Name: micropatterning and assembly of 3d microvessels
Uploaded: 2016-09-09T15:00:00
Description: Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

The authors would like to acknowledge the Lynn and Mike Garvey Imaging Laboratory at the Institute for Stem Cell and Regenerative Medicine as well as the Washington Nanofabrication Facility at the University of Washington. They also acknowledge the financial support of National Institute of Health grants DP2DK102258 (to YZ), and training grants T32EB001650 (to SSK and MAR) and T32HL007312 (to MAR).

1. Microfabrication of Patterned Polydimethylsiloxane (PDMS) with Network Design
<ol>
	<li>Wafer Fabrication to Create a Negative Template of the Network Design
	<ol>
		<li>Create a network pattern using any computer-aided design (CAD) software. Ensure that the diagonal dimension between the inlet and outlet match the distance between the inlet and outlet reservoirs on the housing devices in future steps (see 2.1.1). 
		Note: The design of the pattern itself is custom depending on the specific res...

<ol>
	<li>Rubanyi, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endothelium+in+cardiovascular+homeostasis+and+diseases.">The role of endothelium in cardiovascular homeostasis and diseases.</a> J. Cardiovasc. Pharmacol. 22, 37-44 (1993).</li><li>van Hinsbergh, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelium:+vascular+control+of+haemostasis.">The endothelium: vascular control of haemostasis.</a> Eur. J. Obstet. Gynecol. Reprod. Biol. 95 (2), 198-201 (2001).</li><li>Chiu, J. -. J., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Disturbed+Flow+on+Vascular+Endothelium:+Pathophysiological+Basis+and+Clinical+Perspectives.">Effects of Disturbed Flow on Vascular Endothelium: Pathophysiological Basis and Clinical Perspectives.</a> Physiol. Rev. 91, 327-387 (2011).</li><li>Qi, Y., Jiang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGF-BB+and+TGB-b1+on+cross-talk+between+endothelial+and+smooth+muscle+cells+in+vascular+remodeling+induced+by+low+shear+stress.">PDGF-BB and TGB-b1 on cross-talk between endothelial and smooth muscle cells in vascular remodeling induced by low shear stress.</a> Proc. Natl. Acad. Sci. U. S. A. 108, 1908-1913 (2011).</li><li>Sozzani, S., Del Prete, A., Bonecchi, R., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+as+effector+and+target+molecules+in+vascular+biology.">Chemokines as effector and target molecules in vascular biology.</a> Cardiovasc. Res. 107 (3), 364-372 (2015).</li><li>Huh, D., Hamilton, G. A., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+3D+cell+culture+to+organs-on-chips.">From 3D cell culture to organs-on-chips.</a> Trends Cell Biol. 21 (12), 745-754 (2011).</li><li>Staton, C. a., Reed, M. W. R., Brown, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int. J. Exp. Pathol. 90, 195-221 (2009).</li><li>Greek, R., Menache, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Reviews+of+Animal+Models:+Methodology+versus+Epistemology.">Systematic Reviews of Animal Models: Methodology versus Epistemology.</a> Int. J. Med. Sci. 10, 206-221 (2013).</li><li>van der Worp, H. B., Howells, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+Animal+Models+of+Disease+Reliably+Inform+Human+Studies.">Can Animal Models of Disease Reliably Inform Human Studies.</a> PLoS Med. 7 (3), e1000245 (2010).</li><li>Leong, X. -. F., Ng, C. -. Y., Jaarin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+in+Cardiovascular+Research:+Hypertension+and+Atherosclerosis.">Animal Models in Cardiovascular Research: Hypertension and Atherosclerosis.</a> Biomed Res. Int. 2015, 528757 (2015).</li><li>Pries, A. R., Secomb, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+Microvascular+Networks+Work:+Angiogenesis,+Remodeling,+and+Pruning.">Making Microvascular Networks Work: Angiogenesis, Remodeling, and Pruning.</a> Physiology. 29, 446-455 (2014).</li><li>D'Amore, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+Of+Angiogenesis.">Mechanisms Of Angiogenesis.</a> Annu. Rev. Physiol. 49, 453-464 (1987).</li><li>Geudens, I., Gerhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+cell+behaviour+during+blood+vessel+formation.">Coordinating cell behaviour during blood vessel formation.</a> Development. 138, 4569-4583 (2011).</li><li>Ribatti, D., Nico, B., Crivellato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+vascular+system:+a+historical+overview.">The development of the vascular system: a historical overview.