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 border="1">
	<tbody>
 	<tr>
 	<th>Name of Reagent/Material</th>
 <th>Supplier</th>
 <th>Catalogue Number</th>
 </tr>
 <tr>
 	<td>FBS (Fetal Bovine Serum) Heat-Inactivated </td>
 <td>HyClone</td>
 <td>SH30071</td>
 </tr>
 <tr>
 	<td>DMEM</td>
 <td>GIBCO</td>
 <td>11885</td>
 </tr>
 <tr>
 	<td>rhFGF-basic</td>
 <td>R&amp;B</td>
 <td>234-FSE</td>
 </tr>
 <tr>
 	<td>rrPDGF-BB</td>
 <td>R&amp;B</td>
 <td>520-BB</td>
 </tr>
 <tr>
 	<td>Penicilin G</td>
 <td>Sigma</td>
 <td>PENNA</td>
 </tr>
 <tr>
 	<td>Copper(II) Sulfate</td>
 <td>Sigma</td>
 <td>C8027</td>
 </tr>
 <tr>
 	<td>Gylcine</td>
 <td>Sigma</td>
 <td>C8790</td>
 </tr>
 <tr>
 	<td>L-Alanine</td>
 <td>Sigma</td>
 <td>A7469-25G</td>
 </tr>
 <tr>
 	<td>L-Proline</td>
 <td>Sigma</td>
 <td>P5607-25G</td>
 </tr>
 <tr>
 	<td>Ascorbic Acid</td>
 <td>Sigma</td>
 <td>A4544-25G</td>
 </tr>
 <tr>
 	<td>HEPES</td>
 <td>Sigma</td>
 <td>H3375-100G</td>
 </tr>
 <tr>
 	<td>Silicone Stopper</td>
 <td>Cole-Parmer</td>
 <td>06298-24</td>
 </tr>
 <tr>
 	<td>Masterflex tubes L/S</td>
 <td>Cole-Parmer</td>
 <td>06508-16, 06508-18</td>
 </tr>
 <tr>
 	<td>Masterflex pump</td>
 <td>Cole-Parmer</td>
 <td>7553-80</td>
 </tr>
 <tr>
 	<td>Dacron cuff</td>
 <td>Maquet</td>
 <td>174406</td>
 </tr>
 <tr>
 	<td>PGA felt</td>
 <td>Concordia</td>
 <td>MO000877-01</td>
 </tr>
 <tr>
 	<td>4-0 1.5 metric Surgipro II suture</td>
 <td>Syneture</td>
 <td>VP-557-X</td>
 </tr>
 <tr>
 	<td>6-0 0.7 metric Dexon suture</td>
 <td>Syneture</td>
 <td>7538-11</td>
 </tr>
 <tr>
 	<td>0.22&mu;m PTFE filters</td>
 <td>Whatman</td>
 <td>6780-2502</td>
 </tr>
 <tr>
 	<td>Three Way Stop-cock</td>
 <td>Edwards Lifesciences</td>
 <td>593WSC</td>
 </tr>
 <tr>
 	<td>Pressure Transducer</td>
 <td>Edwards Lifesciences</td>
 <td>PX212</td>
 </tr>
 <tr>
 	<td>IV bags</td>
 <td>Baxter</td>
 <td>R4R2110</td>
 </tr>

 <tr>
 	<td>Saline dilution set</td>
 <td>Arrow</td>
 <td>W20030</td>
 </tr>
 <tr>
 	<td>Silicone tubing</td>
 <td>Saint-Gobain</td>
 <td>F05027</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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JoVE Journal

Bioengineering

16.9K vues.  Yale University. Notre groupe a développé un système de culture bioréacteur qui imite les contraintes pulsatiles physiologiques du système cardiovasculaire pour régénérer implantables petit diamètre des greffons vasculaires.

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

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

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 ...

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. ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

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

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

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

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

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.