An affordable method to obtain cultured endothelial cells from peripheral blood
Article first published online: 1 OCT 2013
© 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Journal of Cellular and Molecular Medicine
Volume 17, Issue 11, pages 1475–1483, November 2013
How to Cite
Bueno-Betí, C., Novella, S., Lázaro-Franco, M., Pérez-Cremades, D., Heras, M., Sanchís, J. and Hermenegildo, C. (2013), An affordable method to obtain cultured endothelial cells from peripheral blood. Journal of Cellular and Molecular Medicine, 17: 1475–1483. doi: 10.1111/jcmm.12133
- Issue published online: 23 DEC 2013
- Article first published online: 1 OCT 2013
- Manuscript Accepted: 14 AUG 2013
- Manuscript Received: 24 MAY 2013
- Spanish Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III - FEDER-ERDF. Grant Numbers: FIS PI08/0272, PI10/00518
- Red Cardiovascular. Grant Numbers: RD12/0042/0052, RD12/0042/0010
- Consellería de Educación, Generalitat Valenciana. Grant Number: ACOMP/2013/171
- Atracció de Talent
- endothelial progenitor cells;
- cell culture;
The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media-2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell-like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above-mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols.