从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞
Summary
我们介绍从骨骼肌活检组织获得的分离、培养、特征化和区分人类原发性肌肉祖细胞（hMPCs）的技术。通过这些方法获得和特征化的hMC可用于随后解决与人类肌发生和骨骼肌再生相关的研究问题。
Abstract
使用原始人体组织和细胞是研究生物和生理过程，如骨骼肌再生过程的理想选择。在使用人类原发性成人干细胞，特别是从骨骼肌活检中提取的人类肌肉祖细胞（hMPCs）方面，存在着公认的挑战，包括从采集的组织中产生的细胞产量低和大量供体异质性。不同文化之间的生长和死亡参数。虽然在实验设计中引入异质性需要更大的样本大小来检测显著影响，但它也使我们能够识别在 hMPC 扩展容量中产生可变性的机制，从而使我们能够更好地了解骨骼肌再生的异质性。区分文化扩张能力的新机制有可能导致治疗的发展，以改善骨骼肌再生。
Introduction
骨骼肌是人体最大的器官系统，占全身质量的30-40%。除了在运动中公认的作用外，骨骼肌还维持体温和姿势，并在全身营养平衡中起着核心作用。涉及人类参与者、动物和细胞培养模型的研究对于解决骨骼肌生物学和再生相关问题都很有价值。人类原发性肌肉祖细胞（hMPCs）的分离和培养提供了一个可靠的模型，允许将细胞培养技术和操作应用于人体样本。使用hMPCs的一个优点是，它们保留了每个捐赠者2，3的遗传和代谢表型。维持供体表型使研究人员能够检查造血过程的个体间变异。例如，我们采用了hMPC表征方法，以识别hMPC人口扩容能力4中与年龄和性别相关的差异。
该协议的目的是详细说明分离、培养、表征和区分骨骼肌活检组织的hMPC的技术。在以前描述hMP和识别hMPC隔离5、6的潜在细胞表面制造商的工作的基础上，该协议通过将隔离与hMP的表征联系起来，填补了知识方面的重要空白。此外，该协议中包含的详细分步说明使 hMPC 隔离和特性可供广大科学受众访问，包括以前对 hMPC 经验有限的受众。我们的协议是最早描述使用成像细胞仪来跟踪细胞群的协议之一。新设计的成像细胞仪是最先进的、高通量的和基于微板的，能够在几分钟内对培养容器的每个孔中的所有细胞进行活细胞成像、细胞计数和多通道荧光分析。该系统允许快速量化整个细胞群的增殖和生存能力的动态变化，而对培养的干扰最小。例如，我们能够在体外连续几天对汇合进行客观的汇合测量，以确定从不同捐赠者获得的每个培养物的生长动力学。文献中的许多协议，特别是涉及微量体分的的协议，要求细胞在开始分化或治疗之前达到定义的汇合水平7。我们的方法允许客观地确定文化容器中每个孔的汇合，使研究人员能够以不偏不倚、非主观的方式开始治疗。
过去，使用初级 hMPC 的一个主要限制是低产量，限制了可用于实验的细胞数量。我们和其他人已经表明，骨骼肌活检组织的MPC的收率是每毫克组织1~15个MpCs（图1）8。由于我们的协议允许在用荧光活性细胞分拣（FACS）进行纯化之前对细胞进行四个通道，我们的冷冻hMPC产量来自少量活检组织（50-100毫克），足以满足研究目标，需要多个实验。我们的 FACS 协议产生 ±80% 纯（Pax7 阳性）MPC 总体，因此我们的协议针对产量和纯度进行了优化。
Protocol
Representative Results
Discussion
初级hMPCs是一种重要的研究模型，用于了解骨骼肌生物学和再生过程。此外，hMPC有潜力用于治疗。然而，在研究和治疗中使用原发性hMPCs存在公认的挑战，包括对来自人类的细胞的了解有限10。捐助文化之间的扩展能力也有很大差异，这限制了使用hMPC的可能性，并可能影响研究结果11。例如，最近已经证明，使用该协议中描述的方法分离的hMPCs产生的细胞在扩展?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢康奈尔大学生物技术资源中心成像设施对荧光活化细胞分类的帮助。我们还感谢莫莉·盖勒在参与者招募方面的帮助，并感谢埃里卡·本德进行骨骼肌活检。最后，我们感谢与会者的时间和参与。这项工作得到了国家卫生研究院国家老龄研究所的支持，奖励编号为R01AG058630（至B.D.C.和A.E.T.），由格伦医学研究基金会和美国青少年学院老龄研究基金（至B）支持。.华盛顿特区），由康奈尔妇女总统委员会（到A.E.T.）。
Materials
| 0.25% Trypsin, 2.21 mM EDTA | Corning | 25-053-Cl | Trypsin used for removing adherent hMPCs from cell culture vessels |
| 10 cm cell culture plate | VWR | 664160 | Plates used for culturing hMPCs |
| 15 mL Falcon tube | Falcon | 352196 | 15 mL conical tubes used throughout the hMPC isolation and culturing protocols |
| 24 well cell culture plate | Grenier Bio-One | 662 160 | Plates used for culturing hMPCs |
| 7-AAD Viability Staining Solution | eBioscience | 00-6993-50 | Viability stain for identifying living cells during FACS sorting |
| Alexa Fluor 488 anti-human CD29, Clone: TS2/16 | BioLegend | 303016 | Conjugated antibody for FACS |
| Black 96-well cell culture plate | Grenier Bio-One | 655079 | 96-well cell culture plate ideal for fluorescent imaging using the Celigo S |
| Celigo S | Nexcelcom Bioscience | Imaging cytometer used to track hMPC cultures | |
