This work is funded by National Institutes of Health Grant R01 EB-008836 and R01 HL083895 (both to L.E.N.). We could like to thank Daryl Smith, the University Glassblower, for making the bioreactors for our research.

Autoclave
Assemble and autoclave the tubing for the flow system and bioreactor components (bioreactor itself and the silicone stopper lid) as instructed in Figure 1 and Figure 2. Feeding tube has a male connector on one end and an open end on the other side. Three short tubing segments are inserted through a silicone cap for gas exchange.
1. Sewing PGA Mesh
<ol>
	<li>Cut PGA mesh to 1.1cm x ~8cm sheet (dependent on bioreactor size).</li>
	<li>Clean silicone tubing (3mm inner diameter) with distilled water (dH20) and air dry before usage.</li>
	<li>Use Dexon 6.0 suture to sew PGA mesh around the clean silicone tubing starting with three surgical knots followed by single stitches.</li>
</ol>
2. PGA Scaffolds Surface Treatment
<ol>
	<li>Dip PGA scaffolds in 1M NaOH for 1-2min and record the treatment time and use the same time for all PGA scaffolds.</li>
	<li>Rinse PGA scaffolds in dH2O baths for 2 minutes 3 times.</li>
	<li>Pat PGA scaffolds dry with Kimwipes between each dip in dH2O baths.</li>
	<li>Dry PGA scaffolds under tissue culture hood to air dry for 15 minutes with blower on.</li>
</ol>
3. Sewing Dacron arms
<ol>
	<li>Use Prolene 4.0 suture to sew small pieces of Dacron cuffs (1cm) onto each end of the PGA mesh with an overlap of 2-3mm. Note: be careful not to puncture silicone tubing (Figure 3).</li>
	<li>Use the same suture to sew three stitches around the free end of Dacron cuffs. Make sure to leave enough sutures at the free ends of Prolene suture for step 5.1 (Figure 3). </li>
</ol>
4. Assembly of Bioreactor (Day before beginning of bioreactor culture)
<ol>
	<li>Soak sewn mesh and silicone tubing, surgical instruments, and a thin wire in a 70% ethanol bath for 20-30 minutes.</li>
	<li>Open the autoclave pouches containing the bioreactor and submerge it in 70% ethanol bath for at least 30 minutes prior to assembling. Make sure to flush the bioreactor thoroughly with ethanol. Exposure of all bioreactor components to 70% ethanol is an addition sterilization step. This step also removes endotoxin, which is not removed by autoclaving and is deleterious to the vascular cells.</li>
	<li>Pull silicone tubing through side-arms using thin wire to pull it through.</li>
	<li>Take bioreactor out of ethanol bath (keep mesh submerged in ethanol). Fix the PGA scaffold inside the bioreactor by fastening Dacron cuffs over the flared glass lips by tightening and tying down the Prolene sutures (Figure 3).</li>
	<li>Connect one side of bioreactor side arms to connectors via silicone tubing.</li>
	<li>Pull other side of silicone tubing with enough tension and insert the remaining two connectors into silicone tubing. Hold on to silicone tubing tightly when inserting connectors!</li>
	<li>Reinsert bioreactor into ethanol bath and flush with ethanol by gently pulling connectors out of the side arms.</li>
	<li>Flip bioreactor over and allow a soak for 10 minutes.</li>
	<li>Flip bioreactor right-side up and soak for an additional 10 minutes.</li>
	<li>Drain all ethanol.</li>
	<li>Set up three large petri dishes (10 cm) in series and place bioreactor in the center dish to hold the excess ethanol dripped from the ends of the tubes.</li>
	<li>Flush bioreactor and PGA mesh with tissue culture water using a 5ml or 10ml pipette. Also flush tissue culture water into silicone tubing.</li>
	<li>Thoroughly drain all excess water into petri dishes on either side of the bioreactor.</li>
	<li>Dry bioreactor overnight in hood with blower on and UV OFF.</li>
	<li>Additional Notes: make sure sterile stir bar is in bioreactor. Make sure not to "hover" over the bioreactor from this point forward, to avoid contamination. Cut up plenty of parafilm strips and soak them in a small 70% ethanol bath (large petri dish works well).</li>
</ol>
5. Day 1: Bioreactor Setup 
<ol>
	<li>Place a sterile petri dish over opening of each bioreactor to protect the PGA scaffold inside from contaminants.</li>
