इंजीनियरिंग जैविक आधार संवहनी Grafts का प्रयोग एक काँपने के गुणवाला bioreactor
Summary
हमारे समूह का एक bioreactor संस्कृति प्रणाली है कि हृदय प्रणाली के शारीरिक काँपने के गुणवाला तनाव implantable छोटे व्यास संवहनी grafts पुनर्जीवित करने के लिए mimics विकसित किया है.
Abstract
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.
Protocol
Discussion
इंजीनियर वाहिकाओं की गुणवत्ता के बड़े भाग में टिशू कल्चर में इस्तेमाल किया SMCs की गुणवत्ता से तय है. एसएमसी phenotype के महत्वपूर्ण पहलुओं सिकुड़ा आकारिकी, कम बीतने संख्या, और bioreactor अंदर पैदा करने की क्षमता शाम?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
यह काम स्वास्थ्य अनुदान EB-R01 ०,०८,८३६ के राष्ट्रीय संस्थानों और HL083895 R01 द्वारा वित्त पोषित है (दोनों और LEN के लिए). हम हमारे शोध के लिए बायोरिएक्टर बनाने के लिए, विश्वविद्यालय कंचेरा, डेरिल स्मिथ का शुक्रिया अदा करना सकता है.
Materials
| Name of Reagent/Material | Supplier | Catalogue Number |
|---|---|---|
| FBS (Fetal Bovine Serum) Heat-Inactivated | HyClone | SH30071 |
| DMEM | GIBCO | 11885 |
| rhFGF-basic | R&B | 234-FSE |
| rrPDGF-BB | R&B | 520-BB |
| Penicilin G | Sigma | PENNA |
| Copper(II) Sulfate | Sigma | C8027 |
| Gylcine | Sigma | C8790 |
| L-Alanine | Sigma | A7469-25G |
| L-Proline | Sigma | P5607-25G |
| Ascorbic Acid | Sigma | A4544-25G |
| HEPES | Sigma | H3375-100G |
| Silicone Stopper | Cole-Parmer | 06298-24 |
| Masterflex tubes L/S | Cole-Parmer | 06508-16, 06508-18 |
| Masterflex pump | Cole-Parmer | 7553-80 |
| Dacron cuff | Maquet | 174406 |
| PGA felt | Concordia | MO000877-01 |
| 4-0 1.5 metric Surgipro II suture | Syneture | VP-557-X |
| 6-0 0.7 metric Dexon suture | Syneture | 7538-11 |
| 0.22μm PTFE filters | Whatman | 6780-2502 |
| Three Way Stop-cock | Edwards Lifesciences | 593WSC |
| Pressure Transducer | Edwards Lifesciences | PX212 |
| IV bags | Baxter | R4R2110 |
| Saline dilution set | Arrow | W20030 |
| Silicone tubing | Saint-Gobain | F05027 |
Riferimenti
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- Niklason, L. E., Yeh, A. T., Calle, E. A., Bai, Y., Valentín, A., Humphrey, J. D. Enabling Tools for Engineering Collagenous Tissues Integrating Bioreactors, Intravital Imaging, and Biomechanical Modeling. Proceedings of the National Academy of Sciences of the United States of America. 107, 3335-3339 (2010).
- Gong, Z., Calkins, G., Cheng, E. -. c., Krause, D., Niklason, L. E. Influence of Culture Medium on Smooth Muscle Cell Differentiation from Human Bone Marrow-Derived Mesenchymal Stem Cells. Tissue Engineering Part A. 15, 319-330 (2009).
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