Preservation and Enumeration of Endothelial Progenitor and Endothelial Cells From Peripheral Blood for Clinical Trials

Abstract

Aims

Endothelial progenitor cells (EPCs) are markers of vascular repair. Increased numbers of circulating endothelial cells (ECs) are associated with endothelial damage.

Materials & Methods

We enumerated EPC-EC by using Enrichment kit with addition of anti-human CD146-PE/Cy7 from peripheral blood mononuclear cell (PBMC) isolated either by red blood cell (RBC) lysing solution or by Ficoll centrifugation, and from fresh and preserved samples. PBMCs were quantified by flow cytometry.

Results

RBC lysis yielded higher percentage of PBMC (p = 0.0242) and higher numbers of PBMC/ml (p = 0.0039) than Ficoll. Absolute numbers of CD34⁺CD133⁺VEGFR2⁺ and CD146⁺CD34⁺VEGFR2⁺ were higher (p = 0.0117 for both), when isolated by RBC lysis than by Ficoll, when no difference in other subsets was found. Cryopreservation at -160°C and -80°C and short-term preservation at room temperature decreased EPC-EC.

Conclusions

Our data support use of fresh samples and isolation of PBMC from human blood by RBC lysis for enumeration of EPC and EC.

Keywords::

• angiogenesis
• cryopreservation
• endothelial cells
• endothelial progenitor cells
• flow cytometry

Acknowledgements

Authors would like to thank Tianxia Wu, NINDS, for reviewing of statistical analysis.

Financial & competing interests disclosure

Support for this work included funding from Department of Defense in the Center for Neuroscience and Regenerative Medicine and from the Intramural Research Program of NIH, NINDS. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

Support for this work included funding from Department of Defense in the Center for Neuroscience and Regenerative Medicine and from the Intramural Research Program of NIH, NINDS. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.