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 research goals of the user (see Figure 1 for example patterns).</li>
		<li>Generate a mask of the network pattern using high resolution printing or order a chrome photomask from a commercial supplier. A more detailed protocol of the photolithography process is available elsewhere 35.</li>
		<li>Clean an 8&#34; silicon wafer for 5 min at 100 W with oxygen plasma treatment.</li>
		<li>Pour enough negative photoresist (SU-8 2,100 works well) onto the wafer such that the resist forms a dollop with diameter of 2.5 cm. Using a spin coater, spin the wafer at 500 rpm with 100 rpm/sec ramp and maintain for 10 sec. Then ramp up to 1,600 rpm with 300 rpm/sec and maintain for 30 sec. Note: The speed and ramp need to be optimized for each laboratory condition to yield a resist thickness of 150 &#181;m.</li>
		<li>Soft-bake the wafer at 65 &#176;C for 7 min, ramp up to 95 &#176;C with 360 &#176;C/hr, and maintain for 45 min. Slowly cool down to room temperature on the hot plate. Imprint the vessel pattern onto the coated wafer by exposure to 365 nm UV light under a chrome photomask, with exposure triple that of the manufacturer&#39;s recommendation (350 mJ/cm2 for a SU-8 thickness of around 150 &#181;m). 
		Note: Since SU-8 is a negative resist, the exposed regions (everything except the pattern design) will become insoluble to the developer in step 1.1.7.</li>
		<li>Hard-bake the exposed wafer at 65 &#176;C for 5 min and ramp up to 95 &#176;C with 360 &#176;C/hr and maintain for 25 min, then allow it to slowly cool down to RT on the hot plate.</li>
		<li>Immerse the wafer in SU-8 developer for 10 - 15 min to wash away unexposed resist, then clean with isopropyl alcohol and dry under a compressed nitrogen flow. Inspect the pattern under a light microscope to ensure the SU-8 resist is well bonded to the wafer (look for unwanted peeling and bubbles).</li>
		<li>Measure the thickness of the pattern features using a profilometer according to manufacturer&#39;s instructions 36. During measurement acquisition, avoid regions of the structure that are essential for network function (i.e., inlet and outlet conduits) as a contact based profilometer could compromise the structural integrity of the patterned features on the wafer. Alternatively, use a non-contact method (i.e., optical profilometer) to avoid this issue altogether.</li>
	</ol>
	</li>
	<li>Generation of Patterned and Flat Molds for Collagen Molding 
	Note: Handle silanes within a chemical fume hood.
	<ol>
		<li>Place the wafer in a desiccator with 100 &#181;l of trichloro(3,3,3-trifluoropropyl)silane for 2 hr to silanize the surface.</li>
		<li>Transfer silanized wafer into a 120 x 120 mm square Petri dish. Pour mixed and de-gassed PDMS elastomer and curing agent (10:1 w/w ratio) over the wafer to achieve 4 - 6 mm thickness. Pour additional PDMS into a separate 120 x 120 mm square Petri dish to generate flat molds without patterns. Cure at 65 &#176;C for 2 hr.</li>
		<li>Remove from oven and allow the PDMS to cool to room temperature. Using a scalpel carefully cut a square around the SU-8 and slowly peel off the PDMS mold from the wafer. Trim edges to 30 mm x 30 mm. For flat molds, cut cured PDMS without an imprinted pattern into square pieces about 40 mm x 40 mm.</li>
	</ol>
	</li>
</ol>
2. Housing Devices
<ol>
	<li>Fabrication of Top and Bottom Housing Pieces
	<ol>
		<li>Fabricate vessel housing using poly(methyl methacrylate) (PMMA). To fabricate, order the parts from a standard machine shop with computer numerical controlled (CNC) milling capabilities. See Figure 1 for a schematic of the top and bottom pieces.</li>
		<li>Design the top housing piece (Figure 1D) to include a 20 mm x 20 mm well on the underside of the device with a depth of 1 mm, two collagen injection ports (4 mm diameters) on the top of the device located at the corners of the square well, two inlet and outlet reservoirs with 6 mm diameters, and four screw holes (3 mm diameter) at the four corners of the device. 
		Note: Additional holes may be drilled on the periphery of the piece for handling purposes.</li>
		<li>Design the bottom housing piece (Figure 1E) to include a square hole in the middle (dimensions 15 mm x 15 mm) with a surrounding 25 mm x 25 mm shelf with a depth of 0.25 mm. Drill 4 screw holes with 4-40 thread. Ensure the screw holes in the bottom and top pieces will align together during future assembly steps.</li>
	</ol>
	</li>
</ol>
3. Microvessel Device Fabrication
<ol>
	<li>Preparation of Materials
	<ol>
		<li>Wash and autoclave two sets of tweezers, a stainless steel spatula, a small flat screw driver, stainless steel screws, and several stainless steel dowel pins. For the fabrication of the vessels, also autoclave micropatterned PDMS molds, PDMS flat squares, glass coverslips (22 x 22 mm), and cotton pieces.</li>
		<li>Sterilize the PMMA housing pieces in bleach for at least 1 hr, then rinse with autoclaved water in the tissue culture hood twice, and allow to air dry in sterile tissue culture dishes (150 mm x 25 mm).</li>
