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An affordable method to obtain cultured endothelial cells from peripheral blood.

Bueno-Betí C, Novella S, Lázaro-Franco M, Pérez-Cremades D, Heras M, Sanchís J, Hermenegildo C - J. Cell. Mol. Med. (2013)

Bottom Line: 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.Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC.

View Article: PubMed Central - PubMed

Affiliation: Research Foundation, Hospital Clínico of Valencia - INCLIVA, Valencia, Spain.

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Endothelial progenitor cells' (EPC) isolation conditions. (A) Effect of anticoagulant in the success of EPC cultures and (B) the time at which it was possible to observe the first EPC colonies. Blood samples were collected in tubes containing three different anticoagulants: ethylenediaminetetraacetic acid (EDTA), heparin and sodium citrate. EPC initial cultures were followed up to 40 days of culture (**P < 0.01 versus EDTA by Chi-squared test; n = 10). Effect of blood volume in the success of EPC cultures: bars represent (C) the percentages of EPC cultures obtained from mononuclear cells obtained from different blood volumes and (D) the time at which it was possible to observe the first EPC colonies (*P < 0.05 versus 10 ml; n = 10).
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fig01: Endothelial progenitor cells' (EPC) isolation conditions. (A) Effect of anticoagulant in the success of EPC cultures and (B) the time at which it was possible to observe the first EPC colonies. Blood samples were collected in tubes containing three different anticoagulants: ethylenediaminetetraacetic acid (EDTA), heparin and sodium citrate. EPC initial cultures were followed up to 40 days of culture (**P < 0.01 versus EDTA by Chi-squared test; n = 10). Effect of blood volume in the success of EPC cultures: bars represent (C) the percentages of EPC cultures obtained from mononuclear cells obtained from different blood volumes and (D) the time at which it was possible to observe the first EPC colonies (*P < 0.05 versus 10 ml; n = 10).

Mentions: Only 20% of EDTA-recovered blood samples gave rise to EPC cultures. However, when heparin or sodium citrate was used for blood recovery, 80% of EPC cultures were successfully obtained (P < 0.01; Fig. 1A). No significant differences were found with regard to EPC appearance mean time between anticoagulants (Fig. 1B). Moreover, 100% of cultures derived from citrated samples showed different morphology, were negative for the uptake of Ac-LDL and unable to bind Ulex-lectin, thus demonstrating they were not EPC (Fig. S1). Only heparin allows obtaining high yields of homogeneous EPC cultures with the shorter appearance mean time. Thus, rest of the experiments were performed with blood obtained with heparin as anticoagulant.


An affordable method to obtain cultured endothelial cells from peripheral blood.

Bueno-Betí C, Novella S, Lázaro-Franco M, Pérez-Cremades D, Heras M, Sanchís J, Hermenegildo C - J. Cell. Mol. Med. (2013)

Endothelial progenitor cells' (EPC) isolation conditions. (A) Effect of anticoagulant in the success of EPC cultures and (B) the time at which it was possible to observe the first EPC colonies. Blood samples were collected in tubes containing three different anticoagulants: ethylenediaminetetraacetic acid (EDTA), heparin and sodium citrate. EPC initial cultures were followed up to 40 days of culture (**P < 0.01 versus EDTA by Chi-squared test; n = 10). Effect of blood volume in the success of EPC cultures: bars represent (C) the percentages of EPC cultures obtained from mononuclear cells obtained from different blood volumes and (D) the time at which it was possible to observe the first EPC colonies (*P < 0.05 versus 10 ml; n = 10).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117560&req=5

fig01: Endothelial progenitor cells' (EPC) isolation conditions. (A) Effect of anticoagulant in the success of EPC cultures and (B) the time at which it was possible to observe the first EPC colonies. Blood samples were collected in tubes containing three different anticoagulants: ethylenediaminetetraacetic acid (EDTA), heparin and sodium citrate. EPC initial cultures were followed up to 40 days of culture (**P < 0.01 versus EDTA by Chi-squared test; n = 10). Effect of blood volume in the success of EPC cultures: bars represent (C) the percentages of EPC cultures obtained from mononuclear cells obtained from different blood volumes and (D) the time at which it was possible to observe the first EPC colonies (*P < 0.05 versus 10 ml; n = 10).
Mentions: Only 20% of EDTA-recovered blood samples gave rise to EPC cultures. However, when heparin or sodium citrate was used for blood recovery, 80% of EPC cultures were successfully obtained (P < 0.01; Fig. 1A). No significant differences were found with regard to EPC appearance mean time between anticoagulants (Fig. 1B). Moreover, 100% of cultures derived from citrated samples showed different morphology, were negative for the uptake of Ac-LDL and unable to bind Ulex-lectin, thus demonstrating they were not EPC (Fig. S1). Only heparin allows obtaining high yields of homogeneous EPC cultures with the shorter appearance mean time. Thus, rest of the experiments were performed with blood obtained with heparin as anticoagulant.

Bottom Line: 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.Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC.

View Article: PubMed Central - PubMed

Affiliation: Research Foundation, Hospital Clínico of Valencia - INCLIVA, Valencia, Spain.

Show MeSH