| Cell Strainer | VWR | 352350 | Cell strainer to eliminate large pieces of debris during muscle biopsy processing |
| Collagen Type I (Rat Tail) | Corning | 354236 | Collagen for coating cell culture plates |
| Collagenase D | Roche | 11 088 882 001 | Used for degradation of collagen and other connective tissue in the skeletal muscle biopsy tissue |
| Dimethyl Sulfoxide | VWR | WN182 | Used for cryopreservation of hMPCs |
| Dispase II | Sigma Life Sciences | D4693 | A protease used for enzymatic digestion of skeletal muscle biopsy tissue |
| Dulbecco's Modified Eagle Medium Low Glucose powder | Gibco | 31600-034 | Low glucose DMEM for muscle biopsy processing |
| Dulbecco's Phosphate Buffered Saline | Gibco | 21600-010 | PBS for muscle biopsy processing |
| EDTA Disodium Salt Dihydrate | J.T. Baker | 4040-01 | Required for FACS buffer |
| Fetal Bovine Serum | VWR | 89510-186 | Fetal bovine serum used for hMPC growth media |
| Ham's F12 | Gibco | 21700-026 | Base media for hMPCs |
| Heat Inactivated Equine Serum | Gibco | 26-050-070 | Horse serum used to make hMPC differentiation media |
| Hemocytometer | iNCyto | DHC-N0105 | Used to count cells |
| Hibernate A | Gibco | A1247501 | Media for preserving skeletal muscle biopsy tissue |
| Hoechst 33342, trihydrochloride, trihydrate | Life Technologies | H21492 | DNA stain for identifying all cells using the Celigo S |
| Isopropanol | Fisher Scientific | A416P-4 | Used for controlled rate freezing of hMPCs |
| Moxi buffer | Orflo | MXA006 | Buffer for automated cell counter |
| Moxi Cassettes | Orflo | MXC002 | Cassesttes for automated cell counter |
| Moxi z Mini Automated Cell Counter | Orflo | Automated cell counter | |
| Mr. Frosty Freezing Container | Thermo Fisher Scientific | 5100-0001 | Commerically available controlled rate cell freezing container |
| Normal Goat Serum (10%) | Thermo Fisher Scientific | 50062Z | Goat serum used in FACS buffer |
| PE-Cy7 Mouse Anti-human CD56 , Clone: B159 | BD Pharmingen | 557747 | Conjugated antibody for FACS |
| Penicillin/Streptomycin 100X Solution | Corning | 30-002-CI | Antibiotics added to culture media |
| Propidium iodide | Thermo Fisher Scientific | P3566 | DNA stain for identifying dead cells using the Celigo S |
| Recombinant Human basic fibroblast growth factor | Promega | G5071 | Supplement in hMPC growth media to prevent spontaneous differentiation |
| Recovery Cell Culture Freezing Medium | Gibco | 12648-010 | Media used to cryoperseve muscle biopsy slurries |
| Sodium Bicarbonate | Fisher Scientific | S233-3 | Added to Ham's F12 |
| Sterile Round Bottom 5 mL tubes | VWR | 60818-565 | Tubes used for FACS |
| UltraComp eBeads | eBioscience | 01-2222-42 | Compensation beads fort calibrating flow FACS settings |
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