The authors would like to thank our fellow Lam lab colleagues Ammar Chishti and Bijal Patel for their kind assistance with some of the histology and cell culture. Funding was provided by the Wayne State University Nanomedicine Fellowship (CBP), Start-Up Funds and Cardiovascular Research Institute Seed Grant (MTL).

1. Cell Culture Preparation
<ol>
	<li>Utilize human aortic smooth muscle cells purchased commercially.</li>
	<li>Maintain cells in smooth muscle cell growth media composed of 88.6% 231 media, 0.1% each of recombinant human insulin (rH-insulin), recombinant human fibroblast growth factor (rH-FGF), recombinant human epidermal growth factor (rH-FGF), and ascorbic acid; and 5% each of fetal bovine serum (FBS) and L-glutamine; and 1% antibiotic/antimycotic. 
	NOTE: Each growth factor, FBS and L-glutamine are purchased as a vascular media growth kit.</li>
	<li>Change media every 48 hours until the cells are about 90% confluent and ready for tissue seeding.</li>
	<li>Store cells in an incubator in between media changes for expansion.</li>
</ol>
2. Preparation of 3D Printed Inserts and Custom Silicone Molded Plates
<ol>
	<li>Use a commercial 3D printer (e.g., Replicator Mini) for 3D printing the plate inserts.
	<ol>
		<li>Use open source 3D design software such as Blender to create the 3D models of the printed outer-shells and posts.</li>
		<li>Export the model&#39;s driver file via a .stl format allowing for portability to the 3D printer&#39;s software.</li>
		<li>Produce printed posts and outer shells using poly(lactic acid) filament (PLA) loaded into the 3D printer.</li>
		<li>Following printing, perform a 30-minute soak in a 70%-100% ethanol solution to sterilize each insert.</li>
	</ol>
	</li>
	<li>Prepare a 1:10 curing agent to base polymer mixture of poly(dimethylsiloxane) (PDMS) silicone polymer and allow the mixture to degas at room temperature for 10 min.
	<ol>
		<li>Define petri dish sizes used as small (35 mm), intermediate (60 mm), and large (100 mm).</li>
		<li>Add 2 mL, 4 mL and 6 mL of uncured silicone to each small, intermediate and large plate, respectively, and create a thin layer across the entire bottom of the petri dish.</li>
		<li>Create posts for the small plate by pouring PDMS into a 100 mm plate to a height of 7 mm and allow to cure on a hot plate at 60 &#176;C for about 2-3 hours. Then use a 5 mm biopsy punch to punch out cylindrical posts. Use a small amount of uncured PDMS to secure each PDMS cylinder and to the center of each small plate.</li>
		<li>For the intermediate and large plates, prior to complete curing of the PDMS at the bottom of the plate, place the 3D printed posts 10 mm and 20 mm in diameter centrally into each intermediate and large plates, respectively. For the large plates, additionally place a 3D printed outer shell about 66.7 mm in diameter equidistance from the post.
		<ol>
			<li>Allow each dish to cure in open air on a hot plate at 60 &#176;C for about 2-3 hours, allowing 18 hours for degassing of the polymer. Fix printed components to the plate in the proper region and orientation as seen in Figure 1.</li>
		</ol>
		</li>
		<li>Add a solution of 70% ethanol with 30% distilled water for 30 minutes to the inside of all plates for sterilization and then cover each plate.</li>
		<li>Carefully aspirate the ethanol from each plate and allow to air dry.</li>
		<li>Arrange each plate in a biological safety cabinet (BSC) next to its lid, face-up. Expose plates and lids to UV light under the BSC for 30 minutes to complete sterilization. Sterile technique is performed with every step after UV exposure.</li>
	</ol>
	</li>
</ol>
3. Preparation of Fibrin Hydrogel, Seeding with Smooth Muscle Cells and Maintenance of Plates
<ol>
	<li>Mix a solution of fibrin gel containing growth media + 0.01% TGF-&#946;1 in the amounts of 0.5 mL, 1.1 mL and 1.81 mL for small, intermediate and large plate sizes, respectively.
	<ol>
		<li>Add 40 &#181;L, 88.4 &#181;L and 145 &#956;L of thrombin, from a stock of 100 U/mL, to the media of each small, intermediate and large plate, respectively. Gently swirl each plate by hand to ensure that thrombin is evenly mixed within the media.</li>
		<li>Next, add 160 &#181;L, 354 &#181;L and 580 &#956;L fibrinogen, from a stock of 20 mg/mL, drop-wise circularly to the thrombin-media mixture to each small, intermediate and large plate, respectively. Gently swirl by hand to ensure mixing and distribution of the hydrogel into an even layer.</li>
		<li>Allow hydrogel to cure for 10-15 minutes at room temperature.</li>
	</ol>
	</li>
	<li>Trypsinize smooth muscle cells expanded in 150 mm cell culture plates and centrifuge according to standard protocols. The resulting pellet should be resuspended in 3 mL of differentiation media consisting of 98% - 231 media, 1% FBS and 1% antibiotic/antimycotic.
	<ol>