	<li>Assemble flow system to bioreactor as indicated in Figure 4 and parafilm all the connection joints.
		<ol>
			<li>Wipe connectors first with alcohol wipes.</li>
			<li>Attach injection port to third, unused arm of bioreactor.</li>
			<li>Remove walrus tubing and tie off blue end of saline dilution set as close to Y-junction as possible. Pull tube clamp in place to ensure no liquid transfer to this part of the tube</li>
			<li>Remove IV bag, &amp; attach walrus tubing (red) to far end of IV bag. Make sure to wipe insertion port with alcohol wipe first.</li>
			<li>Attach walrus to one side of flow system (via white end tube). </li>
			<li>Insert 3-way stopcock into flow system.</li>
			<li>Remove pressure transducer &amp; connect to three-way stopcock.</li>
			<li>Attach other end of pressure transducer to middle opening in bag.</li>
		</ol>
	</li>
	<li>Connect the bioreactor to the flow system via the Y-junction. Use a 60ml syringe to add 350ml of 1% fungizone (mix 5ml fungizone with 495ml of PBS) to IV bag.</li>
	<li>Squeeze IV bag to flush the flowing system by adjusting the stop cocks to allow flow of PBS. Note: check inside bioreactor to ensure there is no leaking.</li>
	<li>Re-suspend 8 x106 SMCs (about one confluent T75) in 1.25ml of medium and seed onto each PGA scafffold. Make sure cell suspension has been uniformly dripped onto the PGA mesh-Dacron junction as well as onto the bottom side of PGA mesh.</li>
	<li>Wipe rim of bioreactor with alcohol wipe via rotating the bioreactor sideways avoid hovering.</li>
	<li>Silicone stopper lid assembly (Figure 2)
		<ol>
			<li>Peel autoclave bag back carefully, making sure not to expose bottom of lid.</li>
			<li>Attach injection port to feeding tube at the male connector.</li>
			<li>Attach PTFE 0.20 &mu;m filters to each of the three air ports. </li>
			<li>Be careful not to expose/touch bottom of lid during this process.</li>
			<li>Parafilm the injection port.</li>
		</ol>
	</li>
	<li>Insert the silicone stopper lid into glass bioreactor and make sure that the feeding tube inside the bioreactor does not touch the seeded PGA scaffolds. Parafilm around the lid. </li>
	<li>Place bioreactor with the flow system inside the incubator (on its side) and rotate the bioreactor every 5 minutes for 25-30 minutes.</li>
	<li>Fill the bioreactor chamber with 400ml of our 4-10 culture media as described in (Table 1). This culture medium is "optimized" for porcine engineered arteries.</li>
</ol>
6. Day 6-7: Turning on the Pump, and First Feeding
<ol>
	<li>Grow the seeded scaffolds statically without any pulsatile pumping through the silicone tubing for 6-7 days. There is no need for medium change or vitamin C supplementation during this time.</li>
	<li>Make sure there are no leaks of PBS or kinking of flow system tubing before turning on the pump.</li>
	<li>Turn on the pump of the flow system and make sure to adjust pump setting so that the pressure reads approximately 270/-30mmHg.</li>
	<li>Record pressures daily throughout culture and maintain the pressure at 270/-30mmHg. Pressure transducer can be connected to a computer to read and monitor the pressure.</li>
</ol>
First Feeding
<ol start="5">
	<li>Assemble injection port &amp; PTFE filter onto feeding lids for both medium change and medium waste deposal purposes.</li>
	<li>Place the feeding tube firmly on the pump and insert one end to feeding port of the bioreactor and the other end to the feeding lid. Make sure to wipe the injection ports with ethanol wipes.</li>
	<li>Use a dual-directional Masterflex pump to pump out 200ml of medium. Then use a new feeding tube to pump 200ml of fresh medium back into the bioreactor. Always start with a very slow speed, especially when pumping medium back to the bioreactor.</li>
	<li>Change medium and supplement ascorbic acid 2x/week. To add ascorbic acid, use a 30ml syringe to take out 25ml of medium and place it aside in the tissue hood. Dissolve 25mg of ascorbic acid in 5ml of PBS and filter it through a 0.22 &mu;m filter. First inject the sterile ascorbic acid into the feeding tube and add back the 25ml medium taken out earlier. The medium recipe is given in Table 1.</li>
</ol>
7.	Representative Results:

