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PNAS 110 (17): 6712-6717

Copyright © 2013 by the National Academy of Sciences.


Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro

Duc-Huy T. Nguyena,1, Sarah C. Stapletona,1, Michael T. Yangb, Susie S. Chab, Colin K. Choib, Peter A. Galieb, and Christopher S. Chena,b,2

Departments of aChemical and Biomolecular Engineering and bBioengineering, University of Pennsylvania, Philadelphia, PA 19104

Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved March 15, 2013 (received for review December 10, 2012)

Abstract: Angiogenesis is a complex morphogenetic process whereby endothelial cells from existing vessels invade as multicellular sprouts to form new vessels. Here, we have engineered a unique organotypic model of angiogenic sprouting and neovessel formation that originates from preformed artificial vessels fully encapsulated within a 3D extracellular matrix. Using this model, we screened the effects of angiogenic factors and identified two distinct cocktails that promoted robust multicellular endothelial sprouting. The angiogenic sprouts in our system exhibited hallmark structural features of in vivo angiogenesis, including directed invasion of leading cells that developed filopodia-like protrusions characteristic of tip cells, following stalk cells exhibiting apical–basal polarity, and lumens and branches connecting back to the parent vessels. Ultimately, sprouts bridged between preformed channels and formed perfusable neovessels. Using this model, we investigated the effects of angiogenic inhibitors on sprouting morphogenesis. Interestingly, the ability of VEGF receptor 2 inhibition to antagonize filopodia formation in tip cells was context-dependent, suggesting a mechanism by which vessels might be able to toggle between VEGF-dependent and VEGF-independent modes of angiogenesis. Like VEGF, sphingosine-1-phosphate also seemed to exert its proangiogenic effects by stimulating directional filopodial extension, whereas matrix metalloproteinase inhibitors prevented sprout extension but had no impact on filopodial formation. Together, these results demonstrate an in vitro 3D biomimetic model that reconstitutes the morphogenetic steps of angiogenic sprouting and highlight the potential utility of the model to elucidate the molecular mechanisms that coordinate the complex series of events involved in neovascularization.

Key Words: 3D culture • microfabrication • microfluidics • gradient • fluid flow

Author contributions: D.-H.T.N., S.C.S., and C.S.C. designed research; D.-H.T.N., S.C.S., M.T.Y., S.S.C., and P.A.G. performed research; M.T.Y. contributed new reagents/analytic tools; D.-H.T.N., S.C.S., and P.A.G. analyzed data; and D.-H.T.N., S.C.S., M.T.Y., C.K.C., and C.S.C. wrote the paper.

1D.-H.T.N. and S.C.S. contributed equally to this work.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at

2To whom correspondence should be addressed. E-mail: chrischen{at}

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