工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激
Summary
工程肌肉组织再生医学具有巨大的潜力，作为疾病模型，也可作为肉类的替代来源。在这里，我们描述的工程的肌肉构造，在这种情况下，从小鼠成肌细胞的祖细胞，通过电脉冲刺激。
Abstract
工程的肌肉组织，可用于几个不同的用途，包括用于生产的组织在体外作为一种疾病模型， 如学习压力性溃疡，再生医学和肉类替代1。首次报道的3D肌肉结构已作了很多年前在该领域的先驱，是Vandenburgh和他的同事2,3。在肌肉组织工程取得的进展不仅是广大的生化因子，干细胞和祖细胞知识增益的结果，但研究人员所获得的洞察力，特别是基于物理因素发挥重要作用，在控制细胞的行为，组织的发展。国家的最先进的工程肌肉构造目前包括人口细胞的水凝胶结构。在我们的实验室中，这些通常由鼠成肌细胞的祖细胞，分离出小鼠后肢肌肉或小鼠成肌细胞系C2C12，MIXED与胶原/基底膜的混合物，并镀两个锚固点之间，模仿肌肉韧带。其他细胞可能被视为良好， 例如 ，替代的细胞系，例如L6大鼠成肌细胞4，新生儿肌源性祖细胞5，来自成年肌肉组织从其他物种如人类6甚至诱导式多能性干细胞（iPS细胞）7的细胞。细胞收缩引起的细胞的取向沿长轴的构建体8,9和文化约一个星期后的肌肉祖细胞的分化。此外，应用程序可以增强电刺激分化的过程中，在一定程度上8。由于其有限的尺寸（8×2×0.5 mm）的完整的组织可以使用共焦显微镜分析，监视例如生存能力，分化和细胞对准。根据特定的应用要求的enginee红色的肌肉组织会有所不同， 例如用于再生医学需要向上缩放的组织大小和血管，而作为一个肉替代的词条到其他物种是必要的。
Protocol
1。小鼠成肌细胞的祖细胞或C2C12细胞培养隔离细胞根据最初由谢费尔和他的同事10出版和购买适于由Collins 等人 11和博南等12的协议，并在液氮中存储这些。这需要小鼠， 如之C57B1 / 6。在其他实验室所使用的替代方法， 例如李瑛等人发表在杂志的可视化实验的方法13。对于试剂和设备，被称为第7和第8页上的表。从一…
Representative Results
最终产品将是肌肉如在图3中所示的构造。组织的大小将是约8毫米长，2毫米宽，0.5毫米厚。在分化过程中的电刺激将改变的表达，肌球蛋白重链异构体，但不大大提高的分化的方法，诱导分化培养基8，但也可以应用于电刺激可以肌肉的功能的过程结束时，判断是否，因为将能够充分发展的肌节的肌肉收缩时的电脉冲。 分化和成熟的也可以使用定量PCR来分析?…
Discussion
肌肉组织工程具有巨大的潜力作为一种疾病模型，药物筛选，在再生医学中的使用和肉类生产。然而，这些应用程序的要求有所不同。我们选择与胶原蛋白和基质胶的组合，因为胶原蛋白可以用于细胞取向和成肌细胞前体细胞，因为需要基底膜衍生的蛋白质的存在下，如在之前的2D研究12确定。此外，纤维蛋白凝胶已在我们的实验室测试，似乎不支持C2C12肌母细胞的祖细胞，胶原蛋白?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
要感谢亚斌吴培养的组织图2给出了，照片是由巴特面包车Overbeeke。 SenterNovem，授予ISO 42022的工作的财政支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Matrigel-growth factor reduced | Beckton and Dickinson | ||
| DMEM (high glucose)* | Gibco | 42430 | |
| Advanced DMEM | Gibco | 12491 | |
| Horse serum | Gibco | 65050-122 | |
| Fetal bovine serum | Greiner | 758075 | |
| 0.45 and 0.22 μm syringe filter* | Whatmann (Schleicher and Scheull) | 10462100 | |
| L-glutamine | Gibco | 25030024 | |
| Penicillin/streptomycin | Gibco | 10378016 | |
| Amphotericin | Gibco | 15290-018 | |
| Culture plastic | Greiner | Includes culture flasks and pipettes | |
| Chick embryo extract | United States Biological | C3999 | |
| Pasteur pipette* | Hilgenberg | Pasteur pipettes, with constriction, with cotton, open tip L: 230 mm with tip diameter of 0,9 – 1,1 mm | |
| Pasteur pipette* | Hilgenberg | Pasteur pipettes, with constriction, with cotton, open tip L: 230 mm with tip diameter of 1,4 – 1,6 mm | |
| Pasteur pipette | VWR | 612-1702 | |
| Collagenase type I* | Sigma | C0130-16 | |
| 40 μm cell strainer* | BD Falcon | 352340 | |
| 19G needle | |||
| Elastomer | Dow Corning corporation | 3097358-1004 | Silastic MDX 4-4210# |
| Curing agent | Dow Corning corporation | Silastic MDX 4-4210# | |
| Velcro | Regular store | You can buy this at a regular store, only use the soft side | |
| Collagen type I, rat tail | BD Biosciences | 3544236 | |
| C-Pace EP Culture Pacer | Ionoptix | ||
| 6-well culture dishes for electrical stimulation | Beckton Dickinson-Falcon | BD Falcon #353846 | |
| C-Dish culture dish electrodes | Ionoptix | ||
| * Needed for the isolation of cells (point 1.1) # Together in one kit |
Riferimenti
- Langelaan, M. L. P., Boonen, K. J. M., Polak, R. B., et al. Meet the new meat: tissue engineered skeletal muscle. Trends Food Sci. Tech. 21 (2), 59-66 (2010).
- Shansky, J., Chromiak, J., Tatto, M., Vandenburgh, H. A simplified method for tissue engineering skeletal muscle organoids in vitro. In Vitro Cell Dev. Biol. Animal. 33 (9), 659-661 (1997).
- Vandenburgh, H., Del Tatto, M., Shansky, J., et al. Tissue-engineered skeletal muscle organoids for reversible gene therapy. Hum. Gene Ther. 7 (17), 2195-2200 (1996).
- Yaffe, D. Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Natl. Acad. Sci. U.S.A. 61 (2), 477-483 (1968).
- Rando, T. A., Blau, H. M. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J. Cell Biol. 125 (6), 1275-1287 (1994).
- Koning, M., Werker, P. M. N., vander Schaft, D. W. J., Bank, R. A., Harmsen, M. C. MicroRNA-1 and MicroRNA-206 Improve Differentiation Potential of Human Satellite Cells: A Novel Approach for Tissue Engineering of Skeletal Muscle. Tissue Eng. Part A. , (2011).
- Darabi, R., Pan, W., Bosnakovski, D., et al. Functional myogenic engraftment from mouse iPS cells. Stem Cell Rev. 7 (4), 948-957 (2011).
- Langelaan, M. L. P., Boonen, K. J. M., Rosaria-Chak, K. Y., et al. Advanced maturation by electrical stimulation: Differences in response between C2C12 and primary muscle progenitor cells. J. Tissue Eng. Regen. Med. 5 (7), 529-539 (2011).
- van der Schaft, D., van Spreeuwel, A. C., van Assen, H. C., Baaijens, F. Mechanoregulation of vascularization in aligned tissue engineered muscle; a role for VEGF. Tissue Eng. Part A. , (2011).
- Shefer, G., Wleklinski-Lee, M., Yablonka-Reuveni, Z. Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway. J. Cell. Sci. 117 (Pt. 22), 5393-5404 (2004).
- Collins, C. A., Olsen, I., Zammit, P. S., et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell. 122 (2), 289-301 (2005).
- Boonen, K. J. M., Rosaria-Chak, K. Y., Baaijens, F. P. T., van der Schaft, D. W. J., Post, M. J. Essential environmental cues from the satellite cell niche: optimizing proliferation and differentiation. Am. J. Physiol. Cell Physiol. 296 (6), C1338-C1345 (2009).
- Li, Y., Pan, H., Huard, J. Isolating Stem Cells from Soft Musculoskeletal Tissues. J. Vis. Exp. (41), e2011 (2010).
- Boonen, K. J. M., Langelaan, M. L. P., Polak, R. B., et al. Effects of a combined mechanical stimulation protocol: Value for skeletal muscle tissue engineering. J. Biomech. 43 (8), 1514-1521 (2010).
- Gawlitta, D., Boonen, K. J. M., Oomens, C. W. J., Baaijens, F. P. T., Bouten, C. V. C. The influence of serum-free culture conditions on skeletal muscle differentiation in a tissue-engineered model. Tissue Eng. Part A. 14 (1), 161-171 (2008).
- Koning, M., van Luijn, M., van der Schaft, D. W. J., et al. Human skeletal muscle formation and engraftment In vivo is independent of preconditioning In vitro with HUVEC. , (2013).
- Levenberg, S., Rouwkema, J., Macdonald, M., et al. Engineering vascularized skeletal muscle tissue. Nat. Biotechnol. 23 (7), 879-884 (2005).
- Koffler, J., Kaufman-Francis, K., Yulia, S., et al. Improved vascular organization enhances functional integration of engineered skeletal muscle grafts. Proc. Natl. Acad. Sci. U.S.A. 108 (36), 14789-14794 (2011).
Tags
IngegneriaMurine Myoblast Progenitor CellsElectrical StimulationDisease ModelRegenerative MedicineMeat Alternative3D Muscle ConstructsBiochemical FactorsStem CellsPhysical FactorsCell-populated Hydrogel ConstructsCollagen/MatrigelMuscle LigamentsAlternative Cell LinesL6 Rat MyoblastsNeonatal Muscle Derived Progenitor CellsInduced Pluripotent Stem Cells (iPS Cells)Cell Contractility
Citazione di questo articolo
van der Schaft, D. W. J., van Spreeuwel, A. C. C., Boonen, K. J. M., Langelaan, M. L. P., Bouten, C. V. C., Baaijens, F. P. T. Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation. J. Vis. Exp. (73), e4267, doi:10.3791/4267 (2013).