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

Författarna vill tacka för det Lynn och Mike Garvey Imaging Laboratory vid Institutet för Stem Cell och regenerativ medicin samt Washington Nanoteknik Facility vid University of Washington. De erkänner också ekonomiskt stöd från National Institute of Health ger DP2DK102258 (till YZ) och utbildningsbidrag T32EB001650 (till SSK och MAR) och T32HL007312 (MAR).

1. Mikro av Mönstrad Polydimetylsiloxan (PDMS) med nätverksdesign <ol><li> Wafer Fabrication att skapa en negativ mall för nätverksdesign <ol><li> Skapa ett nätverk mönster med någon datorstödd konstruktion (CAD) programvara. Säkerställa att den diagonala dimensionen mellan inloppet och utloppet är anpassat till avståndet mellan inlopps- och utloppsreservoarerna på bostadsenheter i framtida steg (se 2.1.1). Obs: Utformningen av mönstret i sig är anpassad beroende på de specifika...

<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 mikrokärl är ett in vitro-modell där fysiologiska egenskaper såsom luminal geometri, hydrodynamiska krafter, och multi-cellulära interaktioner är närvarande och avstämbar. Denna typ av plattform är kraftfull i att det ger möjlighet att modellera och studera endothelial beteende i en mängd olika sammanhang där in vitro odlingsbetingelser i kan matchas med den för mikro i fråga. Till exempel, de mekanismer som driver endotelceller processer, såsom angiogenes, är kända...

Mikrovaskulaturen i varje organ hjälper till att definiera den vävnadsmikromiljö, underhålla vävnadshomeostas och reglera inflammation, permeabilitet, trombos och fibrinolys 1,2. Mikrokärlendotel, i synnerhet, är gränssnittet mellan blodflödet och den omgivande vävnaden och spelar därför en viktig roll i att modulera vaskulär och organfunktion som svar på stimuli såsom hydrodynamiska krafter och cirkulerande cytokiner och hormoner 3 - 5. Att förstå de detaljerade interaktioner mellan endotelet, blod, och den omgivande vävnaden mikro är viktig för att studera vaskulär biologi och sjukdomsprogression. Däremot har framsteg i att studera dessa interaktioner hindrats av begränsade in vitro verktyg som inte rekapitulera in vivo mikrovaskulära struktur och funktion 6,7. Som ett resultat har området och terapeutisk avancemang relied tungt på kostsamma och tids-krävande djurmodeller som ofta misslyckas med att översätta till framgång i människor 8 - 10. Medan in vivo-modeller är ovärderliga för studier av sjukdomsmekanismer och vaskulära funktioner, de är komplexa och ofta saknar exakt kontroll av enskilda cellulära, biokemiska och biofysiska signaler. Kärl hela kroppen har en mogen hierarkisk struktur i samband med expansiva kapillärbäddar, vilket ger optimerad perfusion och näringstransport samtidigt 11. Inledningsvis kärlformer som en primitiv plexus som omorganiserar till ett hierarkiskt förgrenad nätverk under tidig utveckling 12,13. Även om många av de som är inblandade i dessa processer signaler väl förstått 14-16, är det fortfarande svårfångade hur en sådan vaskulär mönstring bestäms 15. I sin tur, rekapitulera denna process in vitro för att konstruera organiserade vaskulära nätverk har been svåra. Många befintliga in vitro-plattformar för att modellera vaskulatur, såsom tvådimensionella endoteliala cellkulturer, saknar viktiga egenskaper såsom flercelliga närhet, tredimensionellt luminal geometri, flöde, och extracellulära matrisen. Tube bildningsanalyser i 3D hydrogeler (kollagen eller fibrin) 17 - 19 eller invasionsanalyser 20,21 har använts för att studera endotelfunktion i 3D och deras interaktioner med andra vaskulära 17,22 eller vävnadscelltyper 23. Men monterade lumen i dessa analyser saknar sammankoppling, hemodynamiska flöde och lämpliga perfusion. Vidare benägenheten för vaskulär regression i dessa rörbildning analyser 24 förhindrar långtidsodling och mognad vilket begränsar graden av funktionella studier som kan utföras. Det finns således en växande behov av att konstruera in vitro plattformar mikrovaskulära nätverk som på lämpligt sätt kan modellera svdothelial egenskaper och är i stånd att långtidsodling. En mängd av vaskulära modifieringstekniker har vuxit fram under årens lopp för medicinska tillämpningar för att ersätta eller bypass påverkas fartyg hos patienter med kärlsjukdom. Fartyg med stor diameter tillverkade av syntetiska material såsom polyetylentereftalat (PET), och polytetrafluoretylen (ePTFE) har haft betydande terapeutisk framgång med långtidsöppenhet (genomsnitt 95% öppenhet över 5 år) 25. Även liten diameter syntetiska transplantat (&lt;6 mm) vanligtvis möter komplikationer såsom intimal hyperplasi och thrombopoiesis 26-28, vävnadstekniska transplantat med liten diameter som gjorts med biologiskt material har gjort betydande framsteg 29,30. Trots framsteg av detta slag har konstruerade fartyg på mikro förblev en utmaning. Att adekvat modellera mikrovaskulaturen, är det nödvändigt att generera komplexa nätverksmönster med suflig mekanisk styrka för att bibehålla öppenhet och med en matriskomposition som gör det möjligt för både närings permeation för parenkymala celler och cellulära ombyggnad. Detta protokoll presenterar en ny konstgjord perfusable fartyg nätverk som härmar en infödd in vivo miljö med en avstämbar och kontrollerbar mikro 31-34. Den beskrivna metoden genererar Engineered mikrokärl med diametrar i storleksordningen 100 nm. Engineered mikrokärl tillverkas genom perfusion endotelceller genom en mikroflödessystem kanal som är inbäddad i mjuk typ I-kollagen hydrogel. Detta system har kapacitet att generera mönstrade nätverk med öppen luminal struktur, replikera flera cellulära interaktioner, modulera extracellulär matriskomposition, och tillämpa fysiologiskt relevanta hemodynamiska krafter.