</a> Methods Mol. Biol. 1214, 1-14 (2015).</li><li>Ribatti, D., Nico, B., Crivellato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+molecular+aspects+of+physiological+vascular+morphogenesis.">Morphological and molecular aspects of physiological vascular morphogenesis.</a> Angiogenesis. 12 (2), 101-111 (2009).</li><li>Bautch, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-directed+blood+vessel+patterning:+From+cells+to+organism.">VEGF-directed blood vessel patterning: From cells to organism.</a> Cold Spring Harb. Perspect. Med. 2 (9), 1-12 (2012).</li><li>Stratman, A. N., Schwindt, A. E., Malotte, K. M., Davis, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-derived+PDGF-BB+and+HB-EGF+coordinately+regulate+pericyte+recruitment+during+vasculogenic+tube+assembly+and+stabilization.">Endothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization.</a> Blood. 116, 4720-4730 (2010).</li><li>Bach, T. L., Barsigian, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VE-Cadherin+mediates+endothelial+cell+capillary+tube+formation+in+fibrin+and+collagen+gels.">VE-Cadherin mediates endothelial cell capillary tube formation in fibrin and collagen gels.</a> Exp. Cell Res. 238 (238), 324-334 (1998).</li><li>Kubow, K. E., Conrad, S. K., Horwitz, a. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+microarchitecture+and+myosin+II+determine+adhesion+in+3D+matrices.">Matrix microarchitecture and myosin II determine adhesion in 3D matrices.</a> Curr. Biol. 23 (17), 1607-1619 (2013).</li><li>Potapova, I. A., Gaudette, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cells+Support+Migration,+Extracellular+Matrix+Invasion,+Proliferation,+and+Survival+of+Endothelial+Cells+In+Vitro.">Mesenchymal Stem Cells Support Migration, Extracellular Matrix Invasion, Proliferation, and Survival of Endothelial Cells In Vitro.</a> Stem Cells. 25 (7), 1761-1768 (2007).</li><li>Bayless, K. J., Davis, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine-1-phosphate+markedly+induces+matrix+metalloproteinase+and+integrin-dependent+human+endothelial+cell+invasion+and+lumen+formation+in+three-dimensional+collagen+and+fibrin+matrices.">Sphingosine-1-phosphate markedly induces matrix metalloproteinase and integrin-dependent human endothelial cell invasion and lumen formation in three-dimensional collagen and fibrin matrices.</a> Biochem. Biophys. Res. Commun. 312 (4), 903-913 (2003).</li><li>Hellstr&#246;m, M., Gerhardt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+pericytes+leads+to+endothelial+hyperplasia+and+abnormal+vascular+morphogenesis.">Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis.</a> J. Cell Biol. 152 (3), 543-553 (2001).</li><li>Tulloch, N. L., Muskheli, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Engineered+Human+Myocardium+With+Mechanical+Loading+and+Vascular+Coculture.">Growth of Engineered Human Myocardium With Mechanical Loading and Vascular Coculture.</a> Circ. Res. 109, 47-59 (2011).</li><li>Davis, G. E., Saunders, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+balance+of+capillary+tube+formation+versus+regression+in+wound+repair:+role+of+matrix+metalloproteinases+and+their+inhibitors.">Molecular balance of capillary tube formation versus regression in wound repair: role of matrix metalloproteinases and their inhibitors.</a> J. Investig. dermatology Symp. 11 (1), 44-56 (2006).</li><li>Kannan, R. Y., Salacinski, H. J., Butler, P. E., Hamilton, G., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+prosthetic+bypass+grafts:+a+review.">Current status of prosthetic bypass grafts: a review.</a> J. Biomed. Mater. Res. B. Appl. Biomater. 74, 570-581 (2005).</li><li>Nerem, R. M., Seliktar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Tissue+Engineering.">Vascular Tissue Engineering.</a> Annu. Rev. Biomed. Eng. 3 (1), 225-243 (2001).</li><li>Melchiorri, A. J., Hibino, N., Fisher, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+and+techniques+to+enhance+the+in+situ+endothelialization+of+small-diameter+biodegradable+polymeric+vascular+grafts.">Strategies and techniques to enhance the in situ endothelialization of small-diameter biodegradable polymeric vascular grafts.</a> Tissue Eng. Part B. Rev. 19 (4), 292-307 (2013).</li><li>Abbott, W. M., Callow, A., Moore, W., Rutherford, R., Veith, F., Weinberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+performance+standards+for+arterial+prostheses.">Evaluation and performance standards for arterial prostheses.