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Spheroid-based human endothelial cell microvessel formation in vivo

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Abstract

The study of angiogenic endothelial cells (ECs) has in recent years greatly stimulated multiple fields of vascular biology research. A number of cellular models and numerous complex developmental, manipulatory and tumor animal models have been developed to study angiogenesis in vitro and in vivo. To connect the versatility of cellular assays with the complexity of readouts of in vivo experimentation, we have developed an endothelial transplantation assay. This assay is based on grafting ex vivo generated EC spheroids (2 d) in a suitable matrix in immunocompromised mice, to give rise to a 3D network of capillaries (20 d). This vasculature connects to the mouse vasculature, is perfused and matures by recruiting mouse mural cells. Here we describe the detailed protocol for this assay, including generation of spheroids, injection into mice, excision and processing of resulting plugs, and quantification by immunohistochemical analysis of the resulting vasculature.

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Figure 1: Flow chart of the in vivo angiogenesis assay.
Figure 2: Preparation of spheroids.
Figure 3: Injection and dissection of a matrigel–fibrin plug.
Figure 4: Human neovessel formation originating from transplanted endothelial cell (EC) spheroids.
Figure 5: Characterization of the human vascularization in vivo 20 d after implantation.
Figure 6: Co-grafting of different tumor cell populations (HT29, colon cancer; LLC, Lewis lung carcinoma; A375, melanoma) with human umbilical vein endothelial cell (HUVEC) spheroids to show the contribution of co-grafted endothelial cell (EC) to tumor angiogenesis.

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Acknowledgements

This work was supported by grants from the German Research Council (DFG, AU83/10-1) and the European Union [LSHG-CT-2004-503573]. HGA is supported by an endowed chair from the Aventis Foundation. Holger Weber is an employee of ProQinase GmbH.

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Author notes

  1. Anna M Laib and Arne Bartol: These authors contributed equally to this study.

Authors and Affiliations

  1. Joint Research Division Vascular Biology of the Medical Faculty Mannheim (CBTM), University of Heidelberg, and the German Cancer Research Center Heidelberg (DKFZ), Im Neuenheimer Feld, Heidelberg, Germany

    Anna M Laib, Arne Bartol & Hellmut G Augustin

  2. Friedrich-Miescher-Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland

    Abdullah Alajati

  3. Department of Physiology and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld, Heidelberg, Germany

    Thomas Korff

  4. ProQinase GmbH, Breisacher Straße, Freiburg, Germany

    Holger Weber

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  1. Anna M Laib
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  2. Arne Bartol
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  3. Abdullah Alajati
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  4. Thomas Korff
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  5. Holger Weber
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  6. Hellmut G Augustin
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Correspondence to Hellmut G Augustin.

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Laib, A., Bartol, A., Alajati, A. et al. Spheroid-based human endothelial cell microvessel formation in vivo. Nat Protoc 4, 1202–1215 (2009). https://doi.org/10.1038/nprot.2009.96

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