Versatile synthetic alternatives to Matrigel for vascular toxicity screening and stem cell expansion

Abstract

The physiological relevance of Matrigel as a cell-culture substrate and in angiogenesis assays is often called into question. Here, we describe an array-based method for the identification of synthetic hydrogels that promote the formation of robust in vitro vascular networks for the detection of putative vascular disruptors and that support human embryonic stem cell expansion and pluripotency. We identified hydrogel substrates that promote endothelial-network formation by primary human umbilical vein endothelial cells and by endothelial cells derived from human-induced pluripotent stem cells, and used the hydrogels with endothelial networks to identify angiogenesis inhibitors. The synthetic hydrogels showed superior sensitivity and reproducibility over Matrigel when known inhibitors were evaluated, as well as in a blinded screen of a subset of 38 chemicals, selected according to predicted vascular disruption potential, from the Toxicity ForeCaster library of the United States Environmental Protection Agency. We propose that the identified synthetic hydrogels are suitable alternatives to Matrigel for common cell-culture applications.

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Figure 1: An endothelial-cell-culture system identifies environments that enable endothelial-network formation on synthetic PEG-based hydrogels.
Figure 2: Visualization and quantification of endothelial networks on synthetic hydrogels and Matrigel.
Figure 3: Concentration-dependent inhibition of endothelial-network formation by the vascular inhibitors Vatalanib (PTK787), Semaxinib (SU5416) and sFlt-1, measured as percent change in network area compared with vehicle controls.
Figure 4: Concentration-dependent inhibition of endothelial-network formation by the vascular inhibitors anti-VEGF, Prinomastat hydrochloride and Sutent, measured as percent change in network area compared with vehicle controls.
Figure 5: Identification of vascular inhibitors from a subset of candidate chemical compounds from the ToxCast library with the use of synthetic hydrogel versus Matrigel systems.
Figure 6: Material-dependent maintenance of hESC pluripotency.

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Acknowledgements

The authors acknowledge funding from the National Institutes of Health (NIH; R01HL093282-01A1, R21EB016381-01, 1UH2TR000506-01, T32HL007889, T32HL07936, R01EB10039, R24 EY022883, R01 EY026078, P30 EY016665, P30 CA 014520 and 5 P30 CA 014520-01), the Biotechnology Training Program (NIGMS5T32GM08349), the National Science Foundation (GE-0718123), the University of Wisconsin-Madison Graduate Engineering Research Scholars program, the Environmental Protection Agency (STAR grant no. 83573701), the Chemical Safety for Sustainability Research Program, the Office of Research and Development, the Virtual Tissue Models Project and the National Center for Computational Biology, the University of Wisconsin-Madison Molecular and Environmental Toxicity Center Training Program (NIH T32 ES007015), the Gates Millennium Scholars Program and the Retina Research Foundation, and an unrestricted departmental award from Research to Prevent Blindness. N.S. was a recipient of the Research to Prevent Blindness Stein Innovation Award. Mechanical testing data were obtained using the Ares LS2 rheometer at the University of Wisconsin-Madison Soft Materials Laboratory. This study made use of the National Magnetic Resonance Facility at Madison, which is supported by NIH grant P41GM103399 (NIGMS; old number: P41RR002301). Equipment was purchased with funds from the University of Wisconsin-Madison, the NIH (P41GM103399, S10RR02781, S10RR08438, S10RR023438, S10RR025062 and S10RR029220), the NSF (DMB-8415048, OIA-9977486 and BIR-9214394) and the United States Department of Agriculture. The authors acknowledge M. L. Dombroe for assistance with the HUVEC cultures and the laboratory of O. Mezu-Ndubuisi for providing mice for use in the aortic ring sprouting assays. The US Environmental Protection Agency (EPA), through its Office of Research and Development, funded and managed part of the research described here. The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the EPA.

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Contributions

E.H.N., W.T.D., C.S.L., M.F., D.G.B., T.B.K. and G.E.A. contributed to the conception and design of the endothelial-network experiments. E.H.N., W.T.D., M.F., M.P.S., C.S.L. and M.A.S. contributed to the execution of the endothelial-network experiments. E.H.N., W.T.D., M.F., M.P.S., C.S.L., D.G.B., M.A.S. and T.B.K. contributed to the analysis and figure preparation of the endothelial-network experiments. N.N.T.L. contributed to the conception, design and execution of the hESC experiments. E.H.N. and N.N.T.L. contributed to the analysis and figure preparation of the hESC experiments. E.H.N. and C.S.L. contributed to the conception, design and execution of the hydrogel characterization experiments. E.H.N., W.T.D., T.B.K. and W.L.M. drafted the manuscript. N.S. and W.L.M. supervised the work throughout data collection and manuscript preparation. W.L.M. approved the final version of the manuscript.

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Correspondence to William L. Murphy.

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Competing interests

C.S.L. is an employee and stockholder of Stem Pharm. W.L.M. is a founder and stockholder of Stem Pharm.

Supplementary information

Supplementary Information

Supplementary discussion, results, methods, figures and references. (PDF 3663 kb)

Supplementary Video 1

Endothelial-network formation by HUVECs over the course of 24 hours after seeding onto PEG hydrogels. (WMV 9353 kb)

Supplementary Video 2

Endothelial-network formation by HUVECs over the course of 24 hours after seeding onto Matrigel. (WMV 6650 kb)

Supplementary Video 3

Endothelial-network formation by iPSC-ECs over the course of 24 hours after seeding onto PEG hydrogels. (WMV 12369 kb)

Supplementary Video 4

Formation process of the thin hydrogel array. (MP4 25323 kb)

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Nguyen, E., Daly, W., Le, N. et al. Versatile synthetic alternatives to Matrigel for vascular toxicity screening and stem cell expansion. Nat Biomed Eng 1, 0096 (2017). https://doi.org/10.1038/s41551-017-0096

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