		<li>Vigorously mix cells by titrating up and down with a 2 mL pipette to break up any cell clumps. Count cells with a hemocytometer and create a cell suspension of 2 x 106 cells/mL, 1.0 x 107 cells/mL and 1.4 x 107 cells/mL for small, intermediate and large plates, respectively.</li>
		<li>Add 1 mL of each cell suspension into a corresponding 50 mL conical labelled small, intermediate and large. Set up an additional 50 mL conical in this manner for each additional tissue ring desired.</li>
		<li>Add differentiation media to each conical to obtain final seeding volumes of 2 mL, 4 mL and 5 mL for each small, intermediate and large vessel, respectively. Then, carefully pipet the cell solution drop-wise on top of the prepared hydrogel in each corresponding plate.</li>
		<li>Place plates into the incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Change differentiation media every 48-72 hours for each plate. In the case of the larger plate, change media initially after 24 hours, then change it every 48-24 hours to compensate for the large cell seeding density.
	<ol>
		<li>After 2-4 days as the rings will have completely contracted in towards the post, add 10 &#181;L, 20 &#181;L and 35 &#956;L of TGF-&#946;1 to each small, intermediate and large ring, respectively. After exposure to TGF-&#946;1 for at least 24 hours, rings are ready to be handled.</li>
	</ol>
	</li>
</ol>
4. Assembly of Vascular Construct and Maintenance
<ol>
	<li>Before the fabrication of the final vascular construct, a specialized container is created to hold the completed vessel.
	<ol>
		<li>For the small vessel, create a tall plate for ring stacking by cutting a 2-inch section off the top of a 50 mL polycarbonate conical tube, and then PDMS glue the cut edge into a 35 mm plate. Use the conical lid as the plate lid.</li>
		<li>For the intermediate and large vessel tall ring stacking plates, cut a 1.75-inch diameter polycarbonate tube into 2.5 inch sections lengthwise to serve as the tall plate walls. For the tall plate bottoms, cut a 0.125-inch-thick polycarbonate sheet into 2-inch diameter circle pieces. Using acrylic solvent cement, bind the polycarbonate tube section to the circular cut piece. Use the lid from a 60 mm petri dish as the lid for the tall plate.</li>
		<li>3D print posts 5, 10 and 20 mm in diameter and 50 mm in length.</li>
		<li>Add 10 mL of uncured silicone to each container. Prior to the complete curing of the PDMS, centrally place each post created in step 4.1.3 into each small, intermediate, and large container.</li>
		<li>Allow to cure on a hot plate set to 60 &#176;C for 2-3 hours.</li>
		<li>Sterilize with a solution of 70% ethanol with 30% distilled water for 30 minutes.</li>
		<li>Carefully aspirate the ethanol from each container and then allow to air dry in the BSC. Next, arrange the containers in the hood with each plate placed next to its lid, face-up. Expose containers to UV light under the BSC for an additional 30 minutes for further sterilization. Use sterile technique with every step after UV exposure.</li>
	</ol>
	</li>
	<li>Using very fine forceps, carefully remove each tightly rolled smooth muscle hydrogel ring from its post and transfer to its corresponding larger container with the tall posts.
	<ol>
		<li>Use a pair of forceps in each hand and lift one side of the ring from the post, and then the other. Be careful to protect and maintain the lumen.</li>
		<li>Perform the transfer with this two handed method, sliding first one side, then the other side of the ring onto the tall post. Using gentle, gradual movements, and working circumferentially, slowly push the ring down onto the tall post. Subsequently stack tissue rings until the desired vessel length has been obtained, with each ring adding approximately 1-2 mm of length to the completed construct.</li>
	</ol>
	</li>
	<li>With the ring stack positioned on the tall 3D printed posts, turn the plate so that the post is parallel with the working surface.
	<ol>
		<li>Using a micropipette, add 40, 80 and 160 &#956;L of thrombin at a concentration of 100 U/mL gently to the outer surface of each small, intermediate and large vessel, respectively. While adding the thrombin, slowly rotate the plate to ensure even coverage of all surfaces of the construct. This will be the base for the fibrin glue utilized to secure the ring stack construct in the initial days after construction.</li>
		<li>Next, add 40, 80 and 160 &#956;L of fibrinogen at a concentration of 20 mg/mL to each small, intermediate, and large construct, respectively, using a micropipette while rotating the construct quickly. The thrombin and fibrinogen quickly set into a firm gel once mixed. Due to the short curing time, apply the fibrinogen as quickly and evenly as possible.</li>
	</ol>
	</li>
	<li>Add 20 mL of differentiation media to each container holding the construct. Place vessels into a 37 &#176;C incubator until needed. Change media every 3-5 days.</li>
</ol>

