Name: isolation and immortalization of patient-derived cell lines from muscle biopsy for disease modeling
Uploaded: 2015-01-18T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling at JoVE.com

This publication is funded through Cure CMD, an Association Contre Les Myopathies (AFM) grant (project 16297), and by the National Institutes of Health (grant numbers K08 AR059750, L40 AR057721 and 2R01NS047727).

注:协议收集人体组织必须审查和批准的机构IRB委员会。收集丢弃，去识别人体组织已经批准了波士顿儿童医院和布里格姆妇女医院IRB委员会。以下描述的方法已经应用于用于从去标识肌细胞的分离，丢弃的组织。所描述的方法适用于组织从患者同意收集材料。 1.细胞分离<ol><li>肌肉活检和肌原细胞的纯化的离解<ol><li>预先称重10cm的组织培养板在组织培养生物安全罩，然后重新称重含有肌肉活检来计算组织的量的板被解离。 </li><li>使用无菌手术刀，肉的肌肉组织和细加几滴无菌1X HBSS以防止组织干燥。 </li><li>添加每个迪为3.5mlspase II和每克肌肉组织胶原酶ð被消化。通过无菌25毫升吸管几次吸取的组织糜和酶溶液。孵育板在组织培养培养箱中于37℃，5％CO 2的15分钟和消化组织直至浆料容易传递虽然灭菌的5毫升吸管。 注:组织离解，通常通过酶消化内45-90分钟达到。请参阅&#39;代表结果"部分了解更多详细信息。 </li><li>添加2倍体积的无菌生长培养基分离的组织，滤波器的通过100微米的细胞过滤过50ml锥形管中，沉淀的细胞10分钟，在329×g离心（〜1100转），室温。请参考材料表介质组合物。 </li><li>重悬沉淀在1体积的无菌生长培养基并加入7倍体积的红血细胞溶解液。过滤通过40微米的细胞过滤的溶液中，然后沉淀吨他的细胞10分钟，在329×g离心，室温。 </li><li>计数细胞在血细胞计数器和重悬细胞于1×HBSS中的0.5％BSA的1×10 6个细胞/ 100微升的浓度。预留〜250,000个细胞在单管，将用作为负（未染色）的控制。预留额外的单色染控制管碘化丙啶和CD56，这是必需的CD56阳性细胞的FACS分选合适的门。请参考细胞分选说明书或FACS分拣核心设施的专家，以确保适当的控制包括咨询。 </li><li>染色的细胞进行排序与5微升/ 10 6细胞抗CD56抗体。在冰上孵育所有样品（包括对照）处理30分钟。 </li><li>在10ml 1×HBSS洗涤样品并沉淀细胞在329×g离心（〜1100转）在冷冻离心机，4℃下10分钟。 </li><li>以1微克/ ml的终浓度添加碘化丙啶至样品为SOR泰德排除死细胞。净化从使用荧光激活细胞分选仪的非肌细胞肌CD56阳性细胞。 </li></ol></li><li>皮肤活检的解离 注:真皮成纤维细胞可以从皮肤拳打患者进行隔离时，肌肉活检不可用。真皮成纤维细胞可以被用作生物材料的许多研究，包括转导的MyoD，以产生肌原细胞。另外，皮肤成纤维细胞可用于产生iPS细胞，这可以分化成为进一步研究各种细胞类型。 <ol><li>运输皮肤活检在输送介质实验室。一旦接收样本，尽快进行原代培养。如果主培养不能在同一天被建立，存储所述样品在室温下过夜。 </li><li>转移的皮肤活检在层流罩中的无菌35mm平皿。 </li><li>冲洗皮肤活检在培养皿用无菌1X PBS以清除血液和碎片。用无菌解剖刀除去脂肪组织。 </li><li>加入2 ml胶原酶溶液和剁碎用手术刀将组织，孵育在37℃下45分钟到1小时，这取决于组织的大小。 </li><li>转移消化的组织到15ml锥形管中，冲洗培养皿以2 ml成纤维细胞培养基中两次，并收集在同一个管中的介质。 </li><li>沉淀细胞，在200×g离心5分钟，在室温下进行。 </li><li>弃去上清液，并用3 ml成纤维细胞培养基洗涤沉淀，再次除去胶原酶，然后沉淀细胞。请参考材料表介质组合物。 </li><li>重复步骤1.2.7一次。 </li><li>重新悬浮的沉淀在5ml成纤维细胞培养基和细胞板到一个T25的无菌组织培养烧瓶中。