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Isolation of endothelial cells from fresh tissues

Nature Protocols volume 3, pages 1085–1091 (2008)Cite this article

Abstract

Here, we present a protocol for the isolation of endothelial cells (ECs) from tissues. ECs make up a minor population of cells in a tissue, but play a major role in tissue homeostasis, as well as in diverse pathologies. To understand the biology of ECs, characterization of this cell population is highly desirable, but requires the availability of purified cells. For this purpose, tissues are mechanically minced and subsequently digested enzymatically with collagenase and dispase. ECs in the resulting single-cell suspension are labeled with Abs against EC surface antigens and separated from the remainder of the cells and debris by capture with magnetic beads or by high-speed cell sorting. Purified ECs are viable and suitable for characterization of diverse cellular properties. This protocol is optimized for human tissues but can also be adapted for use with other species. Depending on the tissue, the procedure can be completed in ∼6 h.

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Figure 1: Isolation of endothelial cells (ECs) from tissues by immunomagnetic separation and fluorescence-activated cell sorting (FACS).
Figure 2: Selection of Abs for endothelial cell (EC) isolation.
Figure 3: Immunofluorescent characterization of isolated endothelial cells (ECs).
Figure 4: Fluorescence-activated cell sorting (FACS) plots of endothelial cell (EC) isolation.
Figure 5: Downstream applications of isolated endothelial cells (ECs).

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Acknowledgements

This work was supported by grants of the transnational University Limburg (tUL), GROW—School for Oncology and Developmental Biology and the sixth EU Framework Programme (Integrated Project 'Angiotargeting'; contract no. 504743). We thank Petra Hautvast for technical assistance.

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Author notes

  1. Karolien Castermans

    Present address: Present address: Interdisciplinary Cluster for Applied Genoproteomics, GIGA-R; Molecular Biology and Genetic Engineering Unit, University of Liège, Belgium.,

Authors and Affiliations

  1. Department of Pathology, Angiogenesis Laboratory, School for Oncology and Developmental Biology (GROW), Maastricht University, Maastricht, The Netherlands

    Judy R van Beijnum, Mat Rousch, Karolien Castermans, Edith van der Linden & Arjan W Griffioen

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Correspondence to Arjan W Griffioen.

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van Beijnum, J., Rousch, M., Castermans, K. et al. Isolation of endothelial cells from fresh tissues. Nat Protoc 3, 1085–1091 (2008). https://doi.org/10.1038/nprot.2008.71

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