<img src="/files/ftp_upload/2646/2646fig1.jpg" alt="Figure 1"/> 
Figure 1. The tubing and connectors for the flow system assembly is shown above.
<img src="/files/ftp_upload/2646/2646fig2.jpg" alt="Figure 2"/> 
Figure 2. The silicone stopper lid assembly is shown above.
<img src="/files/ftp_upload/2646/2646fig3.jpg" alt="Figure 3"/> 
Figure 3. Schematics of bioreactor assembly are shown above. Inside the bioreactor Dacron cuffs are fastened onto the glass arms with the blue suture knots. 
<img src="/files/ftp_upload/2646/2646fig4.jpg" alt="Figure 4"/> 
Figure 4. Flow system connected to tubing and bioreactor is shown above. L/S18 tubing will be pumped by a Masterflex pump and thus driving the flow. The pressure transducer will measure the pressure before entering the bioreactor at upper stream. 
<img src="/files/ftp_upload/2646/2646fig5.jpg" alt="Figure 5"/> 
Figure 5. Image of harvested engineered vessel. Engineered vessels will appear to be opaque and achieve a wall thickness of approximately 250&mu;m after 8-week culture under pulsatile conditions. 
<img src="/files/ftp_upload/2646/2646fig6.jpg" alt="Figure 6"/> 
Figure 6. Haematoxylin and Eosin stained cross-sections of engineered vessels. A and B are 8-week non-pulsed and pulsed vessels, respectively. C and D are 4-week non-pulsed and pulsed vessels, respectively. L indicates the luminal side of the vessels. The scales bar is 100&mu;m.
<img src="/files/ftp_upload/2646/2646fig7.jpg" alt="Figure 7"/> 
Figure 7. Masson&#x2019;s Trichrome stains for collagen (blue) for cross-sections of engineered vessels. A and B are 8-week non-pulsed and pulsed vessels, respectively. C and D are 4-week non-pulsed and pulsed vessels, respectively. Note that the 4-week pulsed vessel shows more collagen than its non-pulsed counterpart. White arrows point to remaining PGA fragments in the vessels. The scales bar is 100&mu;m.
<img src="/files/ftp_upload/2646/2646fig8.jpg" alt="Figure 8"/> 
Figure 8. Immunochemistry staining of SMC markers in bovine engineered arteries. Smooth muscle &alpha;-actin, calponin-1, and smooth muscle myosin heavy chain (SMMHC) are early, intermediate, and late SMC contractile markers, respectively. By the end of 12-week culture, the cells in the vessel wall express SM &alpha;-actin and moderate amounts of Calponin-1 and SMMHC. The scales bar is 20&mu;m.

