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 (3...

<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>

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

Research

JoVE Journal

Bioengineering

16.8K 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.