孵育烧瓶在37℃，5％的CO 2。 </li><li>评价培养成纤维细胞附着和生长在未来1-3天。&lt;BR /&gt;注:一些小的组织块也可以连接到板和成纤维细胞移植这些组织片段。 </li><li>维持培养相同的条件下，直至成纤维细胞生长至约80％汇合。 </li><li>通过胰酶消化收集成纤维细胞和转移到新鲜培养瓶中额外的扩展。通过洗涤的文化在1X PBS 3次游离Ca ++和Mg ++的执行胰酶消化。加入胰蛋白酶-EDTA（见材料表），以将细胞（2毫升/ T25培养瓶中）为2分钟，在37℃。 </li><li>分裂细胞分离到额外的瓶中。如果一些组织片保持附着到原始T25烧瓶，加入5 ml新鲜培养基到该烧瓶中，并更成纤维细胞会迁移出不断。 注:扩展成纤维细胞培养物可以被冷冻下来为P1，并存储在液氮中未来的实验。 </li></ol></li></ol> 2.永生肌细胞<ol><l我&gt;板5000000菲尼克斯亲嗜性包装细胞（PE）的过夜在10cm的无菌组织培养板中的DMEM和培养基199中的4:1的比例，补充有10％小牛血清。 </li><li>料的细胞30分钟前，转染用5ml新鲜培养基补充了10mM咖啡因。 </li><li>从一个MIDI准备（CDK4或hTERT基因的质粒）和均匀的PolyJet的2微克质粒DNA的混合物中，并在室温下孵育15分钟，如制造商推荐的。 </li><li>质粒/混合的PolyJet添加到PE细胞过夜。 </li><li>喂细胞用新鲜的培养基（DMEM培养基和199中的4:1的比例，补充有10％小牛血清）。 12小时后，收集含病毒的上清液，并通过0.45μm孔径过滤器过滤。 </li><li>使用1毫升回收的上清的感染过夜嗜包装细胞系PA317 5和选择后获得稳定的病毒产生细胞系与任一为0.5mg / ml的新霉素（G418），用于CDK4或0.5毫克/毫升潮霉素为端粒酶。 </li><li>准备通过发展稳定的包装细胞融合附近，然后收获上清液每天早晨，傍晚和清晨的三丰收工作病毒上清。 </li><li>过滤病毒上清，要么在-80直接使用或把它们分为1​​ml等份并储存℃下以备后用。需要注意的是病毒上清失去50％的感染效率与每个冻融。记得漂白洗丢弃一切已经触及病毒颗粒之前。 注:稳定PA317病毒产生细胞系可以被冻结，并保持在-150℃下的永久存储（冷冻培养基在10％的DMSO; 90％血清）。 </li><li>板的FACS纯化的肌细胞以5×10 4个细胞/孔在6孔板涂有0.1％明胶的密度（1.1-1.9中的步骤描述）。确保细胞被附着在板继续进行病毒感染之前。 </li><li>加入400微升过滤，女reshly制备病毒上清或冷冻等分试样，以每6孔板过夜（保持2孔作为对照）。 </li><li>通过用2.5ml /孔的新鲜肌肉媒体（4喂食细胞改变介质:; 0.02M的HEPES缓冲液; 1.4毫克/升维生素B12; 1 Dulbecco改进的Eagle培养基（DMEM）和培养基199补充有15％胎牛血清0.03毫克/ L的硫酸锌，0.055毫克/ L的地塞米松，2.5微克/升的肝细胞生长因子与10μg/ L的碱性成纤维细胞生长因子）。丢弃含有漂白剂容器中的病毒颗粒的媒体和吸管。离开细胞在相同培养基中3天，以从感染中恢复，然后用任一400微克/ ml的新霉素（CDK4感染）或300微克/毫升潮霉素（hTERT的感染）治疗供选择。 </li><li>保持在药物选择细胞直至细胞在对照培养皿模具（1-2周）。 </li><li>传代细胞，才成为汇合（60-80％汇合;采用0.05％胰蛋白酶的EDTA），即使是在selecti上期。 Replate细胞多10cm培养皿新鲜的成肌细胞的培养基（如2.11所述）补充与选择的药物。保持永生选择细胞作为异质群体或克隆，以获得完全均匀的遗传背景（转基因的每一个细胞都在相同的插入）。 </li><li>使用以下步骤进行克隆选择: <ol><li>种子的细胞以低密度（ 例如 300到500个细胞于10cm菜），并保持它们为约两周，直到小菌落形成（10-20个细胞）。 </li><li>除去大部分介质在这一点上，只留下一薄膜，防止细胞干燥。 </li><li>将一个克隆环（有机硅真空硅脂一端蘸上）在每个需要的克隆，并添加几滴胰蛋白酶/ EDTA的。 </li><li>收获细胞一旦成为用1毫升提示或巴斯德吸管吸仔细的细胞，并将它们转移到最小可用多孔解放军杀进德（96或48，24或12孔板）。 </li><li>根据需要，以防止局部融合展开克隆。 </li></ol></li></ol>