</a> J. Vasc. Surg. 17 (4), 746-756 (1993).</li><li>Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Arteries+Grown+in+Vitro.">Functional Arteries Grown in Vitro.</a> Science. 284 (5413), 489-493 (1999).</li><li>Niklason, L., Counter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+engineered+from+human+cells+-+Authors'+reply.">Blood vessels engineered from human cells - Authors' reply.</a> Lancet. 366 (9489), 892-893 (2005).</li><li>Zheng, Y., Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+microvessels+for+the+study+of+angiogenesis+and+thrombosis.">In vitro microvessels for the study of angiogenesis and thrombosis.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 9342-9347 (2012).</li><li>Zheng, Y., Chen, J., L&#242;pez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-driven+assembly+of+VWF+fibres+and+webs+in+in+vitro+microvessels.">Flow-driven assembly of VWF fibres and webs in in vitro microvessels.</a> Nat. Commun. 6, 7858 (2015).</li><li>Ligresti, G., Nagao, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Three-Dimensional+Human+Peritubular+Microvascular+System.">A Novel Three-Dimensional Human Peritubular Microvascular System.</a> J. Am. Soc. Nephrol. 27, (2015).</li><li>Roberts, M. A., Tran, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cells+in+dense+collagen+promote+cardiomyocyte+and+microvascular+patterning+in+engineered+human+heart+tissue.">Stromal cells in dense collagen promote cardiomyocyte and microvascular patterning in engineered human heart tissue.</a> Tissue Eng. Part A. , (2016).</li><li>Qin, D., Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+for+micro-+and+nanoscale+patterning.">Soft lithography for micro- and nanoscale patterning.</a> Nat. Protoc. 5 (3), 491-502 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-Step+200+Manual.">Alpha-Step 200 Manual.</a> Tencor Instruments. , (1989).</li><li>Rajan, N., Habermehl, J., Cot&#233;, M. -. F., Doillon, C. J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ready-to-use,+storable+and+reconstituted+type+I+collagen+from+rat+tail+tendon+for+tissue+engineering+applications.">Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications.</a> Nat. Protoc. 1 (6), 2753-2758 (2006).</li><li>Baudin, B., Bruneel, A., Bosselut, N., Vaubourdolle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+isolation+and+culture+of+human+umbilical+vein+endothelial+cells.">A protocol for isolation and culture of human umbilical vein endothelial cells.</a> Nat. Protoc. 2 (3), 481-485 (2007).</li><li>Leung, A. D., Wong, K. H. K., Tien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+expanders+stabilize+human+microvessels+in+microfluidic+scaffolds.">Plasma expanders stabilize human microvessels in microfluidic scaffolds.</a> J. Biomed. Mater. Res. - Part A. 100 (7), 1815-1822 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tousimis+SAMDRI-780+Critical+Point+Drying+Apparatus.">Tousimis SAMDRI-780 Critical Point Drying Apparatus.</a> Tousimis Research Corporation. , (1987).</li><li>Palpant, N. J., Pabon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+&#946;-catenin+signaling+respecifies+anterior-like+endothelium+into+beating+human+cardiomyocytes.">Inhibition of &#946;-catenin signaling respecifies anterior-like endothelium into beating human cardiomyocytes.</a> Development. 142 (18), 3198-3209 (2015).</li><li>Gimbrone, M. a., Topper, J. N., Nagel, T., Anderson, K. R., Garcia-Cardena, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Dysfunction,+Hemodynamic+Forces,+and+Atherogenesis.">Endothelial Dysfunction, Hemodynamic Forces, and Atherogenesis.</a> Thromb. Haemost. 82, 722-726 (1999).</li><li>Wu, M. H., Ustinova, E., Granger, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+binding+to+fibronectin+and+vitronectin+maintains+the+barrier+function+of+isolated+porcine+coronary+venules.">Integrin binding to fibronectin and vitronectin maintains the barrier function of isolated porcine coronary venules.</a> J. Physiol. 532 (3), 785-791 (2001).</li><li>Ribatti, D., Nico, B., Vacca, A., Roncali, L., Dammacco, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+heterogeneity+and+organ+specificity.">Endothelial cell heterogeneity and organ specificity.</a> J. Hematother. Stem Cell Res. 11, 81-90 (2002).</li><li>Shanks, N., Greek, R., Greek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+animal+models+predictive+for+humans.">Are animal models predictive for humans.</a> Philos. Ethics. Humanit. Med. 4, 2 (2009).</li></ol>

