The vascular endothelium forms the inner lining of blood vessels and actively regulates vascular permeability in response to chemical and physical stimuli. Understanding the molecular pathways and mechanisms that regulate the permeability of blood vessels is of critical importance for developing therapies for cardiovascular dysfunction and disease. Recently, we developed a novel microfluidic human engineered microvessel (hEMV) platform to enable controlled blood flow through a human endothelial lumen within a physiologic 3D extracellular matrix (ECM) into which pericytes and other stromal cells can be introduced to recapitulate tissue-specific microvascular physiology. This protocol describes how to design and fabricate the silicon hEMV device master molds (takes ~1 week) and elastomeric substrates (takes 3 d); how to seed, culture, and apply calibrated fluid shear stress to hEMVs (takes 1–7 d); and how to assess vascular barrier function (takes 1 d) and perform immunofluorescence imaging (takes 3 d).
Subscribe to Journal
Get full journal access for 1 year
only $21.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
The custom code used for the current study is provided in the Supplementary Data.
Ayata, C. & Ropper, A. H. Ischaemic brain oedema. J. Clin. Neurosci. 9, 113–124 (2002).
Dongaonkar, R. M., Stewart, R. H., Geissler, H. J. & Laine, G. A. Myocardial microvascular permeability, interstitial oedema, and compromised cardiac function. Cardiovasc. Res. 87, 331–339 (2010).
Hahn, C. & Schwartz, Ma Mechanotransduction in vascular physiology and atherogenesis. Nat. Rev. Mol. Cell Biol. 10, 53–62 (2009).
Heron, M. Deaths: leading causes for 2013. Natl. Vital Stat. Rep. 65, 1–95 (2016).
Chauhan, V. P., Stylianopoulos, T., Boucher, Y. & Jain, R. K. Delivery of molecular and nanoscale medicine to tumors: transport barriers and strategies. Annu. Rev. Chem. Biomol. Eng. 2, 281–298 (2011).
Mehta, D. & Malik, A. B. Signaling mechanisms regulating endothelial permeability. Physiol. Rev. 86, 279–367 (2006).
Tarbell, J. M. Mass transport in arteries and the localization of atherosclerosis. Annu. Rev. Biomed. Eng. 5, 79–118 (2003).
DePaola, N. et al. Electrical impedance of cultured endothelium under fluid flow. Ann. Biomed. Eng. 29, 648–656 (2001).
Tarbell, J. M. Shear stress and the endothelial transport barrier. Cardiovasc. Res. 87, 320–330 (2010).
Caplan, B. B. A., Gerrity, R. G. & Schwartz, C. C. J. EC morphology in focal areas of in vivo evans blue uptake in young pig aorta. Exp. Mol. Pathol. 21, 102–117 (1974).
Bergers, G. & Song, S. The role of pericytes in blood-vessel formation and maintenance. Neuro Oncol. 7, 452–464 (2005).
Wong, K. H. K., Chan, J. M., Kamm, R. D. & Tien, J. Microfluidic models of vascular functions. Annu. Rev. Biomed. Eng. 14, 205–230 (2012).
Huber, D., Oskooei, A., Casadevall Solvas, X., Demello, A. & Kaigala, G. V. Hydrodynamics in cell studies. Chem. Rev. 118, 2042–2079 (2018).
Polacheck, W. J. et al. A non-canonical Notch complex regulates adherens junctions and vascular barrier function. Nature 552, 258–262 (2017).
Alimperti, S. et al. Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and N-cadherin balance in mural cell–endothelial cell-regulated barrier function. Proc. Natl Acad. Sci. USA 114, 8758–8763 (2017).
Mccurley, A. et al. Inhibition of avb5 Integrin attenuates vascular permeability and protects against renal ischemia-reperfusion injury. J. Am. Soc. Nephrol. 28, 1741–1752 (2017).
Chang, B. Mouse models for studies of retinal degeneration and diseases. in Retinal Degeneration: Methods and Protocols (eds. Weber, B. H. F. & Langmann, T.) 27–39 (Humana Press, Totowa, NJ, 2012).
Meijer, E. F. J., Baish, J. W., Padera, T. P. & Fukumura, D. Measuring vascular permeability in vivo. Methods Mol. Biol. 1458, 71–85 (2016).
Gerlowski, L. E. & Jain, R. K. Microvascular permeability of normal and neoplastic tissues. Microvasc. Res. 31, 288–305 (1986).
Yuan, F., Leunig, M., Berk, D. A. & Jain, R. K. Microvascular permeability of albumin, vascular surface area, and vascular volume measured in human adenocarcinoma LS174T using dorsal chamber in SCID mice. Microvasc. Res. 45, 269–289 (1993).
Siflinger-Birnboim, A. et al. Molecular sieving characteristics of the cultured endothelial monolayer. J. Cell. Physiol. 132, 111–117 (1987).
Chien, S. Effects of disturbed flow on endothelial cells. Ann. Biomed. Eng. 36, 554–562 (2008).
Dewey, C. F., Bussolari, S. R., Gimbrone, M. A. & Davies, P. F. The dynamic response of vascular endothelial cells to fluid shear stress. J. Biomech. Eng. 103, 177–185 (1981).
Bischoff, I. et al. Pitfalls in assessing microvascular endothelial barrier function: Impedance-based devices versus the classic macromolecular tracer assay. Sci. Rep. 6, 1–11 (2016).
Crone, C. & Olesen, S. P. Electrical resistance of brain microvascular endothelium. Brain Res. 241, 49–55 (1982).
Tiruppathi, C., Malik, A. B., Del Vecchio, P. J., Keese, C. R. & Giaever, I. Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proc. Natl. Acad. Sci. USA 89, 7919–7923 (1992).
