小鼠骨骼肌的普通散装培养物，以概括生态位和干细胞静止
Summary
骨骼肌由多种细胞类型组成，包括常驻干细胞，每种细胞都对肌肉稳态和再生有特殊贡献。在这里，描述了肌肉干细胞的 2D 培养和肌肉细胞生态位在 离体 环境中保留了许多生理、 体内和环境特征。
Abstract
骨骼肌是身体最大的组织，具有从运动到体温控制的多种功能。它的功能和从损伤中恢复取决于多种细胞类型以及核心肌肉细胞（肌纤维、肌肉干细胞）与其生态位之间的分子信号。大多数实验环境不能保留这种复杂的生理微环境，也不允许对静止的肌肉干细胞进行 离体 研究，这种细胞状态对它们至关重要。在这里，概述了具有其生态位的细胞成分的肌肉干细胞的 离体 培养方案。通过肌肉的机械和酶促分解，获得细胞类型的混合物，并将其放入 2D 培养物中。免疫染色显示，在 1 周内，培养物中存在多个生态位细胞以及具有静止肌肉干细胞特征的 Pax7 阳性细胞。这些独特的特性使该协议成为细胞扩增和产生可用于解决基本和转化问题的静止样干细胞的强大工具。
Introduction
运动、呼吸、新陈代谢、身体姿势和体温维持都依赖于骨骼肌，因此骨骼肌功能障碍会导致使人衰弱的病症（即肌病、肌营养不良等）。1. 鉴于其基本功能和丰富性，骨骼肌引起了全球研究实验室的关注，这些实验室致力于了解支持正常肌肉功能并可作为治疗靶点的关键方面。此外，骨骼肌是研究再生和干细胞功能的广泛使用模型，因为健康的肌肉在完全损伤和退化后可以完全自我修复，这主要是由于其驻留的干细胞2;这些也称为卫星细胞，位于肌纤维外围的基底层下3.
成人骨骼肌的核心细胞是肌纤维（长合胞体多核细胞）和卫星细胞（具有肌源性潜力的干细胞，在损伤激活它们之前处于静止状态）。后者细胞是肌肉再生的中心细胞，在没有它们的情况下，这个过程不会发生4,5,6,7。在它们的直接微环境中，有多种细胞类型和分子因子向它们发出信号。这个生态位在整个发育过程中逐渐建立，直到成年8.成人肌肉含有多种细胞类型（内皮细胞、周细胞、巨噬细胞、纤维脂肪祖细胞-FAPs、调节性T细胞等）9,10和细胞外基质成分（层粘连蛋白，胶原蛋白，纤连蛋白，原纤维蛋白，骨膜蛋白等）11 在健康、疾病和再生的背景下相互相互作用以及与卫星细胞相互作用。
在实验环境中保留这个复杂的生态位是基本的，但具有挑战性。同样困难的是保持或恢复静止状态，这是对卫星小区至关重要的细胞状态9。已经引入了几种方法来部分应对这些挑战，每种方法都有其优点和缺点（详见讨论部分）。在这里，提出了一种可以部分克服这两个障碍的方法。肌肉最初被收获，然后在异质细胞混合物被放入培养物之前通过机械和酶促分解。在培养过程中，检测到生态位的许多细胞类型，并观察到已恢复静止的卫星细胞。作为协议的最后一步，介绍了允许通过使用普遍接受的标记物检测每种细胞类型的免疫荧光步骤。
Protocol
所有实验均符合法国和欧盟的动物法规，特别是指令2010/63/UE。动物被饲养在动物设施的受控和富集环境中，认证编号为A94 028 379和D94-028-028;它们仅由授权的研究人员和动物看护人处理，并由动物饲养人员目视检查其一生中是否有不适迹象。他们在解剖前因颈椎脱位而被安乐死。在动物的一生中没有进行任何介入手术;因此，没有必要获得伦理委员会和法国高等教育、研究和创新部对该程序的批准。…
Representative Results
该协议允许肌肉细胞培养，同时保留卫星细胞和大多数细胞的内源性生态位。图 2 总结了协议的主要步骤，而解剖和消化的基本部分如图 1 所示。建议对后肢肌肉组织进行解剖（图1A-C），因为这组肌肉经过充分研究，并且具有发育起源和分子层次结构14。建议在无菌条件下制备所有混合…
Discussion
成人骨骼肌功能由一组精心编排的细胞相互作用和分子信号支撑。在这里，提出了一种方法，该方法允许在与生理微环境非常相似的 离体 环境中研究这些参数。
一些小组已经报道了体外培养肌原细胞的方法。这些方法旨在分离卫星细胞以研究其肌源祖细胞特性。使用两种主要方法分离纯卫星细胞，从比目鱼肌和/或 EDL 肌肉18的分离纤维培养物?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
在图2中，使用了施维雅Medical Art（https://smart.servier.com/）的模板。FR 实验室得到了法国抗肌病协会 – 通过 TRANSLAMUSCLE 的 AFM（赠款 19507 和 22946）、Fondation pour la Recherche Médicale – FRM（EQU202003010217、ENV202004011730、ECO201806006793）、Agence Nationale pour la Recherche – ANR （ANR-21-CE13-0006-02、ANR-19-CE13-0010、ANR-10-LABX-73） 和 La Ligue Contre le Cancer （IP/SC-17130） 的支持。上述资助者在本研究的设计、收集、分析、解释或报告或本手稿的撰写中没有任何作用。
Materials
| anti-CD31 | BD | 550274 | dilution 1:100 |
| anti-FOSB | Santa Cruz | sc-7203 | dilution 1:200 |
| anti-GFP | Abcam | ab13970 | dilution 1:1000 |