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The authors have nothing to disclose.

细胞作为一种有用的资源建立疾病表型或疾病的体外模型时，肌细胞群的分离和培养是非常有用的。此处所描述的生肌细胞分离过程允许从骨骼肌标本，然后可以被传播，分化，或立即分析成肌细胞和成纤维细胞的分离。成肌细胞的结构和功能可以通过显微镜检查来评价，细胞存活，细胞融合的评价的评价，或者通过RNA或蛋白质表达的分子研究。此外，成肌细胞...

A correction was made to the authors section in: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling
One of the authors names was corrected from:
Stacy C. Cossette
to:
Stacy A. Cossette

A correction was made to the authors section in: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

Erratum: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

Molecular diagnostics has dramatically evolved in the past 20 years. Genomic DNA is now routinely isolated from sputum or cheek swab, while in the past it required a blood draw. With the current fast turnaround time and ease of gene sequencing, many disease mutations are routinely identified with no need of additional testing. In the case of muscle disease diagnostics, identification of dozens of new genes in the past decade responsible for either muscular dystrophy or myopathy have dramatically changed the ways these diseases are diagnosed 1,2. Currently, there are dozens of genes that have been identified as causes of muscular dystrophy and congenital myopathy, although the mechanisms by which many of these genes produce disease remain unclear. In particular, rare diseases constitute a challenge due to the small size of the patient populations. For these cases, as well as for more common diseases, the generation of stable tools that facilitate studies on the mechanism of pathogenesis and screening of therapeutic drugs is highly desirable. 
Despite dramatic progress in DNA diagnostics, muscle biopsies are still performed to establish the primary diagnosis in many patients in whom primary metabolic or muscle disease is suspected. When a muscle biopsy is necessary, it offers the opportunity for additional diagnostic and research tissue collection with minimal additional morbidity risk for the patient. As there are a number of uses for each tissue specimen, it is highly desirable to establish techniques for primary cell culture using surgical tissue that are straightforward, efficient, and require minimal amounts of tissue. Proper triage of muscle or skin biopsies is required to maximize isolation of primary cells from tissue and long-term storage of live material. Additionally, stem cell research and drug screening holds great promise for developing therapies for many diseases using cell-based assays 3,4.
We herein describe methods for primary cell isolation from human muscle or skin biopsies. Additionally, we include a protocol for immortalization of myogenic cells, which is useful for generating large numbers of cells from an individual. These cells can be used for downstream applications, such as custom drug-screenings, which are otherwise unachievable with the overall low number of cells obtained from primary tissue.