	</ol>
	</li>
	<li>Sterile Polyethyleneimine-glutaraldehyde (PEI - GA) Coating
	<ol>
		<li>Treat inner wells of sterilized dry PMMA pieces with plasma for approximately 1 min. Add 1% Polyethyleneimine (PEI) onto the inner wells (the 20 mm square well on the top and the 25 mm square shelf on the bottom) of the PMMA pieces for 10 min. Rinse with sterile H2O and allow to dry in the tissue culture hood. 
		Note: The time needed for plasma treatment is variable depending on the device used - the PEI should spread easily indicating a hydrophilic surface. If not, additional plasma treatment may be necessary.</li>
		<li>Apply 0.1% glutaraldehyde (GA) onto PEI treated surfaces of the PMMA pieces for 30 min. Rinse twice with sterile water and allow to dry. 
		Note: In future steps, collagen will adhere to the coated surface through collagen-glutaraldehyde crosslinking. This helps secure the gel in place within the device during future assembly steps and throughout the device culture period.</li>
	</ol>
	</li>
	<li>Collagen Gel Preparation
	<ol>
		<li>Fabricate vessels using 0.6% - 1% collagen solutions. To make 0.75% collagen for vessel fabrication, mix type I collagen stock solution (1.5% - isolated from rat tails 37) with sodium hydroxide (NaOH),10x Media Supplement (M199), and cell culture media. Keep all solutions on ice.</li>
		<li>Determine the volume of collagen by the following equation, where V&#402;inal is the final volume of gel required (typically, 1 ml of collagen per device is sufficient), Vstock collagen is the volume of stock collagen needed, Cstock collagen is the concentration of the stock collagen, and C&#402;inal collagen is the desired concentration of the collagen in the vessel. 
		<img alt="Equation 1" src="/files/ftp_upload/54457/54457eq1.jpg" /></li>
		<li>Use a 1 ml syringe to transfer the appropriate volume of stock collagen to a new 30 ml conical tube. Determine the volumes of the neutralizing NaOH (VNaOH), M199 10x media supplement (V10X), and cell culture media (V1X) as follows and mix separately in a 15 ml conical tube: 
		<img alt="Equation 2" src="/files/ftp_upload/54457/54457eq2.jpg" /></li>
		<li>Add the mixture of neutralizing reagents (V10X, VNaOH, V1X) to the aliquoted collagen, and use a small spatula to gently mix the solution until a homogeneous gel is obtained. Avoid introducing bubbles by stirring slowly and gently.</li>
		<li>To incorporate cells into the matrix at the desired concentration (e.g., 0.5 - 20 million cells/ml), add the cells to the collagen solution after it is mixed with the neutralizing reagents, and continue to stir until the cells are homogeneously distributed.</li>
		<li>When cells are added, adjust the volume of cell culture media to accommodate the additional volume, as determined by the following equation, where Vcells is the required volume of the cell suspension, C&#402;inal cell density is the desired density of cells in the vessel, Csuspension cell density is the density of cells in the stock solution. 
		<img alt="Equation 3" src="/files/ftp_upload/54457/54457eq3.jpg" /></li>
	</ol>
	</li>
	<li>Collagen Injection - Top Piece
	<ol>
		<li>Place the micropatterned PDMS mold in a 100 mm x 20 mm dish. Plasma clean the PDMS mold for 1 min.</li>
		<li>Align the top PMMA piece on top of the PDMS mold so that the square reservoirs on the mold are directly under the inlet and outlet reservoirs (see Figure 1D - dashed lines). Place the stainless steel dowel pins into the inlet and outlet reservoir holes on the top PMMA piece to maintain clear access from the reservoirs to the pattern.</li>
		<li>Use a 1 ml syringe to extract ~ 0.5 - 0.6 ml collagen. Ensure that no bubbles remain in the syringe.</li>
		<li>Press down lightly with tweezers on the top housing to ensure flush contact between the top piece and the PDMS mold. Inject collagen slowly through an injection port on the top PMMA piece. Check that collagen fills in the 20 mm x 20 mm domain above the pattern and does not leak out.</li>
		<li>Close the dish without jostling the dowel pins and allow to gel in a 37 &#176;C incubator for 30 min.</li>
	</ol>
	</li>
	<li>Collagen Injection - Bottom Piece
	<ol>
		<li>Place a 22 x 22 mm glass coverslip in the center of the bottom piece. Use a 1 ml syringe to dispense ~ 0.25 ml collagen evenly onto the glass slide. Gently lower the PDMS flat square over the collagen, ensuring the PDMS is flat against the PMMA and there are no trapped bubbles. Allow to gel at 37 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Device Assembly
	<ol>
		<li>After gelation, add enough PBS around the flat PDMS piece on the bottom piece to surround the PDMS (about 1 ml). Use tweezers to remove any excess collagen around the edges, and slowly peel off the PDMS piece. Add more PBS on top of the collagen to maintain hydration.</li>