<ol>
	<li>Luciani, G. B., 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=Operative+risk+and+outcome+of+surgery+in+adults+with+congenital+valve+disease.">Operative risk and outcome of surgery in adults with congenital valve disease.</a> ASAIO J. 54 (5), 458-462 (2008).</li><li>Lawson, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+human+acellular+vessels+for+dialysis+access+in+patients+with+end-stage+renal+disease:+two+phase+2+single-arm+trials.">Bioengineered human acellular vessels for dialysis access in patients with end-stage renal disease: two phase 2 single-arm trials.</a> Lancet. 14 (387), 2026-2034 (2016).</li><li>McAllister, T. N., 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=Effectiveness+of+haemodialysis+access+with+an+autologous+tissue-engineered+vascular+graft:+a+multicentre+cohort+study.">Effectiveness of haemodialysis access with an autologous tissue-engineered vascular graft: a multicentre cohort study.</a> Lancet. 373 (9673), 1440-1446 (2009).</li><li>Wystrychowski, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+human+use+of+an+allogeneic+tissue-engineered+vascular+graft+for+hemodialysis+access.">First human use of an allogeneic tissue-engineered vascular graft for hemodialysis access.</a> J Vasc Surg. 60 (5), 1353-1357 (2014).</li><li>Konig, G., 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=Mechanical+properties+of+completely+autologous+human+tissue+engineered+blood+vessels+compared+to+human+saphenous+vein+and+mammary+artery.">Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery.</a> Biomaterials. 30 (8), 1542-1550 (2009).</li><li>Gui, 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=Construction+of+tissue-engineered+small-diameter+vascular+grafts+in+fibrin+scaffolds+in+30+days.">Construction of tissue-engineered small-diameter vascular grafts in fibrin scaffolds in 30 days.</a> Tissue Eng Part A. 20 (9-10), 1499-1507 (2014).</li><li>Sundaram, S., Echter, A., Sivarapatna, A., Qiu, C., Niklason, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-diameter+vascular+graft+engineered+using+human+embryonic+stem+cell-derived+mesenchymal+cells.">Small-diameter vascular graft engineered using human embryonic stem cell-derived mesenchymal cells.</a> Tissue Eng Part A. 20 (3-4), 740-750 (2014).</li><li>Quint, C., Arief, M., Muto, A., Dardik, A., 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=Allogeneic+human+tissue-engineered+blood+vessel.">Allogeneic human tissue-engineered blood vessel.</a> J Vasc Surg. 55 (3), 790-798 (2012).</li><li>Quint, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+tissue-engineered+blood+vessel+as+an+arterial+conduit.">Decellularized tissue-engineered blood vessel as an arterial conduit.</a> Proc Natl Acad Sci U S A. 31 (108), 9214-9219 (2011).</li><li>Dahl, S. 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=Readily+available+tissue-engineered+vascular+grafts.">Readily available tissue-engineered vascular grafts.</a> Sci Transl Med. 2 (68), (2011).</li><li>Syedain, Z. H., Meier, L. A., Lahti, M. T., Johnson, S. L., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+completely+biological+engineered+grafts+following+decellularization+into+the+sheep+femoral+artery.">Implantation of completely biological engineered grafts following decellularization into the sheep femoral artery.