1Department of Biomedical Engineering, Yale University, 2Department of Anesthesiology, Yale University School of Medicine
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Huang, A. H., Niklason, L. E. Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor. J. Vis. Exp. (52), e2646, doi:10.3791/2646 (2011).
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.
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
2. PGA Scaffolds Surface Treatment
3. Sewing Dacron arms
4. Assembly of Bioreactor (Day before beginning of bioreactor culture)
5. Day 1: Bioreactor Setup
6. Day 6-7: Turning on the Pump, and First Feeding
7. Representative Results:
Figure 1. The tubing and connectors for the flow system assembly is shown above.
Figure 2. The silicone stopper lid assembly is shown above.
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.
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.
Figure 5. Image of harvested engineered vessel. Engineered vessels will appear to be opaque and achieve a wall thickness of approximately 250μm after 8-week culture under pulsatile conditions.
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 100μm.
Figure 7. Masson’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 100μm.
Figure 8. Immunochemistry staining of SMC markers in bovine engineered arteries. Smooth muscle α-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 α-actin and moderate amounts of Calponin-1 and SMMHC. The scales bar is 20μm.
|DMEM(DME/low modified)||500 ml|
|FBS (fetal bovine serum) heat inactivated||100 ml|
|HEPES 1.0 M||5ml|
|Vitamin C (dissolved in PBS or DMEM)||25 mg|
|Proline/Glycine/Alanine 25 mg/25 mg/10 mg (dissolved in 5ml of PBS)||5ml|
|CuSO4 1.5 μg (dissolved in1 ml of PBS)||1ml|
|Penicillin G at 10,000 units/ml||5ml|
|PDGF-BB (platelet-derived growth factor-BB) at 10ng/ml||5μg|
|bFGF (basic fibroblast growth factor) at 10ng/ml||5μg|
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 0.2μm filter prior to use.
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 substantial decreases in cellular proliferation and collagen matrix deposition in bioreactor culture.
More than 400,000 coronary artery grafts are need for bypass operations each year, resulting in a high demand for small-diameter (<6mm) vascular grafts12. The ultimate goal is to engineer functional small-diameter vessels that mimic the physiological functions of native tissue. Our pulsatile bioreactor system is a promising mean to constitute functional arterial grafts that could restore and replace diseased arteries. In a chemically and mechanically controlled environment, the bioreactor enables us to engineer implantable small-diameter vessels that exhibit strong suture retention and excellent mechanical properties. The pulsatile bioreactor system can be used to reconstruct arteries from bovine3, porcine6,7, and human cells9 in various sizes and dimensions, thus making this system generalizable and versatile. In addition, we have modified our bioreactor to allow real-time imaging of vascular extracellular matrix, non-invasively8. A combined approach of our bioreactor, non-invasive microscopy imaging techniques, and advanced biomechanical assessments will help us understand and evaluate ECM deposition and the changes in vascular mechanical properties over time.
Therefore, this bioreactor system provides a unique approach to engineer biological-based vascular grafts, which may play an important role in autologous vascular grafts regeneration in future clinical applications10,11.
No conflicts of interest declared.
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.
|FBS (Fetal Bovine Serum) Heat-Inactivated||Hyclone||SH30071|
|DMEM||GIBCO, by Life Technologies||11885|
|Masterflex tubes L/S||Cole-Parmer||06508-16, 06508-18|
|4-0 1.5 metric Surgipro II suture||Syneture||VP-557-X|
|6-0 0.7 metric Dexon suture||Syneture||7538-11|
|0.22Ám PTFE filters||Whatman, GE Healthcare||6780-2502|
|Three Way Stop-cock||Edwards Lifesciences||593WSC|
|Pressure Transducer||Edwards Lifesciences||PX212|
|IV bags||Baxter Internationl Inc.||R4R2110|
|Saline dilution set||Arrow International||W20030|