<table border="1">
	<tbody>
 	<tr>
 	<td>Component</td>
 <td>Amount</td>
 </tr>
 <tr>
 	<td>DMEM(DME/low modified) </td>
 <td>500 ml</td>
 </tr>
 <tr>
 	<td>FBS (fetal bovine serum) heat inactivated</td>
 <td>100 ml</td>
 </tr>
 <tr>
 	<td>HEPES 1.0 M </td>
 <td>5ml</td>
 </tr>
 <tr>
 	<td>Vitamin C (dissolved in PBS or DMEM)</td>
 <td>25 mg</td>
 </tr>
 <tr>
 	<td>Proline/Glycine/Alanine 25 mg/25 mg/10 mg (dissolved in 5ml of PBS)</td>
 <td>5ml</td>
 </tr>
 <tr>
 	<td>CuSO4 1.5 &mu;g (dissolved in1 ml of PBS)</td>
 <td>1ml</td>
 </tr>
 <tr>
 	<td>Penicillin G at 10,000 units/ml </td>
 <td>5ml</td>
 </tr>
 <tr>
 	<td>PDGF-BB (platelet-derived growth factor-BB) at 10ng/ml</td>
 <td>5&mu;g</td>
 </tr>
 <tr>
 	<td>bFGF (basic fibroblast growth factor) at 10ng/ml</td>
 <td>5&mu;g</td>
 </tr>
 </tbody>
</table>
Table 1. Components of "4-10" medium are shown in the above table. With the exception of PDGF-BB and bFGF, all other components are to be filtered through a 0.2&mu;m filter prior to use.

<ol>
	<li>Risau, W., Flamme, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculogenesis.">Vasculogenesis.</a> Annu. Rev. Cell Dev. Biol. 11, 73-91 (1995).</li><li>Fankhauser, F., Bebie, H., Kwasniewska, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influcence+of+mechanical+Forices+and+Flow+Mechanisms+on+Vessel+Occlusion.">The Influcence of mechanical Forices and Flow Mechanisms on Vessel Occlusion.</a> Lasers in Surgery and Medicine. 6, 530-532 (1987).</li><li>Niklason, L. E., Gao, J., Abbott, W. M., Hirschi, K. K., Houser, S., Marini, R., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+arteries+grown+in+vitro.">Functional arteries grown in vitro.</a> Science. 284, 489-493 (1999).</li><li>Prabhakar, V., Grinstaff, M. W., Alarcon, J., Knors, C., Solan, A. K., 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=Engineering+porcine+arteries:+Effects+of+scaffold+modification.">Engineering porcine arteries: Effects of scaffold modification.</a> Journal of Biomedical Materials Research Part A. 67A, 303-311 (2003).</li><li>Mitchell, S. L., 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=Requirements+for+growing+tissue-engineered+vascular+grafts.">Requirements for growing tissue-engineered vascular grafts.</a> Cardiovascular Pathology. 12, 59-64 (2003).</li><li>Dahl, S. L. M., Rhim, C., Song, Y. C., 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=Mechanical+properties+and+compositions+of+tissue+engineered+and+native+arteries.">Mechanical properties and compositions of tissue engineered and native arteries.</a> Annals of Biomedical Engineering. 35, 348-355 (2007).</li><li>Quint, C., Kondo, Y., Manson, R. J., Lawson, J. H., 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=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. 108, 9214-9219 (2011).</li><li>Niklason, L. E., Yeh, A. T., Calle, E. A., Bai, Y., Valent&#237;n, A., Humphrey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enabling+Tools+for+Engineering+Collagenous+Tissues+Integrating+Bioreactors,+Intravital+Imaging,+and+Biomechanical+Modeling.">Enabling Tools for Engineering Collagenous Tissues Integrating Bioreactors, Intravital Imaging, and Biomechanical Modeling.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 3335-3339 (2010).</li><li>Gong, Z., Calkins, G., Cheng, E. -. c., Krause, D., 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=Influence+of+Culture+Medium+on+Smooth+Muscle+Cell+Differentiation+from+Human+Bone+Marrow-Derived+Mesenchymal+Stem+Cells.">Influence of Culture Medium on Smooth Muscle Cell Differentiation from Human Bone Marrow-Derived Mesenchymal Stem Cells.</a> Tissue Engineering Part A. 15, 319-330 (2009).</li><li>Gong, Z. D., 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=Small-diameter+human+vessel+wall+engineered+from+bone+marrow-derived+mesenchymal+stem+cells+(hMSCs.">Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs.</a> Faseb Journal. 22, 1635-1648 (2008).</li><li>Poh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+engineered+from+human+cells.">Blood vessels engineered from human cells.</a> Lancet. 365, 2122-2124 (2005).</li><li>American Heart Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biostatistical fact sheet: cardiovascular procedures. , (2002).</li></ol>

No conflicts of interest declared.

