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Microfabrication of AngioChip, a biodegradable polymer scaffold with microfluidic vasculature

Nature Protocols volume 13, pages 1793–1813 (2018)Cite this article

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Abstract

Microengineered biomimetic systems for organ-on-a-chip or tissue engineering purposes often fail as a result of an inability to recapitulate the in vivo environment, specifically the presence of a well-defined vascular system. To address this limitation, we developed an alternative method to cultivate three-dimensional (3D) tissues by incorporating a microfabricated scaffold, termed AngioChip, with a built-in perfusable vascular network. Here, we provide a detailed protocol for fabricating the AngioChip scaffold, populating it with endothelial cells and parenchymal tissues, and applying it in organ-on-a-chip drug testing in vitro and surgical vascular anastomosis in vivo. The fabrication of the AngioChip scaffold is achieved by a 3D stamping technique, in which an intricate microchannel network can be embedded within a 3D scaffold. To develop a vascularized tissue, endothelial cells are cultured in the lumen of the AngioChip network, and parenchymal cells are encapsulated in hydrogels that are amenable to remodeling around the vascular network to form functional tissues. Together, these steps yield a functional, vascularized network in vitro over a 14-d period. Finally, we demonstrate the functionality of AngioChip-vascularized hepatic and cardiac tissues, and describe direct surgical anastomosis of the AngioChip vascular network on the hind limb of a Lewis rat model.

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Fig. 1
Fig. 2: POMaC polymer synthesis and purification.
Fig. 3: Microfabrication of AngioChip scaffold with 3D stamping technique.
Fig. 4: Assembly of an AngioChip bioreactor with seeding of endothelial cells and parenchymal tissues.
Fig. 5: Endothelialized lumen of AngioChip scaffold and sprouting of endothelial cells.
Fig. 6: Characterization of self-assembled tissues after 7 d of cultivation.
Fig. 7: Direct surgical anastomosis of vascularized cardiac AngioChip scaffolds to hind limbs of an adult Lewis rat.

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Acknowledgements

This work was made possible by a National Sciences and Engineering Research Council of Canada (NSERC) Postgraduate Scholarship–Doctoral, awarded to B.F.L.L.; a Canadian Institutes of Health Research (CIHR) Vanier Canada Graduate Scholarship, awarded to L.D.H.; and a CIHR Banting Postdoctoral Fellowship, awarded to B.Z. This work was also funded by CIHR Operating Grants (MOP-126027 and MOP-137107), an NSERC Discovery Grant (RGPIN-2015-05952), an NSERC Steacie Fellowship (SMFSU 4620), a Heart and Stroke Foundation Grant-in-Aid (G-16-00012), an NSERC-CIHR Collaborative Health Research Grant (CHRPJ 4937), an NSERC Strategic Grant (STPGP 5066) to M.R., and National Institutes of Health grant 2R01 HL076485.

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Author notes

  1. These authors contributed equally: Boyang Zhang, Benjamin Fook Lun Lai.

Authors and Affiliations

  1. Institute for Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

    Boyang Zhang, Benjamin Fook Lun Lai, Locke Davenport Huyer, Miles Montgomery & Milica Radisic

  2. Department of Chemistry, Tsinghua University, Beijing, China

    Ruoxiao Xie

  3. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

    Locke Davenport Huyer, Miles Montgomery & Milica Radisic

  4. Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada

    Milica Radisic

  5. The Heart and Stroke/Richard Lewar Centre of Excellence, Toronto, Ontario, Canada

    Milica Radisic

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Contributions

B.Z. and M.R. designed the research; B.Z., L.D.H., and M.M. performed the research; B.Z. and M.R. analyzed the data; B.Z. and R.X. prepared the figures for the paper; B.Z., B.F.L.L., and R.X. prepared the supplementary video; B.Z., B.F.L.L., and M.R. wrote the manuscript.

Corresponding author

Correspondence to Milica Radisic.

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Competing interests

M.R. and B.Z. are among the co-founders of TARA Biosystems and they hold equity in this company. The AngioChip is licensed to TARA Biosystems. The remaining authors declare no competing interests.

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Related links

Key references using this protocol

Zhang, B. et al. Nat. Mater. 15, 669–678 (2016) https://doi.org/10.1038/nmat4570

Lai., B. F. L. et al. Adv. Funct. Mater. 27, 1703524 (2017) https://doi.org/10.1002/adfm.201703524

Zhang, B. et al. Sci. Adv. 1, e1500423 (2015) https://doi.org/10.1126/sciadv.1500423

Integrated supplementary information

Supplementary Figure 1 Bioreactor design.

All units are in millimeters unless specified otherwise. All bioreactor components are made of polycarbonate.

Supplementary Figure 2 Endothelialized AngioChip network.

Confocal fluorescent images of various location of the AngioChip network populated with human umbilical cord endothelial cells stained for VE-cadherin (red) to identify the intercellular junctions (red) on day 2 after cell seeding. Scale bar, 50 μm. Reproduced with permission from Zhang et al.36, Macmillan Publishers Limited.

Supplementary Figure 3 Angiogenic vascular sprouting from AngioChip.

Confocal fluorescent images of GFP-labeled human umbilical cord endothelial cells (green) sprouting from the AngioChip networks through the built-in micro-holes on the channel walls in the presence of thymosin β4 on day 2 after cell seeding. Scale bar, 50μm.

Supplementary Figure 4 Tissue integration of AngioChip implants.

Smooth muscle actin (SMA) stained histology cross-section of the AngioChip cardiac tissue implants after 1 week with the direct surgical anastomosis in the configuration of artery-to-vein graft. Scale bar: 200μm (top image) and 100μm (bottom image). Reproduced with permission from Zhang et al.36, Macmillan Publishers Limited.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4

Supplementary Data

CAD file containing AngioChip design. Dimensions of four different AngioChips with increasing complexity. The default unit for dimensions in AutoCAD is the millimeter. The thickness of all layers is specified in micrometers

Supplementary Video 1

Step-by-step video protocol on the fabrication of AngioChip scaffolds. Step number in the video corresponds to the step number in the main protocol

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Zhang, B., Lai, B.F.L., Xie, R. et al. Microfabrication of AngioChip, a biodegradable polymer scaffold with microfluidic vasculature. Nat Protoc 13, 1793–1813 (2018). https://doi.org/10.1038/s41596-018-0015-8

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