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Isolation of Functional Human Endothelial Cells from Small Volumes of Umbilical Cord Blood

Abstract

Endothelial cells (ECs) isolated from endothelial progenitor cells in blood have great potential as a therapeutic tool to promote vasculogenesis and angiogenesis and treat cardiovascular diseases. However, current methods to isolate ECs are limited by a low yield with few colonies appearing during isolation. In order to utilize blood-derived ECs for therapeutic applications, a simple method is needed that can produce a high yield of ECs from small volumes of blood without the addition of animal-derived products. For the first time, we show that human ECs can be isolated without the prior separation of blood components through the technique of diluted whole blood incubation (DWBI) utilizing commercially available human serum. We isolated ECs from small volumes of blood (~10 mL) via DWBI and characterized them with flow cytometry, immunohistochemistry, and uptake of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). These ECs are functional as demonstrated by their ability to form tubular networks in Matrigel, adhere and align with flow under physiological fluid shear stress, and produce increased nitric oxide under fluid flow. An average of 7.0 ± 2.5 EC colonies that passed all functional tests described above were obtained per 10 mL of blood as compared to only 0.3 ± 0.1 colonies with the traditional method based on density centrifugation. The time until first colony appearance was 8.3 ± 1.2 days for ECs isolated with the DWBI method and 12 ± 1.4 days for ECs isolated with the traditional isolation method. A simplified method, such as DWBI, in combination with advances in isolation yield could enable the use of blood-derived ECs in clinical practice.

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Acknowledgments

We thank the NIH for its support through grant 1R21-HL109897-01 and the American Heart Association (AHA) for its support through grant #12BGIA11070002 to Hardean E. Achneck. We are further grateful for support from the National Science Foundation Graduate Research Fellowship and AHA predoctoral fellowship 12PRE11180003 to Alexandra E. Jantzen. We thank Tracy Cheung and Cristina Fernandez of Duke University Department of Biomedical Engineering for conducting hUCB-EC isolation experiments.

Conflict of interest

The authors declare no conflicts of interest. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.

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Correspondence to Hardean E. Achneck.

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Associate Editor Michael S. Detamore oversaw the review of this article.

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Kang, S.D., Carlon, T.A., Jantzen, A.E. et al. Isolation of Functional Human Endothelial Cells from Small Volumes of Umbilical Cord Blood. Ann Biomed Eng 41, 2181–2192 (2013). https://doi.org/10.1007/s10439-013-0807-5

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Keywords

  • Late-outgrowth endothelial progenitor cell, EPC
  • ECFC
  • EOC
  • Shear stress
  • Nitric oxide
  • Human serum
  • Platelet lysate
  • Cell differentiation
  • Cell isolation