Srinivasan, B. et al. TEER measurement techniques for in vitro barrier model systems. J. Lab. Autom. 20, 107–126 (2015).
Griep, L. M. et al. BBB on CHIP: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function. Biomed. Microdevices 15, 145–150 (2013).
Zheng, Y. et al. In vitro microvessels for the study of angiogenesis and thrombosis. Proc. Natl. Acad. Sci. USA 109, 9342–9347 (2012).
Zervantonakis, I. K. et al. Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function. Proc. Natl. Acad. Sci. USA 109, 13515–13520 (2012).
Farahat, Wa et al. Ensemble analysis of angiogenic growth in three-dimensional microfluidic cell cultures. PLoS ONE 7, e37333 (2012).
Morgan, J. P. et al. Formation of microvascular networks in vitro. Nat. Protoc. 8, 1820–1836 (2013).
Sobrino, A. et al. 3D microtumors in vitro supported by perfused vascular networks. Sci. Rep. 6, 1–11 (2016).
Kim, S., Chung, M., Ahn, J., Lee, S. & Jeon, N. L. Interstitial flow regulates the angiogenic response and phenotype of endothelial cells in a 3D culture model. Lab Chip 16, 4189–4199 (2016).
Chen, M. B. et al. On-chip human microvasculature assay for visualization and quantification of tumor cell extravasation dynamics. Nat. Protoc. 12, 865–880 (2017).
Nguyen, D.-H. T. et al. Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc. Natl. Acad. Sci. USA 110, 6712–6717 (2013).
Linville, R. M., Boland, N. F., Covarrubias, G., Price, G. M. & Tien, J. Physical and chemical signals that promote vascularization of capillary-scale channels. Cell. Mol. Bioeng. 9, 73–84 (2016).
Geraldo, S. et al. Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo? Eur. J. Cell Biol. 91, 930–937 (2012).
Galie, P.a et al. Fluid shear stress threshold regulates angiogenic sprouting. Proc. Natl. Acad. Sci. USA 111, 7968–7973 (2014).
Capes, D. F. et al. Fluctuations in syringe-pump infusions: association with blood pressure variations in infants. Am. J. Health Syst. Pharm. 52, 1646–1653 (1995).
Deen, W. M. Analysis of Transport Phenomena (Oxford University Press, New York, 1998).
Adamson, R. H., Lenz, J. F. & Curry, F. E. Quantitative laser scanning confocal microscopy on single capillaries: permeability measurement. Microcirculation 1, 251–265 (1994).
Dejana, E., Orsenigo, F. & Lampugnani, M. G. The role of adherens junctions and VE-cadherin in the control of vascular permeability. J. Cell Sci. 121, 2115–2122 (2008).
Sung, K. E. et al. Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices. Biomaterials 30, 4833–4841 (2009).
Cines, D. B. et al. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91, 3527–3561 (1998).
Song, J. W. & Munn, L. L. Fluid forces control endothelial sprouting. Proc. Natl. Acad. Sci. USA 108, 15342–15347 (2011).
Polacheck, W. J., German, A. E., Mammoto, A., Ingber, D. E. & Kamm, R. D. Mechanotransduction of fluid stress guides 3D cell migration. Proc. Natl. Acad. Sci. USA 111, 2447–2452 (2014).
Trappmann, B. et al. Matrix degradability controls multicellularity of 3D cell migration. Nat. Commun. 8, 1–8 (2017).
Funamoto, K. et al. A novel microfluidic platform for high-resolution imaging of a three-dimensional cell culture under a controlled hypoxic environment. Lab Chip 12, 4855–4863 (2012).
Armulik, A. et al. Pericytes regulate the blood–brain barrier. Nature 468, 557–561 (2010).
Daneman, R., Zhou, L., Kebede, A. A. & Barres, B. A. Pericytes are required for blood–brain barrier integrity during embryogenesis. Nature 468, 562–566 (2010).
Bautch, V. L. Stem cells and the vasculature. Nat. Med. 17, 1437–1443 (2011).
Au, P., Tam, J., Fukumura, D. & Jain, R. K. Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood 111, 4551–4558 (2008).
This work was supported in part by grants from the National Institutes of Health (EB00262, EB08396, UH3EB017103, and HL115553) and the National Science Foundation Center for Engineering MechanoBiology (CMMI15-48571). W.J.P. acknowledges support from a Ruth L. Kirchstein National Research Service Award (F32 HL129733) and from the NIH through the Organ Design and Engineering Training program (T32 EB16652); M.L.K. acknowledges support from the Hartwell Foundation and from the National Institutes of Health (K99-CA226366-01A1); and J.B.T. acknowledges support from the NIH through the Translational Research in Biomaterials Training Program (T32 EB006359).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Polacheck, W. J. et al. Nature 552, 258–262 (2017): https://doi.org/10.1038/nature24998
Alimperti, S. et al. Proc. Natl. Acad. Sci. USA 114, 8758–8763 (2017): https://doi.org/10.1073/pnas.1618333114
McCurley, A. et al. J. Am. Soc. Nephrol. 28, 1741–1752 (2017): https://doi.org/10.1681/ASN.2016020200
About this article
Cite this article
Polacheck, W.J., Kutys, M.L., Tefft, J.B. et al. Microfabricated blood vessels for modeling the vascular transport barrier. Nat Protoc 14, 1425–1454 (2019). https://doi.org/10.1038/s41596-019-0144-8
The effects of luminal and trans-endothelial fluid flows on the extravasation and tissue invasion of tumor cells in a 3D in vitro microvascular platform
Microfluidic and Organ-on-a-Chip Approaches to Investigate Cellular and Microenvironmental Contributions to Cardiovascular Function and Pathology
Frontiers in Bioengineering and Biotechnology (2021)
ACS Central Science (2020)