The quality of engineered vessels is in large part dictated by the quality of the SMCs used in tissue culture. The critical aspects of SMC phenotype include contractile morphology, low passage number, and the ability to proliferate inside the bioreactor. We recommend that the passage number be no greater than P3 at the time of cell seeding onto the polymer scaffold. Moreover, it is crucial to confirm that the SMC sources are mycoplasma free prior to use. We have observed that mycoplasma-contaminated cells lead to substan...

<table><tbody><tr><td>FBS (Fetal Bovine Serum) Heat-Inactivated </td><td>Hyclone</td><td>SH30071</td><td></td></tr><tr><td>DMEM</td><td>GIBCO, by Life Technologies</td><td>11885</td><td></td></tr><tr><td>rhFGF-basic</td><td>R&amp;amp;D Systems</td><td>234-FSE</td><td></td></tr><tr><td>rrPDGF-BB</td><td>R&amp;amp;D Systems</td><td>520-BB</td><td></td></tr><tr><td>Penicilin G</td><td>Sigma-Aldrich</td><td>PENNA</td><td></td></tr><tr><td>Copper(II) Sulfate</td><td>Sigma-Aldrich</td><td>C8027</td><td></td></tr><tr><td>Gylcine</td><td>Sigma-Aldrich</td><td>C8790</td><td></td></tr><tr><td>L-Alanine</td><td>Sigma-Aldrich</td><td>A7469-25G</td><td></td></tr><tr><td>L-Proline</td><td>Sigma-Aldrich</td><td>P5607-25G</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A4544-25G</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375-100G</td><td></td></tr><tr><td>Silicone Stopper</td><td>Cole-Parmer</td><td>06298-24</td><td></td></tr><tr><td>Masterflex tubes L/S</td><td>Cole-Parmer</td><td>06508-16, 06508-18</td><td></td></tr><tr><td>Masterflex pump</td><td>Cole-Parmer</td><td>7553-80</td><td></td></tr><tr><td>Dacron cuff</td><td>Maquet</td><td>174406</td><td></td></tr><tr><td>PGA felt</td><td>Concordia</td><td>MO000877-01</td><td></td></tr><tr><td>4-0 1.5 metric Surgipro II suture</td><td>Syneture</td><td>VP-557-X</td><td></td></tr><tr><td>6-0 0.7 metric Dexon suture</td><td>Syneture</td><td>7538-11</td><td></td></tr><tr><td>0.22&amp;mu;m PTFE filters</td><td>Whatman, GE Healthcare</td><td>6780-2502</td><td></td></tr><tr><td>Three Way Stop-cock</td><td>Edwards Lifesciences</td><td>593WSC</td><td></td></tr><tr><td>Pressure Transducer</td><td>Edwards Lifesciences</td><td>PX212</td><td></td></tr><tr><td>IV bags</td><td>Baxter Internationl Inc.</td><td>R4R2110</td><td></td></tr><tr><td>Saline dilution set</td><td>Arrow International</td><td>W20030</td><td></td></tr><tr><td>Silicone tubing</td><td>Saint-Gobain</td><td>F05027</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor at JoVE.com

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

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 ...

engineering-biological-based-vascular-grafts-using-pulsatile

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Bioengineering

17.0K Views.  Yale University. 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.

Video: Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

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 ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

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. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

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.

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 ...

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

Engineering

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.

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, ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

A Novel 3D Printed Bioreactor for Ex Vivo Culture of Blood Vessels in Perfusion

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Author Spotlight: EasyFlow - An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

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Elastomeric PGS Scaffolds in Arterial Tissue Engineering

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

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Micropatterning and Assembly of 3D Microvessels

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

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

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor