組織工学：層状細胞シートの送達のための多細胞3D足場の構築
Summary
For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.
Abstract
そのような大人の人間の心のような多くの組織は、十分に損傷後に再生することができません。組織工学における2,3の戦略は、回復と修復に体を支援するために技術革新を提案する。例えば、TEアプローチは、心筋梗塞（MI）後の心臓リモデリングを減弱、おそらくほぼ正常プレMIレベルに総心臓機能を高めることができるかもしれません。4、心臓組織の正常な再生がの適切な送達を含む任意の機能的な組織と同様に移植された細胞/組織移植片の統合と生存に有利な環境合図で複数の細胞タイプ。送達ビヒクル、細胞の生存に及ぼす影響、材料強度、及び細胞 – 組織組織の促進として評価可溶性シグナル、細胞間相互作用、およびマトリックス材料：操作された組織が含む複数のパラメータに対処すべきである。のみこれらの必須要素を無視し、移植片細胞の直接注入を利用する研究。2,5,6これらの成分を組み合わせた組織の設計は、まだ開発されなければならない。ここでは、「組織」における新たな血管の形成を増強するための標的臓器の細胞型および内皮細胞を含む生物学的由来の材料の2つの異なる種類のパターン化された細胞シートの積層を使用して統合された設計の例を提示する。これらの研究は、心臓のような組織の発生に焦点を当てているが、この組織の設計は、最小限の設計および材料の変更と心臓以外の多くの臓器に適用することができ、再生治療のための既製品であることを意味する。プロトコルは5詳細な手順が含まれています。温度感受性ポリ（N -isopropylacrylamide）（PNIPAAM）が被覆組織培養皿に使用される。その後、組織特異的細胞は強い横癒着による細胞シートを形成するためにコーティングされたプレート/マイクロパターン表面の表面上で培養される。第三に、ベースマトリックスは、血管新生permissiを多孔性マトリックスを組み合わせることにより、組織のために作成されヒドロゲルおよび内皮ve細胞。最後に、細胞シートは、完全な構築物を作るPNIPAAM被覆皿から持ち上げ、ベース要素に転送される。
Introduction
Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types.
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.
Protocol
Representative Results
Discussion
プロトコルにおける重要なステップは次のとおりです。温度応答性高分子の板表面を被覆し、プレートを冷却した後、細胞シートを操作する。異なるセルは、異なる物理的特性を示すので、接着性のような、昇降時間は、各異なる細胞型について最適化されるべきである。このプロトコルの第2、及び最も重要な挑戦的な成分は、細胞シート、組織アセンブリのための方法の重要な態様での操…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)
Materials
| Table of Reagents: | |||
| Reagent | Company | Catalogue number | Comments |
| Calcein-AM | Invitrogen | C3099 | Cell tracker / live dye |
| Lysotracker Red | Invitrogen | L7528 | Cell tracker |
| Neutral Red | Sigma | N7005 | Visible Cell dye |
| pNIPAAM | Sigma Aldrich | 412780250 | Poly(N-isopropylacrylamide) |
| Toluene | Sigma Aldrich | 244511-1L | |
| Hexane | Sigma Aldrich | 296090-1L | |
| RAOSMC | Lonza | R-ASM-580 | Rat Aortic Smooth Muscle Cells |
| SmGM2 | Lonza | CC-4149 | Smooth Muscle Media |
| HUVEC | Invitrogen | C-003-5C | Human Venous Endothelial Cells |
| HyStem | Glycosan/Biotime | ———— | |
| Isopropyl alcohol | VWR International | BDH1133-4LP | |
| Trypsin | Corning Cellgro | 25-053-C1 | |
| PBS | Gibco | 14287-072 | |
| FBS | Gibco | 16140-071 | |
| Table of Specific Equipment: | |||
| Equipment | Company | Catalogue number | Comments (optional) |
| Filter paper | Ahlstrom | 6310-0900 | |
| Buchner Funnel | Sigma Aldrich | Z247308 | |
| UpCell Plates | Nunc | 2014-11 | |
| UV light. | Jelight Company | UVO Cleaner Model No.42 |
Referências
- Ohashi, K., Okano, T. Functional tissue engineering of the liver and islets. Anat Rec (Hoboken). 297, 73-82 (2014).
- Chen, Q. Z., Harding, S. E., Ali, N. N., Lyon, A. R., Boccaccini, A. R. Biomaterials in cardiac tissue engineering: Ten years of research survey. Mat Sci Eng R. 59, 1-37 (2008).
- Jakob, P., Landmesser, U. Current status of cell-based therapy for heart failure. Curr Heart Fail Rep. 10, 165-176 (2013).
- Tongers, J., Losordo, D. W., Landmesser, U. Stem and progenitor cell-based therapy in ischaemic heart disease: promise, uncertainties, and challenges. Eur Heart J. 32, 1197-1206 (2011).
- Etzion, S., et al. Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction. J Mol Cell Cardiol. 33, 1321-1330 (2001).
- Masuda, S., Shimizu, T., Yamato, M., Okano, T. Cell sheet engineering for heart tissue repair. Adv Drug Deliv Rev. 60, 277-285 (2008).
- Koh, G. Y., Soonpaa, M. H., Klug, M. G., Field, L. J. Strategies for myocardial repair. J Interv Cardiol. 8, 387-393 (1995).
- Li, R. K., et al. Construction of a bioengineered cardiac graft. J Thorac Cardiovasc Surg. 119, 368-375 (2000).
- Muller-Ehmsen, J., et al. Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function. Circulation. , 105-1720 (2002).
- Reinecke, H., Zhang, M., Bartosek, T., Murry, C. E. Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation. , 100-193 (1999).
- Roell, W., et al. Cellular cardiomyoplasty improves survival after myocardial injury. Circulation. 105, 2435-2441 (2002).
- Soonpaa, M. H., et al. Potential approaches for myocardial regeneration. Ann N Y Acad Sci. 752, 446-454 (1995).
- Akins, R. E. Can tissue engineering mend broken hearts. Circ Res. 90, 120-122 (2002).
- Goodell, M. A., et al. Stem cell plasticity in muscle and bone marrow. Ann N Y Acad Sci. 938, 208-218 (2001).
- Menasche, P., et al. Myoblast transplantation for heart failure. Lancet. 357, 279-280 (2001).
- Murry, C. E., Wiseman, R. W., Schwartz, S. M., Hauschka, S. D. Skeletal myoblast transplantation for repair of myocardial necrosis. J Clin Invest. 98, 2512-2523 (1172).
- Orlic, D., et al. Transplanted adult bone marrow cells repair myocardial infarcts in mice. Ann N Y Acad Sci. 938, 221-229 (2001).
- Elia, R., et al. Silk-hyaluronan-based composite hydrogels: a novel, securable vehicle for drug delivery. J Biomater Appl. 27, 749-762 (2013).
- Kai, D., et al. Stem cell-loaded nanofibrous patch promotes the regeneration of infarcted myocardium with functional improvement in rat model. Acta Biomater. , (2014).
- Hong, H. J., et al. Tracheal reconstruction using chondrocytes seeded on a poly(l-lactic-co-glycolic acid)-fibrin/hyaluronan. J Biomed Mater Res A. , (2014).
- Serpooshan, V., et al. The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction. Biomaterials. 34, 9048-9055 (2013).
- Turner, W. S., et al. Cardiac tissue development for delivery of embryonic stem cell-derived endothelial and cardiac cells in natural matrices. J Biomed Mater Res B Appl Biomater. 100, 2060-2072 (2012).
- Sato, M., Yamato, M., Hamahashi, K., Okano, T., Mochida, J. Articular cartilage regeneration using cell sheet technology. Anat Rec (Hoboken). 297, 36-43 (2014).
- Sawa, Y., Miyagawa, S. Present and future perspectives on cell sheet-based myocardial regeneration therapy. Biomed Res Int. 2013, 583912 (2013).
- Demirbag, B., Huri, P. Y., Kose, G. T., Buyuksungur, A., Hasirci, V. Advanced cell therapies with and without scaffolds. Biotechnol J. 6, 1437-1453 (2011).
- Song, J. J., Ott, H. C. Organ engineering based on decellularized matrix scaffolds. Trends Mol Med. 17, 424-432 (2011).
- Badylak, S. F., et al. The use of extracellular matrix as an inductive scaffold for the partial replacement of functional myocardium. Cell Transplant. 15, S29-S40 (2006).
- Wang, Y., et al. Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds. Hepatology. 53, 293-305 (2011).
- Gilbert, T. W., et al. Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix. Biomaterials. 29, 4775-4782 (2008).
- Luna, J. I., et al. Multiscale biomimetic topography for the alignment of neonatal and embryonic stem cell-derived heart cells. Tissue Eng Part C Methods. 17, 579-588 (2011).
