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

micropatterning and assembly of 3d microvessels

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

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

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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

Research

JoVE Journal

Bioengineering

11.6K Views.  University of Washington. 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.

Video: Micropatterning and Assembly of 3D Microvessels

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

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

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

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

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

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

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

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

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

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

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

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

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

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

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

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

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

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

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

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

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