<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 plattformar för att studera endotelceller och kärlbiologi är i stort sett begränsade till 2D endotelceller kultur, flödeskammare med polymer eller glasbaserade substrat, och hydrogel-baserade rör bildningsanalyser. Dessa analyser, medan informativ, inte rekapitulera lumen geometri, ordentlig extracellulära matrisen och flercelliga närhet, som spelar nyckelroller i att modulera vaskulär funktion. Detta manuskript beskriver en formsprutningsmetod för att generera Engineered fartyg med diametrar av storleksordningen 100 ^ m. Mikrokärl tillverkas genom ympning endotelceller i en mikroflödessystem kanal inbäddad i en infödd typ I-kollagen hydrogel. Genom att införliva parenkymceller i kollagenmatrisen före kanalbildning, kan specifika vävnadsmikromiljöer modelleras och studeras. Ytterligare moduleringar av hydrodynamiska egenskaper och media sammansättning möjliggör styrning av komplexa kärlfunktionen inom det önskade mikromiljö.Denna plattform gör det möjligt för studier av perivaskulär cell rekrytering, blod endotel interaktioner, flöde svar och vävnads mikrovaskulära interaktioner. Engineered mikrokärl erbjuder möjligheten att isolera påverkan från enskilda komponenter i en kärl nisch och exakt kontrollera kemiska, mekaniska och biologiska egenskaper för att studera vaskulär biologi i både hälsa och sjukdom.

Den konstruerade fartyg plattform skapar funktionell mikrocirkulation inbäddad i en naturlig kollagen typ I matris och möjliggör strikt kontroll av den cellulära, biofysiska och biokemiska miljö in vitro. Att tillverka modifierade mikrokärl, de mänskliga navelvenendotelceller (HUVEC) perfusion genom kollagen inbäddade mikroflödes nätverk där de fäster för att bilda en patent lumen och sammanflytande endotel. Såsom illustreras i figur 1A-C, kan kär...

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

Micropatterning and Assembly of 3D Microvessels

Detta manuskript presenterar en formsprutnings metod för att konstruera mikrokärl som rekapitulera fysiologiska egenskaper endotel. Den mikroflödesbaserad process skapar patent 3D vaskulära nätverk med tailorable förhållanden, såsom flöde, cellulära sammansättning, geometri, och biokemiska gradienter. Tillverkningsprocessen och exempel på möjliga tillämpningar beskrivs.

Micropatterning och montering av 3D mikrokärl

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

Beredning av 3D koUagengeler och mikro för studien av 3D interaktioner<em&gt; In Vivo</em

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

Automatiserad robot vätskehantering montering av modulära DNA enheter

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

Syntes av katjoniserad Magnetoferritin för ultrasnabb magnetiseringen av celler

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

Montering och spårning av mikrobiell gemenskapsutveckling inom en Microwell Array-plattform

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 av brosk och hud vävnad analoger utnyttja en roman passiv enhet blandningsteknik för 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

Tillverkning av anpassade agaros brunnar för Cell sådd och vävnad Ring självmontering med 3D-tryckt formar

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

Temperaturkontrollerad montering och karakterisering av ett droppgränssnitt 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

Kontroll av cellgeometri genom infraröd laserassisterad mikropappersbehandling

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

En mikromönsteranalys för mätning av cellens kiralitet

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

Gelatinmetakryloylgranulära hydrogelställningar: Mikrogeltillverkning med hög genomströmning, frystorkning, kemisk montering och 3D-bioprinting

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