		<li>To prepare the top piece, use tweezers to pick up the top PMMA piece and flip it over so that the PDMS mold is on top. Add a few drops of PBS to the interface, then quickly and firmly remove the PDMS mold from the top PMMA piece.</li>
		<li>Gently remove stainless steel dowel pins from the other side of PMMA housing using another pair of tweezers. Add several more drops of PBS onto the micropatterned collagen. Flip the PMMA top piece over so that the collagen faces down and place 4 screws into the corner holes.</li>
		<li>Gently lay the top piece on top of the bottom piece, aligning the screws with the corner holes on the bottom piece. Do not allow the two pieces to slide against each other. Use a screwdriver to tighten the screws using a gentle touch. Do not overtighten.</li>
		<li>Aspirate the PBS surrounding the PMMA surfaces and place a small piece of cotton under one edge of the device. Using a pipette, remove any PBS from the reservoirs and replace with cell culture media. Allow the device to gel together in a 37&#176;C incubator for at least 1 hr.</li>
	</ol>
	</li>
	<li>Cell Seeding
	<ol>
		<li>Culture human umbilical vein endothelial cells (HUVECs) in endothelial growth media at 37 &#176;C (CO2, O2) to confluency 38. Whenever possible, use between passages 4 and 7.</li>
		<li>Trypsinize endothelial cells by washing the culture flask with 6 ml of PBS and then adding 2 ml of 0.05% trypsin at 37 &#176;C to dissociate the cells. Leave the cells in trypsin until most of the focal adhesions have detached and the cells are rounded up (this typically takes about 2 - 3 min). Stop the trypsin reaction by adding 4 ml of endothelial growth media containing fetal bovine serum (FBS). Wash the flask with the media several times to ensure the cells are detached from the flask and collect in a conical tube.</li>
		<li>Count the cells using a hemocytometer and resuspend them at a density of 10 million cells per ml. Remove all cell culture media from the inlet and outlet reservoirs. Using a 200 &#181;l gel loading tip, add 10 &#181;l of cell suspension into the center of the inlet reservoir. The cells should begin to flow into the network immediately and coat the network.</li>
		<li>Add 200 &#181;l media equally to both reservoirs and let cells attach and spread within the network for at least 1 hr.</li>
	</ol>
	</li>
	<li>Device Culture
	<ol>
		<li>Culture the device with gravity driven flow through the network by adding 200 &#181;l of cell culture media to the inlet reservoir and 50 &#181;l to the outlet reservoir. With this method of culture, replace media every 12 hr to maintain endothelial viability. 
		Note: If continuous flow culture conditions are desirable (to maintain constant shear stress, collect media at the outlet, etc.) a syringe pump may be used instead of gravity driven culture.</li>
		<li>Allow the seeded endothelial cells at least 24 hr to attach and stabilize before applying continuous flow. Prior to continuous flow setup, maintain the devices with gravity driven flow conditions. 
		NOTE: The media conditions for applied flow differ from gravity driven culture. The addition of a plasma expander, such as dextran, to the media stabilizes the engineered microvessels and prevents vascular collapse during sustained perfusion 39.</li>
		<li>To make media for continuous flow setup, dissolve 3.5% Dextran (70 kDa) in endothelial growth media for optimal vessel culture. Using sterile technique, fill 10 ml syringes with endothelial growth media with 3.5% Dextran.</li>
		<li>Prepare a sterile culture dish for flow by melting holes in the side wall of a Petri dish using a soldering iron.</li>
		<li>Attach 12&#34; silicon tubing (1/32&#34; ID) to a Luer lock coupling (Female, 1/16&#34; ID), and a 1/16&#34; straight tube-to-tube connector with a 1&#34; segment of tubing on the other end. Insert a 1/4&#34; 20G blunt needle (detached from the needle hub) to the free end of the 1&#34; segment. Also prepare a 3&#34; segment of tubing attached to a tube-to-tube 90&#176; elbow connector joint (to be fitted in the housing inlet). In subsequent steps, the longer tubing will be threaded through the prepared dish and attached to the 3&#34; segment via the 20 G needle. Outlet tubing should mirror inlet tubing, with a free end instead of a Luer lock coupling. Autoclave all segments prior to use.</li>
		<li>Thread autoclaved inlet and outlet tubing through holes in the prepared dish. Attach 3&#34; segments to inner 20 G needles. Attach the Luer lock coupling to the media-filled syringe and perfuse media through the tubing using the syringe pump set at a high flow rate. Ensure that there are no bubbles in the tubing or connectors, as these will impede flow to the vessel.</li>
		<li>Insert the connector joint into the housing inlet. Set the syringe pump to the desired flow rate and begin perfusion. 
		NOTE: This can be adjusted depending on the vessel geometry and experimental conditions of applied shear stresses. For a 100 &#181;m diameter channel, a flow rate of 3 &#181;l/min works well.</li>