Engineered microvessels are an in vitro model where physiological characteristics such as luminal geometry, hydrodynamic forces, and multi-cellular interactions are present and tunable. This type of platform is powerful in that it offers the ability to model and study endothelial behavior in a variety of contexts where the in vitro culture conditions can be matched to that of the microenvironment in question. For example, the mechanisms driving endothelial processes, such as angiogenesis, are known to o...

The microvasculature in each organ helps define the tissue microenvironment, maintain tissue homeostasis and regulate inflammation, permeability, thrombosis, and fibrinolysis 1,2. Microvascular endothelium, in particular, is the interface between blood flow and the surrounding tissue and therefore plays a critical role in modulating vascular and organ function in response to stimuli such as hydrodynamic forces and circulating cytokines and hormones 3-5. Understanding the detailed interactions between the endothelium, blood, and the surrounding tissue microenvironment is important for the study of vascular biology and disease progression. However, progress in studying these interactions has been hindered by limited in vitro tools that do not recapitulate in vivo microvascular structure and function 6,7. As a result, the field and therapeutic advancement has relied heavily on costly and time-consuming animal models that often fail to translate to success in humans 8-10. While in vivo models are invaluable in the study of disease mechanisms and vascular functions, they are complex and often lack precise control of individual cellular, biochemical, and biophysical cues.
Vasculature throughout the body possesses a mature hierarchical structure in conjunction with expansive capillary beds, providing optimized perfusion and nutrient transport simultaneously 11. Initially, vasculature forms as a primitive plexus which reorganizes to a hierarchically branched network during early development 12,13. Although many of the signals involved in these processes are well understood 14-16, it remains elusive how such vascular patterning is determined 15. In turn, recapitulating this process in vitro to engineer organized vascular networks has been difficult. Many existing in vitro platforms to model vasculature, such as two dimensional endothelial cell cultures, lack important characteristics such as multi-cellular proximity, three dimensional luminal geometry, flow, and extracellular matrix. Tube formation assays in 3D hydrogels (collagen or fibrin) 17-19 or invasion assays 20,21 have been used to study endothelial function in 3D and their interactions with other vascular 17,22 or tissue cell types 23. However, assembled lumens in these assays lack interconnectivity, hemodynamic flow, and appropriate perfusion. Furthermore, the propensity for vascular regression in these tube formation assays 24 prevents long term culture and maturation which limits the degree of functional studies that can be performed. Thus, there is a burgeoning need to engineer in vitro platforms of microvascular networks that can appropriately model endothelial characteristics and are capable of long term culture.
A variety of vascular engineering techniques have emerged over the years for medical applications to replace or bypass affected vessels in patients with vascular disease. Large diameter vessels made from synthetic materials such as polyethylene terephthalate (PET), and polytetrafluoroethylene (ePTFE) have had considerable therapeutic success with long term patency (average 95% patency over 5 years) 25. Although small diameter synthetic grafts (&#60; 6 mm) typically face complications such as intimal hyperplasia and thrombopoiesis 26-28, tissue engineered small diameter grafts made with biological material have made significant progress 29,30. Despite advancements of this kind, engineered vessels on the microscale have remained a challenge. To adequately model the microvasculature, it is necessary to generate complex network patterns with sufficient mechanical strength to maintain patency and with a matrix composition that allows for both nutrient permeation for parenchymal cells and cellular remodeling.
This protocol presents a novel artificial perfusable vessel network that mimics a native in vivo setting with a tunable and controllable microenvironment 31-34. The described method generates engineered microvessels with diameters on the order of 100 &#181;m. Engineered microvessels are fabricated by perfusing endothelial cells through a microfluidic channel that is embedded within soft type I collagen hydrogel. This system has the capacity to generate patterned networks with open luminal structure, replicate multi-cellular interactions, modulate extracellular matrix composition, and apply physiologically relevant hemodynamic forces.