| anti-Ki67 | Abcam | ab16667 | dilution 1:1000 |
| anti-MyHC | DSHB | MF20-c | dilution 1:400 |
| anti-MYOD | Active Motif | 39991 | dilution 1:200 |
| anti-MYOG | Santa Cruz | sc-576 | dilution 1:150 |
| anti-Pax7 | Santa Cruz | sc-81648 | dilution 1:100 |
| anti-PDGFRα | Invitrogen | PA5-16571 | dilution 1:50 |
| b-FGF | Peprotech | 450-33 | concentration 4 ng/mL |
| bovine serum albumin (BSA) – used for digestion | Sigma Aldrich | A7906-1006 | concentration 0.2% |
| BSA IgG-free, protease-free – used for staining | Jackson ImmunoResearch | 001-000-162 | concentration 5% |
| cell strainer 40 um | Dominique Dutscher | 352340 | |
| cell strainer 70 um | Dominique Dutscher | 352350 | |
| cell strainer 100 um | Dominique Dutscher | 352360 | |
| Collagenase | Roche | 10103586001 | concentration 0.5 U/mL |
| Dimethyl sulfoxide (DMSO) | Euromedex | UD8050-05-A | |
| Dispase | Roche | 4942078001 | concentration 3 U/mL |
| Dissection forceps size 5 | Fine Science Tools | 91150-20 | |
| Dissection forceps size 55 | Fine Science Tools | 11295-51 | |
| Dissection scissors (big, straight) | Fine Science Tools | 9146-11 | ideal for chopping |
| Dissection scissors (small, curved) | Fine Science Tools | 15017-10 | |
| Dissection scissors (small, straight) | Fine Science Tools | 14084-08 | |
| Dulbecco's Modified Eagle's Medium (DMEM) | ThermoFisher | 41966-029 | |
| EdU Click-iT kit | ThermoFisher | C10340 | |
| Fetal bovine serum – option 1 | Eurobio | CVF00-01 | |
| Fetal bovine serum – option 2 | Gibco | 10270-106 | |
| Matrigel | Corning Life Sciences | 354234 | coating solution |
| Parafilm | Dominique Dutscher | 090261 | flexible film |
| Penicillin streptomycin | Gibco | 15140-122 | |
| Paraformaldehyde – option 1 | PanReac AppliChem ITW Reagents | 211511.1209 | concentration 4% |
| Paraformaldeyde – option 2 | ThermoFisher | 28908 | concentration 4% |
| Shaking water bath | ThermoFisher | TSSWB27 | |
| TritonX100 | Sigma Aldrich | T8532-500 ML | concentration 0.5% |
| Wild-type mice | Janvier | C57BL/6NRj |
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Cite This Article
Zaidan, L., Geara, P., Borok, M. J., Machado, L., Mademtzoglou, D., Mourikis, P., Relaix, F. Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence. J. Vis. Exp. (196), e65433, doi:10.3791/65433 (2023).