</a> Tissue Eng Part A. 20 (11-12), 1726-1734 (2014).</li><li>Syedain, Z. H., Meier, L. A., Bjork, J. W., Lee, A., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantable+arterial+grafts+from+human+fibroblasts+and+fibrin+using+a+multi-graft+pulsed+flow-stretch+bioreactor+with+noninvasive+strength+monitoring.">Implantable arterial grafts from human fibroblasts and fibrin using a multi-graft pulsed flow-stretch bioreactor with noninvasive strength monitoring.</a> Biomaterials. 32 (3), 714-722 (2011).</li><li>Meier, L. A., 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=Blood+outgrowth+endothelial+cells+alter+remodeling+of+completely+biological+engineered+grafts+implanted+into+the+sheep+femoral+artery.">Blood outgrowth endothelial cells alter remodeling of completely biological engineered grafts implanted into the sheep femoral artery.</a> J Cardiovasc Transl Res. 7 (2), 242-249 (2014).</li><li>Pinnock, C. B., Meier, E. M., Joshi, N. N., Wu, B., Lam, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Customizable+engineered+blood+vessels+using+3D+printed+inserts.">Customizable engineered blood vessels using 3D printed inserts.</a> Methods. S1046-2023 (15), 30184-30185 (2015).</li><li>Blakely, A. M., Manning, K. L., Tripathi, A., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-Pick,+Place,and+Perfuse:+A+New+Instrument+for+Three-Dimensional+Tissue+Engineering.">Bio-Pick, Place,and Perfuse: A New Instrument for Three-Dimensional Tissue Engineering.</a> Tissue Eng Part C Methods. 21 (7), 737-746 (2015).</li><li>Gwyther, T. A., 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=Engineered+vascular+tissue+fabricated+from+aggregated+smooth+muscle+cells.">Engineered vascular tissue fabricated from aggregated smooth muscle cells.</a> Cells Tissues Organs. 194 (1), 13-24 (2011).</li><li>Fearon, W. F., 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=Changes+in+coronary+arterial+dimensions+early+after+cardiac+transplantation.">Changes in coronary arterial dimensions early after cardiac transplantation.</a> Transplantation. 27 (6), 700-705 (2007).</li><li>Erbel, R., Eggebrecht, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+dimensions+and+the+risk+of+dissection.">Aortic dimensions and the risk of dissection.</a> Heart. 92 (1), 137-142 (2006).</li><li>Ha, D. M., 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=Transforming+growth+factor-beta+1+produced+by+vascular+smooth+muscle+cells+predicts+fibrosis+in+the+gastrocnemius+of+patients+with+peripheral+artery+disease.">Transforming growth factor-beta 1 produced by vascular smooth muscle cells predicts fibrosis in the gastrocnemius of patients with peripheral artery disease.</a> J Transl Med. 14, 39 (2016).</li><li>Skalli, O., 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=Alpha-smooth+muscle+actin,+a+differentiation+marker+of+smooth+muscle+cells,+is+present+in+microfilamentous+bundles+of+pericytes.">Alpha-smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes.</a> J Histochem Cytochem. 37 (3), 315-321 (1989).</li><li>von der Ecken, 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=Structure+of+the+F-actin-tropomyosin+complex.">Structure of the F-actin-tropomyosin complex.</a> Nature. 519 (7541), 114-117 (2015).</li></ol>