<table border="0" cellpadding="0" cellspacing="0" width="1363">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Wafer Fabrication</td>
			<td style="width:225px;"></td>
			<td style="width:140px;"></td>
			<td style="width:540px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AutoGlow Plasma System</td>
			<td>AutoGlow</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Headway Spin Coater</td>
			<td>Headway Research, Inc&#160;</td>
			<td colspan="2">PWM32 Spin Coater&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ABM Contact Aligner</td>
			<td>AB-M</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha Step Profilometer</td>
			<td>Tencor</td>
			<td>Alpha Step 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 Developer</td>
			<td>Microchem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 Resist</td>
			<td>Microchem</td>
			<td>SU-8 2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8&#34; silicon wafer</td>
			<td>Wafer World Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tabletop Micro Pattern Generator</td>
			<td>Heidelberg Instruments</td>
			<td>&#956;PG 101</td>
			<td>For generation of photomask</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot plate</td>
			<td>VWR</td>
			<td>97042-646</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ispropyl alcohol</td>
			<td>Avantor Performance Materials</td>
			<td>9088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (120 x 120 mm, square)</td>
			<td>Sigma-Aldrich</td>
			<td>Z617679</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trichloro(3,3,3-trifluoropropyl)silane</td>
			<td>Sigma-Aldrich</td>
			<td>MKBG3805V</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Polydimethylsiloxane (PDMS) elastomer base and curing agent</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td>Mixed at 10:1 (w/w)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vacuum desiccator</td>
			<td>Sigma-Aldrich</td>
			<td>Z119024-1EA</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Oven</td>
			<td>VWR</td>
			<td>9120976</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Device Fabrication and Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">poly(methyl methacrylate) (PMMA)</td>
			<td>Plexiglas</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corona Treater</td>
			<td>Electro-Technic Products, Inc.</td>
			<td>BD-20</td>
			<td>Handheld device for plasma treatment of PMMA devices and PDMS molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soldering Iron</td>
			<td>Weller&#160;</td>
			<td>WTCPS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless Steel Truss Head Slotted Machine Screw</td>
			<td>McMaster-Carr&#160;</td>
			<td>91785A096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless steel dowel pins</td>
			<td>McMaster-Carr&#160;</td>
			<td>93600A060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers&#160;</td>
			<td>Miltex</td>
			<td>24-572</td>
			<td>Any similar tweezers may be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spatula (Micro Spoon)</td>
			<td>Electron Microscopy Services</td>
			<td>62410-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screw driver</td>
			<td></td>
			<td></td>
			<td>Any flat head screwdriver may be used, autoclaved</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass coverslips (22 x 22 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bleach</td>
			<td>Clorox</td>
			<td>4460030966</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (150 X 25mm)</td>
			<td>Corning</td>
			<td>430599</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (100 X 20 mm)</td>
			<td>Corning</td>
			<td>2909</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cotton, cut into 1 cm x 3 cm pieces</td>
			<td></td>
			<td></td>
			<td>Autoclaved</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethyleneimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>P3143</td>
			<td>Dilute to 1% in cell culture grade water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G6257</td>
			<td>Dilute to 0.1% in cell culture grade water</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sterile H2O</td>