		<li>If desired, collect perfusate with prepared outlet tubing inserted into a sterile 5 ml polystyrene round bottom tube containing 500 &#181;l of media. 
		Note: A small amount of media is added to the collecting tube to prevent bubble formation that can block flow.</li>
		<li>Depending on flow rate, the syringe may need to be refilled after 24 to 36 hr. This is done by removing the connector from the housing inlet, replacing the syringe, and allowing media to perfuse at a high flow rate for several min. This sequence ensures that bubbles are not introduced to the tubing. Reset the pump to the desired flow rate for culture, insert the connector into the housing inlet, and return to the incubator.</li>
	</ol>
	</li>
</ol>
4. Device Analysis
<ol>
	<li>Permeability Analysis
	<ol>
		<li>Use 40 kDa FITC-Dextran to visualize the permeability of the vessel in situ. Place the vessel housing on a confocal microscope and focus at the vessel plane. Add 5 &#181;M 40 kDa FITC-Dextran to the inlet and image at 4X magnification, 25 msec exposure time, and at 1 frame per sec for 10 min.</li>
		<li>Analyze the image series with analysis code to estimate the permeability of Dextran across the vessel wall into collagen (&#181;m/sec) based on a model published by Zheng et. al. 31.</li>
	</ol>
	</li>
	<li>Immunostaining &#38; Confocal Imaging Analysis
	<ol>
		<li>To fix vessels, perfuse with 3.7% formaldehyde through the inlet for 20 min and wash by perfusing PBS three times (20 min per wash). Block nonspecific antibody binding with 2% bovine serum albumin (BSA) and 0.5% Triton X-100 perfused through the network for 1 hr. Perfuse primary antibody solutions overnight at 4 &#176;C and wash three times with PBS. Perfuse secondary antibody solutions for 1 - 2 hr, and wash again with PBS in the same manner.</li>
		<li>Take immunofluorescence images of microvessels in situ using a confocal microscope with a z-step of 1 &#181;m. Image the microvessels at a magnification of 4X, 10X, or 20X. 
		Note: Magnification of 4X will provide global network structure information, while 10X and 20X magnified images will provide cellular details such as elongation, junction formation, etc.</li>
		<li>Analyze the image stacks using ImageJ with Z projections (Image/Stacks/Z-project), cross sections (Image/Stacks/Orthogonal view), and 3D reconstructions (Image/Stacks/3D Project).</li>
	</ol>
	</li>
	<li>Scanning Electron Microscopy Imaging
	<ol>
		<li>For electron microscopy analysis, fix in situ by perfusing half-strength Karnovsky&#39;s solution (2% paraformaldehyde/2.5% glutaraldehyde in 0.2 M cacodylate buffer) overnight. Disassemble the vessel and carefully separate the two pieces. Trim edges and fully immerse in the fixative solution for several days.</li>
		<li>Immerse the thick top portion of the vessel in 25% glutaraldehyde for 20 min and rinse three times with PBS (2 min each). Dehydrate in serial ethanol washes of 50%, 70%, 85% (2 min each) and two washes in 100% ethanol (5 min each).</li>
		<li>Prepare the sample for analysis with further dehydration by critical point drying following the manufacturer&#39;s protocol 40. Sputter coat the vessel with gold-palladium (7 nm) and analyze under a scanning electron microscope with an accelerating voltage of 5 kV, spot size 3.</li>
	</ol>
	</li>
</ol>

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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. 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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>

The authors have nothing to disclose.

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

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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 ...

micropatterning-and-assembly-of-3d-microvessels

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JoVE Journal

Bioengineering

11.7K Views.  University of Washington.  Dit manuscript bevat een spuitgiet methode om microvaten dat fysiologische eigenschappen van endotheel recapituleren ingenieur. De microfluïdische-based proces creëert patent 3D vasculaire netwerken met tailorable omstandigheden, zoals stroom, cellulaire samenstelling, geometrie, en biochemische hellingen. Het fabricageproces en voorbeelden van mogelijke toepassingen worden beschreven. 

Video: Micropatterning and Assembly of 3D Microvessels

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.

The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates.

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material ...

Engineering

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Biology

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

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 compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

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

Micropatterning and Assembly of 3D Microvessels

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