The authors have nothing to disclose.

The Ring Stacking Method presents multiple advantages over current vascular tissue engineered construct techniques. The RSM can be adapted to create human vessels of any size by simply customizing the post and outer shell dimensions. Our method allows for development of polymer-free engineered vessels composed solely of human cells and rapidly degrading support material found in the body&#39;s natural wound healing process. Polymer grafts are known to cause restenosis in the clinic and could become problematic if contain...

In treatment of coronary artery disease (CAD), a patient&#39;s own blood vessels are harvested as graft material for bypass surgery. However, oftentimes, ill patients do not have viable vessels to donate to themselves, and in cases where they do, the donor site causes considerable additional harm and has a serious risk for infection.1 Engineered vascular grafts could fill this need. Scalability is of utmost importance for engineering vessels in order to meet the wide range of patient vessel size requirements. However, present methods for engineering vessels are not easily scalable, and typically require remanufacture of complex molds or polymer scaffolds. Most engineered grafts either utilize a polymer tubular scaffold that is seeded with vascular fibroblasts, smooth muscle, or endothelial cells; or rolling a cell sheet around a mandrel to create a tissue tube. Two engineered vascular grafts in clinical trials are based on a decellularized polymer-ECM platform.2,3,4 Polymer grafts available for use in vascular repair are already known to have issues with patency, which could arise as a major issue with long-term application of a graft with a sustained polymer presence. Tubular molds have been used to fabricate completely cellular vessels,5,6,7,8,9,10,11,12,13 which procedures would require additional design and tool manufacturing for custom molds in order to produce vessels in a variety of sizes.
The method described herein encompasses a novel technique for creating easily scalable engineered vascular grafts using customizable 3D printed inserts and traditional culture plates.14 Cells are seeded into plates with inserts of a central post and outer shell. The post controls lumen diameter and allows the cell monolayer to self-assemble into a ring of tissue. The outer shell controls thickness of the ring, and thus wall thickness of the final vessel. Completed tissue rings are then stacked to form a tubular, vascular graft. The advantage to this method, termed the &#34;Ring Stacking Method,&#34; is that any adherent cell type can be seeded into the plate setup and tissue rings or tubes of any size needed for the desired application can be generated by simply modifying guide inserts. Comparative techniques in tissue engineering creating rings of tissue remain difficult to scale,15,16 requiring remanufacture of molds for each desired size. Additionally, vascular grafts made using this method can be produced in 2-3 weeks, several weeks faster compared to other engineered vessels.6 For the clinic, this time discrepancy can make a significant difference in the treatment of a deteriorating patient.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Human Aortic Smooth Muscle Cells&#160;</td>
			<td>ATCC</td>
			<td>PCS-100-012</td>
			<td>vascular smooth muscle cells</td>
		</tr>
		<tr>
			<td>Medium 231</td>
			<td>Gibco (Life Technologies&#160;</td>
			<td>M-231-500</td>
			<td>media specific to vascular smooth muscle cells</td>
		</tr>
		<tr>
			<td>Human Aortic Smooth Muscle Cell Growth Kit&#160;</td>
			<td>ATCC</td>
			<td>PSC-100-042</td>
			<td>growth factors for maintaining vascular smooth muscle cell viability</td>
		</tr>
		<tr>
			<td>Replicator Mini 3D printer&#160;</td>
			<td>MakerBot&#160;</td>
			<td>N/A</td>
			<td>3D printer</td>
		</tr>
		<tr>
			<td>Poly(lactic acid) 3D ink (PLA)</td>
			<td>MakerBot&#160;</td>
			<td>N/A</td>
			<td>3D printer filament</td>
		</tr>
		<tr>
			<td>Poly(dimethlysiloxane) (PDMS)</td>
			<td>Ellworth Adhesives&#160;</td>
			<td>3097358-1004</td>
			<td>polymer for gluing plate parts</td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Hyclone Labratories, Inc.</td>
			<td>SH30256.01</td>
			<td>fibrin gel component</td>
		</tr>
		<tr>
			<td>Thrombin&#160;</td>
			<td>Sigma Life Sciences</td>
			<td>F3879-5G</td>
			<td>fibrin gel component</td>
		</tr>
		<tr>
			<td>Tranforming Growth Factor-Beta 1&#160;</td>
			<td>PeproTech</td>
			<td>100-21</td>
			<td>growth factor for stimulating collagen production</td>
		</tr>
		<tr>
			<td>Hemocytometer&#160;</td>
			<td>Hausser Scientific Co.</td>
			<td>3200</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Polycarbonate tubing&#160;</td>
			<td>US Plastics&#160;</td>
			<td>PCTUB1.750X1.625</td>
			<td>material for making tall, ring stacking plates</td>
		</tr>
		<tr>
			<td>Polycarbonate sheet&#160;</td>
			<td>Home Depot</td>
			<td>409497</td>
			<td>material for making tall, ring stacking plates</td>
		</tr>
		<tr>
			<td>Adhesive polymer solvent&#160;</td>
			<td>SCIGRIP</td>
			<td>10799</td>
			<td>material for making tall, ring stacking plates</td>
		</tr>
		<tr>
			<td>Instron&#160; 5940</td>
			<td>Instron</td>
			<td>N/A</td>
			<td>tensile testing machine</td>
		</tr>
		<tr>
			<td>U-Stretch</td>
			<td>Cell Scale</td>
			<td>N/A</td>
			<td>tensile testing machine</td>
		</tr>
		<tr>
			<td>Smooth Muscle Actin&#160;</td>
			<td>MA5-11547</td>
			<td>Thermo Fisher</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Tropomyosin</td>
			<td>MA5-11783</td>
			<td>Thermo Fisher</td>
			<td>antibody</td>
		</tr>
	</tbody>
</table>