			<td></td>
			<td></td>
			<td>Autoclaved DI H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Type I collagen, dissolved in 0.1% acetic acid</td>
			<td></td>
			<td></td>
			<td>Isolated from rat tails as described in Rajan et. al. 2006 (ref #37)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 mL syringe</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL conical tubes</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">30 mL conical tubes</td>
			<td>Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 10X Media&#160;</td>
			<td>Life Technologies</td>
			<td>11825-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1N NaOH (sterile)</td>
			<td>Sigma-Aldrich</td>
			<td>415413</td>
			<td>Dilute to 1N in cell culture grade water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HUVECs&#160;</td>
			<td>Lonza</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endothelial growth media</td>
			<td>Lonza</td>
			<td>CC-3124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum (FBS)</td>
			<td>Thermofisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran from Leuconostoc spp. (70kDa)</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td>Hausser Scientific Co.</td>
			<td>3200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel loading tips</td>
			<td>VWR</td>
			<td>37001-152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18G Blunt Fill Needle</td>
			<td>BD&#160;</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20G Stainless Steel Dispensing Needle</td>
			<td>McMaster-Carr</td>
			<td>75165A123</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tygon 1/32&#8221; ID, 3/32&#34; OD Silicon Tubing</td>
			<td>Cole-Parmer</td>
			<td>EW-95702-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1/16&#34; Tube-to-tube Coupling</td>
			<td>McMaster-Carr</td>
			<td>5116K165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">90&#176; Elbow Connectors, Tube-to-Tube</td>
			<td>McMaster-Carr</td>
			<td>5121K901</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luer Lock Coupling (Female, 1/16&#34; ID)</td>
			<td>McMaster-Carr</td>
			<td>51525K211</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Forceps, with Jaw Grips</td>
			<td>Electron Microscopy Services</td>
			<td>72971</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dual Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Polystyrene Round-bottom tube</td>
			<td>Fisher Scientific</td>
			<td>14-959-2A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Device Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8806-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-9284</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-hCD31</td>
			<td>Abcam</td>
			<td>ab32457</td>
			<td>1:25 working dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FITC conjugated anti-von Willebrand Factor antibody</td>
			<td>Abcam</td>
			<td>ab8822</td>
			<td>1:100 working dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit 568 secondary antibody</td>
			<td>Thermofisher Scientific</td>
			<td>A-11011</td>
			<td>1:100 working dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoescht</td>
			<td>Thermofisher Scientific</td>
			<td>H1399</td>
			<td>Resuspended in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium cacodylate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C0250</td>
			<td>To make 0.2M cacodylate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>VWR International</td>
			<td>BDH1164-4LP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40kDa FITC-conjugated Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD40S&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Additional Culture Reagents&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR-99021</td>
			<td>Selleck Chem</td>
			<td>S2924</td>
			<td>Small molecule GSK-3 inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant VEGF</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant bFGF</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
	</tbody>
</table>

