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Perfusion and endothelialization of engineered tissues with patterned vascular networks

Nature Protocols volume 16, pages 3089–3113 (2021)Cite this article

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Abstract

As engineered tissues progress toward therapeutically relevant length scales and cell densities, it is critical to deliver oxygen and nutrients throughout the tissue volume via perfusion through vascular networks. Furthermore, seeding of endothelial cells within these networks can recapitulate the barrier function and vascular physiology of native blood vessels. In this protocol, we describe how to fabricate and assemble customizable open-source tissue perfusion chambers and catheterize tissue constructs inside them. Human endothelial cells are seeded along the lumenal surfaces of the tissue constructs, which are subsequently connected to fluid pumping equipment. The protocol is agnostic with respect to biofabrication methodology as well as cell and material composition, and thus can enable a wide variety of experimental designs. It takes ~14 h over the course of 3 d to prepare perfusion chambers and begin a perfusion experiment. We envision that this protocol will facilitate the adoption and standardization of perfusion tissue culture methods across the fields of biomaterials and tissue engineering.

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Fig. 1: Customizable open-source chambers for sterile perfusion culture.
Fig. 2: Geometric and mechanical requirements for catheterization and perfusion protocol.
Fig. 3: Fabrication and assembly guide for custom perfusion chambers.
Fig. 4: Achieving uniform endothelialization in perfusable networks of cylindrical vessels.
Fig. 5: Assembly guide for connecting vascularized gels for sterile perfusion.
Fig. 6: Establishing a sterile fluidic connection for perfusion culture.
Fig. 7: Potential pitfalls encountered during endothelialization and perfusion.
Fig. 8: Endothelial cells form confluent monolayers within vascular networks under perfusion.

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Data availability

STL files for the 3D printed perfusion chambers shown in Figs. 1 and 3 are available at https://github.com/MillerLabFTW/Perfusion. Raw imaging data for the microscopy images presented are available from the corresponding author.

Code availability

The openSCAD script for generating perfusion chambers is available at https://github.com/MillerLabFTW/Perfusion under a GNU General Public License and will be maintained and updated in that repository. In Supplementary Software 1, we also provide the MATLAB script used to analyze endothelial cell coverage.

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Acknowledgements

We thank the many open-source and related projects that facilitated this work, including Arduino.cc, Blender.org, Fritzing.org, Grabcad.com, NIH ImageJ, Fiji.sc, Slic3r.org and Openscad.org, as well as the creators of computer-aided designs that we downloaded from Grabcad for renderings. We thank S. J. Paulsen, K. D. Janson and S. H. Saxton for helpful discussions and protocol validation. We thank D. W. Sazer for photography assistance. This work was supported in part by the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (J.S.M.), Lung Biotechnology PBC (J.S.M.), the US National Institutes of Health (NIH) (NRSA award HL140905, I.S.K.) and the US National Science Foundation (NSF) (GRFP award 1450681, G.A.C. and B.G.).

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  1. These authors contributed equally: Ian S. Kinstlinger, Gisele A. Calderon.

Authors and Affiliations

  1. Department of Bioengineering, Rice University, Houston, TX, USA

    Ian S. Kinstlinger, Gisele A. Calderon, Madison K. Royse, A. Kristen Means, Bagrat Grigoryan & Jordan S. Miller

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Contributions

I.S.K. and B.G. developed perfusion chambers and protocols. G.A.C. developed the endothelialization workflow; G.A.C., M.K.R. and A.K.M. performed cell seeding and imaging experiments. I.S.K., G.A.C. and J.S.M. wrote the manuscript with input from all authors. J.S.M. supervised the project.

Corresponding author

Correspondence to Jordan S. Miller.

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Competing interests

B.G. and J.S.M. are cofounders of and hold equity stakes in Volumetric.

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Peer review information Nature Protocols thanks Lorenzo Moroni, Mark Skylar-Scott and Ying Zheng for their contribution to the peer review of this work.

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Key references using this protocol

Grigoryan, B. et al. Science 364, 458–464 (2019): https://doi.org/10.1126/science.aav9750

Kinstlinger, I. S. et al. Nat. Biomed. Eng. 4, 916–932 (2020): https://doi.org/10.1038/s41551-020-0566-1

Supplementary information

Supplementary Information

Supplementary Manual and Supplementary Figs. 1 and 2.

Supplementary Software 1

MATLAB code used to analyze data for Fig. 4

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Kinstlinger, I.S., Calderon, G.A., Royse, M.K. et al. Perfusion and endothelialization of engineered tissues with patterned vascular networks. Nat Protoc 16, 3089–3113 (2021). https://doi.org/10.1038/s41596-021-00533-1

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