Skip to main content

High-yield method for isolation and culture of endothelial cells from rat coronary blood vessels suitable for analysis of intracellular calcium and nitric oxide biosynthetic pathways


We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This method makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.


  1. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: Identification by morphologic and immunologic criteria. J Clin Invest 1973;52:2745–2756.

    PubMed  Article  CAS  Google Scholar 

  2. van Leeuwen EBM, Molema G, de Jong KP, van Luyn MJA, Dijk F, Slooff MJH, Ruiters MHJ, van der Meer J. One-step method for endothelial cell isolation from large human blood vessels using fibrin glue. Lab Invest 2000;80:987–989.

    PubMed  Google Scholar 

  3. Wechezak A, Mansfield PB. Isolation and growth characteristics of cell lines from bovine venous endothelium. In Vitro 1973;9:39–45.

    PubMed  Article  CAS  Google Scholar 

  4. Crutchley DJ, Ryan US, Ryan JW. Effects of aspirin and dypiridamole on the degradation of adenosine diphosphate by cultured cells derived from bovine pulmonary artery. J Clin Invest 1980;66:29–35.

    PubMed  Article  CAS  Google Scholar 

  5. Carley WW, Tanoue L, Merker M, Gillis CN. Isolation of rabbit pulmonary microvascular endothelial cells and characterization of their angiotensin converting enzyme activity. Pulm Pharmacol 1990;3:35–40.

    PubMed  Article  CAS  Google Scholar 

  6. Piper HM, Spahr R, Mertens S, Krützfeldt A, Watanabe H. Microvascular endothelial cells from heart. In: Piper HM, editor. Cell culture techniques in heart and vessel research. Berlin-Heidelberg: Springer Verlag; 1990. p.158–177.

    Google Scholar 

  7. Nishida M, Carley WW, Gerritsen ME, Ellingsen O, Kelly RA, Smith TW. Isolation and characterization of human and rat microvascular endothelial cells. Am J Physiol 1993;264:H639-H652.

    PubMed  CAS  Google Scholar 

  8. Grafe M, Auch-Schwelk W, Graf K, Terbeek D, Hertel H, Unkelbach M, Hilderbrandt A, Fleck E. Isolation and characterization of macrovascular and microvascular endothelial cells from human hearts. Am J Physiol 1994;36:H2138-H2148.

    Google Scholar 

  9. Cirillo P, Golino P, Ragni M, Guarino A, Calabro P, Chiarriello M. A simple method for the isolation, cultivation, and characterization of endothelial cells from rabbit coronary circulation. Thromb Res 1999;96:329–333.

    PubMed  Article  CAS  Google Scholar 

  10. Failli P, Nistri S, Quattrone S, Mazzetti L, Bigazzi M, Bani Sacchi T, Bani D. Relaxin up-regulates inducible nitric oxide synthase expression and nitric oxide generation in rat coronary endothelial cells. FASEB J (Dec.14, 2001) Summary: 2002;16:252–254.

    PubMed  CAS  Google Scholar 

  11. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109–142.

    PubMed  CAS  Google Scholar 

  12. Bani D, Failli P, Bello MG, Thiemermann C, Bani Sacchi T, Bigazzi M, Masini E. Relaxin activates the L-arginine-nitric oxide pathway in vascular smooth muscle cells in culture. Hypertension 1998;31:1240–1247.

    PubMed  CAS  Google Scholar 

  13. Tontsch U, Bauer HC. Isolation, characterization and long-term cultivation of porcine and murine cerebral capillary endothelial cells. Microvasc Res 1989;18:325–335.

    Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Daniele Bani.

Additional information

Published: October 28, 2002

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Nistri, S., Mazzetti, L., Failli, P. et al. High-yield method for isolation and culture of endothelial cells from rat coronary blood vessels suitable for analysis of intracellular calcium and nitric oxide biosynthetic pathways. Biol Proced Online 4, 32–37 (2002).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI:

Indexing terms

  • endothelial cells
  • nitric oxide
  • acetylated LDL
  • DAF-2/DA
  • Fura-2AM