micropatterning and assembly of 3d microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

The engineered vessel platform creates functional microvasculature embedded within a natural collagen type I matrix and allows for tight control of the cellular, biophysical and biochemical environment in vitro. To fabricate engineered microvessels, human umbilical vein endothelial cells (HUVECs) are perfused through the collagen-embedded microfluidic network where they attach to form a patent lumen and confluent endothelium. As illustrated in Figure 1A-C, the ve...

Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

Micropatterning and Assembly of 3D Microvessels

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

The overall goal of using engineered micro-vessels is to replicate the structure and function of vessels in the body to study vascular physiology in normal and diseased states. This method can help answer key questions in the field of vascular biology such as how different anathema cells respond to flow, how they influence tissue function, and how vascular diseases initiate and progress over time. The main advantage of this technique is it can be easily modified to mimic different organ systems and disease states.This can be done by changing the cell types, the matrix and media composition, and other variables. Generally individuals new to this method could struggle before they become aware of common mistakes that can lead to poor vessel quality. Visual demonstration of this method is critical because there are several tricks to successful fabrication during the injection molding and assembly steps.For this procedure, have the flat and micro-patterned PDMS molds and the PMMA housing pieces prepared in advance. A description of how to make these parts is provided in the text protocol. Autoclave the PDMS molds along with the screws, pins, forceps, and a spatula.Do not autoclave the PMMA pieces. They must be sterilized in bleach under a hood. After allowing the PMMA pieces to soak in bleach for at least an hour, rinse off the bleach with autoclaved water two times.Then, place the pieces in sterile dishes. Aspirate excess water and let them air dry. With everything cleaned, start the protocol by treating the inner wells of both the top and bottom PMMA pieces using a handheld plasma treater for approximately one minute.Turn the light off to help visualize the coverage of the plasma treatment. Next, add 1%PEI onto the inner wells. If the PEI doesn't spread easily, increase the plasma treatment time.After letting the PEI react for 10 minutes, remove the solution and then rinse the devices off with sterile water and let them air dry. Once dried, apply 0.1%glutaraldehyde onto the treated surfaces and allow the glutaraldehyde to react for half an hour. Then, rinse the pieces with sterile water twice and allow them to air dry.Now, prepare fresh 0.75%neutralized collagen for vessel fabrication by combining the pre allocated stock collagen volume with the neutralization solution of medium and sodium hydroxide. Stir slowly and carefully with a spatula until the solution is homogeneous and is orange in color. Then, if needed, add cells at the desired concentration and continue to stir the solution until the cells are uniformly distributed.In preparation for the assembly process, place the micropatterned PDMS mold into a 100 by 20 millimeter dish and treat the mold with plasma for one minute. Next, align the top PMMA piece onto the PDMS so the square reservoirs line up with the inlet and outlet reservoirs. Then, put stainless steel dowel pins into the inlet and outlet reservoir holes so they will remain open after collagen injection.Now, load the collagen cell mixture into a one milliliter syringe without introducing air bubbles. Then using forceps, gently press down on the top PMMA piece to secure it to the PDMS mold and slowly inject about one half milliliter of collagen cell mixture into the injection port. The collagen must fill the 20 by 20 millimeter area over the pattern and not leak out.Then, carefully close the dish without jostling the dowel pins. Next, into the bottom PMMA piece place a 22 square millimeter glass coverslip in the inner well. Then, dispense about 0.25 milliliters of collagen evenly onto the glass.Complete the bottom half by gently lowering the flat PDMS into the collagen so it is flat against the PMMA with no air bubbles in between. Now, carefully place both halves of the device into a 37 degree Celsius incubator and allow the collagen solution to gel for 30 minutes before proceeding. After gelation, add enough PBS to surround the PDMS on the bottom piece.Then, use forceps to remove any excess collagen around the edges and slowly peel the flat PDMS piece off while keeping the collagen hydrated with PBS. To prepare the top piece, use forceps to lift the PMMA and PDMS assembly and flip it over. Then add a few drops of PBS to the inner face and remove the PDMS mold from the top PMMA piece, peeling quickly and firmly.Next, with a second pair of forceps, gently remove the pins from the other side of the PMMA housing. Ensure the inlet and outlet are clear of excess collagen by moving the pins up and down before their removal. If the