从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征
Summary
The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.
Abstract
修理和骨骼肌的再生需要卫星细胞，这是驻地肌肉干细胞的作用。这些都可以从研究中培养用酶消化人肌肉活检样品和它们的生肌性质进行分离。定量，从酶消化获得的两个主要粘附细胞类型是：（i）卫星细胞（称为生肌细胞或肌细胞前体细胞），初步确定为CD56 +，后来作为CD56 + /结蛋白+细胞和（ii）muscle-成纤维细胞，鉴定为CD56 –和TE-7 +。成纤维细胞增殖非常有效地在文化和混合细胞群，这些细胞可能溢出肌细胞主导文化。从人的肌肉的不同类型的细胞的分离和纯化由此试图调查中培养两种细胞类型的先天行为时一个重要的考虑因素的方法。在这里，我们DESCRIBE基于使用胶原酶细胞轻柔酶消化排序和分散酶，随后通过磁性活化细胞分选（MACS）的一种系统，其不仅提供了高纯度（> 95％生肌细胞）和良好的产率（〜2.8×10 6±8.87 7天后在体外 ），用于在培养实验×10 5个细胞/ g组织。这种方法是基于孵育混合肌源性细胞群与偶联于抗CD56的抗体的磁性微球珠，然后使细胞虽然磁场。 CD56 +细胞势必微珠保留的，而CD56领域–细胞通过列畅通。从分类处理的任何阶段的细胞悬浮液可以被镀和培养。以下给定的介入，细胞形态，并且表达和蛋白质，包括核转录因子的定位可以通过使用免疫荧光标记用特异性抗体和图像P进行定量rocessing和分析软件包。
Introduction
骨骼肌的修复和再生，需要卫星细胞1，肌原干细胞2,3。的作用在体内位于每肌纤维的肌膜和基底层之间的可逆静止状态存在这些细胞中，但被活化增殖，保险丝和区分肌肉组织被破坏，修复和再生3。卫星细胞可以使用酶消化4年轻人和老年人人体肌肉活检标本及其生肌性能隔离随后可以在研究原代培养5。这种隔离方法的关于细胞群体的产量和纯度的效率取决于所使用的方法，可从样品变化进行采样。从酶消化得到的两个主要贴壁细胞类型的卫星细胞（现称为肌细胞或肌肉前体细胞），初步确定为CD56 + /结蛋白细胞，万亩SCLE衍生的成纤维细胞，鉴定为CD56 –和TE7 +细胞5。的成纤维细胞具有快速的增殖速率和不发生不可逆的生长停滞和终末分化时像肌原细胞的细胞 – 细胞接触;因此，在混合人群，成纤维细胞可能溢出肌细胞主导文化。
成纤维细胞经常被视为刺激肌肉生物学家，然而，现在有在成纤维细胞作为值得研究的在他们自己的权利的越来越大的兴趣，特别是因为它们已被证明在肌肉修复6有一个与肌细胞协同作用。从人的肌肉的不同类型的细胞的分离和纯化由此试图调查中培养两种细胞类型的先天行为时一个重要的考虑因素的方法。荧光激活细胞分选（FACS）是一种方法，通过它的细胞可以被分类为进一步研究和/或计数并分析。 FACS已表明可靠丰富人类肌细胞，但细胞的产量进行后续培养迄今尚未高7。定的体细胞，例如与衰老4相关的卫星细胞来源的肌细胞和非常差的增殖和分化的有限复制潜力，更温和的方法是必需的。单肌纤维的文化提供了一个不太积极的，是指获得鼠卫星细胞在他们sublaminal利基及其文化8,9激活后仍驻留的。然而，这常常是不可能的，从人的肌肉活检材料（因为纤维很少能由肌腱到肌腱获得），这意味着，这种技术可能无法访问到兴趣研究人类肌源性细胞许多研究实验室。另外，单纤维的技术仅提供了非常有限的细胞数。
在这里，我们将介绍基于发电机排序系统利用胶原酶细胞TLE酶消化和分散酶，随后通过两个连续的轮磁激活细胞分选（MACS），它不仅提供了高纯度（> 95％生肌细胞）和产率（〜2.8×10 6±8.87×10 5个细胞/ g组织），用于在培养实验。 CD56被认为是金标准表面标记为人类卫星细胞的原位 10和体外 11的识别和提供用于珠附着了理想表面标记候选。在此方法中CD56抗体缀合的氧化铁和超顺磁珠含有多糖类的通过一个高梯度磁性细胞分离柱放置在强磁场12,13结合到细胞和通过。分离柱充满从而起到集中磁力线走向其表面产生强磁场梯度（〜4tesla）铁磁性钢丝绒或铁球矩阵14。在这些列中，即使稍微磁性的细胞被吸引和吸附到其表面14。未结合（CD56 – ）细胞通过柱子而CD56 +细胞标记与磁微珠被保留，直到去除来自磁场12,15。
从分类处理的任何阶段的细胞悬浮液可以在进一步的实验所需的密度进行电镀。以下给定的介入的细胞成分可使用免疫细胞化学来鉴定，成像用宽视场或共聚焦荧光显微镜和分析定量使用图像分析方法，它允许在任何给定的图像中的所有标记细胞的快速客观测量。在我们的实验室，我们已经用这种双重免疫排序方式依次通过图像分析16表明CD56 –人成纤维细胞转分化容易进入脂肪细胞，而细胞肌卫星产地s为这种脂肪细胞转化5高度耐药。
Protocol
Representative Results
Discussion
我们已经描述了immunomagentic分拣过程对人类肌源性前体从肌肉活检材料小样本的选择性富集。这种技术是非常宝贵的在我们的实验室克服人类肌源性文化的流失，成纤维细胞，同时也为了解肌源性祖细胞不同群体的独特的行为。一旦纯化的生肌细胞可以追究变化的蛋白质和/或基因表达，或者用于下行实验。