scaling of engineered vascular grafts using 3d printed guides and the ring stacking method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Demonstrated here is fabrication of 3 different engineered vascular graft sizes (Figure 1), showing that the Ring Stacking Method (RSM) is scalable. To prove applicability, the 3 different vessel sizes chosen correlate to actual human vessel size for the left anterior descending artery (small; lumen diameter = 4 mm)17, descending aorta (intermediate; lumen diameter = 10 mm) and ascending aorta (large; lumen diameter = 20 mm)18</su...

Watch this Scientific Journal Video about Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method at JoVE.com

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

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Research

JoVE Journal

Bioengineering

8.3K Views.  Wayne State University. Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Video: Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

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 ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk stratification in various cardiac disorders. Recently, 3D echocardiography became available in routine cardiology practice; however, high-quality image acquisition and subsequent analysis have a steep learning curve. The present article aims to guide the reader through a detailed 3D protocol by presenting tips and tricks and also by highlighting the potential pitfalls to facilitate the widespread but technically sound use of this important technique concerning the LV. First and foremost, we show the acquisition of a high-quality 3D dataset with optimal spatial and temporal resolution. Then, we present the analytical steps toward a detailed quantification of the LV by using one of the most widely applied built-in software. We will quantify LV volumes, sphericity, mass and also systolic function by measuring ejection fraction and myocardial deformation (longitudinal and circumferential strain). We will discuss and provide clinical examples about the essential scenarios where the transition from a conventional echocardiographic approach to a 3D-based quantification is highly recommended.

In this article, we provide a step-by-step acquisition and analysis protocol for the volumetric assessment and speckle-tracking analysis of the left ventricle by 3D echocardiography, particularly focusing on practical aspects that maximize the feasibility of this technique.