pins fall out on their own, simply use another sterile dowel pin to ensure that the inlet and outlet are clear.Then, drop more PBS onto the micro patterned collagen, flip the piece over, and place screws into the four corner holes. Now, gently lay the top piece on the bottom piece aligning the screws into their holes. Do not let the two pieces slide against each other.Then, gently screw the pieces together using a spatula and a gentle touch so they are not overtightened. It is critical to be gentle and precise during assembly. By applying an ample amount of PBS, the friction between the inner face is reduced.While securing the screws, proceed slowly and stop as soon as any resistance is met. Next, aspirate the PBS surrounding the PMMA surfaces and place a small piece of cotton under one edge of the device. Then, aspirate any PBS from the reservoirs.Now, refill the reservoirs with cell culture medium and incubate the device for at least an hour for gelation. Once the device is assembled, use an endothelial cell suspension at 10 million cells per milliliter to seed the device. Remove the medium in the inlet and outlet reservoirs and using a 20 microleter pipette with gel loading tips, add 10 microliters of the cell suspension into the center of the inlet reservoir.The cells will immediately flow into and coat the network. Once the cells have filled the network and the flow has equilibrated, add 150 microliters of medium into both reservoirs. Then incubate the device for an hour or more so the cells can spread and attach.For long term culture, remove the medium every 12 hours and add back 150 microliters of fresh medium to the inlet reservoir and 50 microliters to the outlet reservoir. After 24 hours of culturing, a syringe pump can be used to establish continuous flow conditions. After bringing the materials to the bio hood, remove the tubing from the autoclave bags and place it in a dish.Then, prepare a sterile flow dish by melting side holes and then threading the tubing through. Prepare a 10 milliliter syringe with culture medium containing 3.5%Dextran. Next, attach the filled syringe to the lure lock at the end of the inlet tubing.Then, secure the syringe to the pump and profuse medium until any trapped air bubbles have exited the tubing. Once the tubing is filled with medium and is clear of bubbles, transfer the device to a new dish and insert the inlet connector joint into the housing inlet. Then, set the syringe pump to the desired flow rate and begin profusion.Prepare the outlet by filling the tubing with cell culture media using an 18 gauge needle and a one milliliter syringe. Clamping it and removing the needle. Then, melt a hole in the cap of the outlet collection tube using a soldering iron.Afterwards, add media to the collection tube, insert the tubing, and attach the connector joint into the housing outlet. Throughout this process, a clamp is used to retain the medium in the tubing. Once completed, transfer the entire setup into an incubator.Depending on the experimental conditions, the syringe may need to be refilled after 24 to 36 hours. During syringe replacement, ensure bubbles are not introduced into the tubing. Human umbilical vein and ethelial cells were profused through the collagen embedded micro fluidic network to form a patent lumen and confluent endothelium.Several vessel geometries were used to establish different flow conditions. Continuous flow culture conditions were used to apply constant shear stress on the endothelium. Devices cultured under gravity driven flow conditions also maintained long term viability and patency.A critical feature of these micro-vessels is their ability to respond to inflammatory stimuli. This was investigated by profusing the micro-vessels with whole blood. Non-stimulated micro-vessels contained non-activated endothelium and remained quiescent.Stimulated micro-vessels became activated and their response initiated thrombus formation. Another interesting application for the system is to study phenotypic changes in vasculature formed from different endothelial cell populations. For example, micro-vessels formed from unique populations of stem cells exhibited different angiogenic sprouting capabilities.After watching this video you should have a good understanding of how to fabricate micro-vessels and use them to study vascular biology and to build complex model organ systems. While attempting this procedure it is important to remember that practice will improve with the rate of successful vessel formation and allow for more consistent results. Once mastered, several devices can be made in three to four hours.Following this procedure, other methods like the profusion of whole blood, Dextran, or pharmaceutical treatments can be performed to assess endothelial quiescence, permeability, drug response, and the general morphology of cells and vessels.

Research

JoVE Journal

Bioengineering

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

A Micropatterning Assay for Measuring Cell Chirality

Chirality is an intrinsic cellular property, which depicts the asymmetry in terms of polarization along the left-right axis of the cell. As this unique ...

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...