除了细胞纯化，我们也详细了快速而直接的荧光显微镜分析的显微照?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors wish to thank Carl Hobbs and Lindsey Majoram for their technical assistance, and Professor Pat Doherty for use of microscopy facilities. Dr. Agley was supported by a studentship from King’s College London. Funding from the Spurrell Trust is also acknowledged.
Materials
| Collagenase D | Roche | 11088866001 | |
| Dispase II | Sigma | D4693-1G | Must be filter sterilized before use |
| Trypsin/EDTA | (Gibco) Invitrogen | 15400-054 | |
| 100 micron cell strainer | BD Biosciences | 352360 | |
| Collagen solution ( 3 mg/ml in 0.1% acetic acid) | Sigma, Dorset, UK | C8919 | |
| Minisart SRP15 syringe filter (0.2 micron) | Sartorius | 17573ACK | Polytetrafluorethylene (PTFE) membrane |
| CD56 human Microbeads | Miltenyi Biotech | 130-050-401 | Be aware of the limited shelf life of microbeads |
| Anti- fibroblast Microbeads, human | Miltenyi Biotech | 130-050-601 | |
| 40 μm Pre-separation filters | Miltenyi Biotech | 130-041-407 | |
| Large Cell Collumns | Miltenyi Biotech | 130-042-202 | These columns come with a flow resistor. Use of the flow resistor is not necessary to obtain the high myogenic purities described here. |
| LS columns | Miltenyi Biotech | 130-042-401 | |
| MiniMacs Seperator | Miltenyi Biotech | 130-042-102 | This separator fits the large cell column but not the LS column. |
| MidiMACS | Miltenyi Biotech | 130-042-302 | |
| MACS multistand | Miltenyi Biotech | 130-042-303 | |
| BSA | Sigma | Must be filter sterilized before use | |
| Oil Red O | Sigma | O0625 | |
| Triethyl phosphate | Sigma | 538728 | |
| Whatman Paper | Sigma | Z241121-1PAK | No. 42, Ashless. To prepare the filter fold the circular filter paper to make a semi circle, then fold the semi-circle in half again to form a cone shape. Fit the cone into a funnel for filtering. |
| ProLong Gold Antifade Reagent | Molecular Probes, Invitrogen | P36930 | This can be purchased with or without DAPI and does not quench initial fluorescence. |
| AxioVision | Carl Zeiss | Contact Zeiss | |
| Adobe Photoshop CS5 Extended | Adobe (purchased from Pugh Computers) | ADPH16982* |
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