Open AccessAn affordable method to obtain cultured endothelial cells from peripheral blood
Author(s) -
BuenoBetí Carlos,
Novella Susana,
LázaroFranco Macarena,
PérezCremades Daniel,
Heras Magda,
Sanchís Juan,
Hermenegildo Carlos
Publication year - 2013
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/jcmm.12133
Subject(s) - vasculogenesis , umbilical vein , andrology , progenitor cell , peripheral blood mononuclear cell , endothelial progenitor cell , cell culture , immunology , stem cell , biology , medicine , microbiology and biotechnology , in vitro , biochemistry , genetics
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 EPC s 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.