'Reilly, M.S., Truit, G., D'Amato, R.J., and Folkman, J. 1995. Inhibition of angiogenesis in vivo by interleukin 12. J. Natl. Cancer Inst. 87:581‐586.
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