<table><tbody><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Tissue&amp;nbsp;culture biosafety hood</td><td>Baker Company, Inc.</td><td>Model SterilGuard Hood</td><td></td></tr><tr><td>Benchtop centrifuge, such as Beckman Model&amp;nbsp;</td><td>Beckman Coulter</td><td>Model Allegra 6R</td><td>If cell sorting is performed, a centrifuge with refrigeration is preferred</td></tr><tr><td>Microscope</td><td>Nikon</td><td>Model Eclipse TS100</td><td></td></tr><tr><td>Tissue culture incubator, connected to a CO&lt;sub&gt;2&lt;/sub&gt; source</td><td>Forma Scientific&amp;nbsp;</td><td>Series II</td><td></td></tr><tr><td>Fluorescence-activated cell sorter (FACS)</td><td>Becton Dickinson</td><td>Model Aria</td><td></td></tr><tr><td>Reagents for isolation of primary myoblasts from tissue</td><td></td><td></td><td></td></tr><tr><td>Dispase II</td><td>Roche Applied Science</td><td>#04942078001</td><td>Prepare a sock solution of 2.4U/ml in DMEM</td></tr><tr><td>Collagenase D&amp;nbsp;</td><td>Roche Applied Science</td><td>#088882001</td><td>Prepare a stock solution of 10 mg/ml</td></tr><tr><td>1x Sterile Hank&amp;rsquo;s Balanced Saline Solution (HBSS), calcium and magnesium free</td><td>GIBCO Life Technologies</td><td>#14185-052</td><td></td></tr><tr><td>Bovine serum albumin, fraction V</td><td>Sigma</td><td>#05470</td><td>Prepare a sterile&amp;nbsp; solution of 1X HBSS 0.5% BSA for FACS sorting</td></tr><tr><td>Sterile growth medium for myoblasts: Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM) with high glucose (4.5 g) supplemented with 30% fetal bovine serum</td><td>GIBCO Life Technologies</td><td>11965-092</td><td>Contains L- glutamine</td></tr><tr><td>RBC lysis solution</td><td>Qiagen</td><td>158904</td><td></td></tr><tr><td>Propidium Iodide stock 10mg/ml</td><td>Sigma</td><td>P4170</td><td>Prepare the stock solution by diluting the powder in sterile distilled water</td></tr><tr><td>Anti-human CD56 antibody for flow cytometry</td><td>Biolegend</td><td>318310</td><td>APC-conjugated antibody, other labels are avaialable</td></tr><tr><td>Reagents for immortalization of primary myoblasts</td><td></td><td></td><td></td></tr><tr><td>Ecotropic packaging cell line PE</td><td>Cell Biolabs</td><td>RV-101</td><td></td></tr><tr><td>Amphotropic packaging cell line PA3174</td><td>Cell Biolabs</td><td>RV-102</td><td></td></tr><tr><td>Pig skin gelatin</td><td>Sigma</td><td>G1890-500g</td><td>Prepare a stock solution of 0.1% gelatin in water. Coat the dish with the solution at 37&amp;deg;C for one hour. Remove the solution and add medium.</td></tr><tr><td>PolyJet&amp;nbsp;</td><td>Signagen</td><td>SL100688</td><td></td></tr><tr><td>G418</td><td>Fisher</td><td>345812</td><td></td></tr><tr><td>Hygromycin</td><td>EMD Biosciences</td><td>400051</td><td></td></tr><tr><td>pBabe plasmids containing mCDK4 and hTERT</td><td>not commercially available</td><td></td><td>Stadler et al, 2013</td></tr><tr><td>Qiagen plasmid midiprep kit</td><td>Qiagen</td><td>12143</td><td></td></tr><tr><td>Medium 199</td><td>Life Technologies&amp;nbsp;</td><td>Medium 199 (31150022)</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>Life Technologies&amp;nbsp;</td><td>DMEM (11965-092)</td><td>Mix 4:1 DMEM:199</td></tr><tr><td>Myoblast growth medium: 4:1 Dulbecco&amp;rsquo;s modified Eagle medium (DMEM) and Medium 199 supplemented with 15% fetal bovine serum; 0.02 M HEPES buffer; 1.4 mg/L vitamin B12; 0.03 mg/L ZnSO4, 0.055 mg/L dexamethasone, 2.5 &amp;mu;g/L hepatocyte growth factor and 10 &amp;mu;g/L beta fibroblast growth factor.</td><td>Life technologie (DMEM, F199); Atlanta Biological (FBS); Invitrogen (Hepes); Fisher (ZincSulfate); Sigma (Vit.B12, Dexamethasone); Chemicon international (HGF); Biopioneer (betaFGF)&amp;nbsp;</td><td>#15630-080 (Hepes); #Z68-500 (ZnSO); #V2876.#D4902 (Vit.B12, Dex); GF116 (HGF); HRP-0011 (bFGF).&amp;nbsp;</td><td>Prepare media and stock solution Vit. B12 (20mg/ml); ZincSulfate (60&amp;micro;g/ml); Dex (55&amp;micro;g/&amp;micro;l) separately for easier use. HGF stock solution (5&amp;micro;g/ml) and FGF (20&amp;micro;g/ml) should be added freshly every week at the final working concentration.</td></tr><tr><td>TrypLE express</td><td>GIBCO Life Technologies</td><td>12605-010</td><td></td></tr><tr><td>Myosin heavy chain antibody for immunostaining</td><td>Developmental Hybridoma Bank</td><td>MF20</td><td>Clone MF20</td></tr><tr><td>Desmin Antibody for immunostaining</td><td>Thermo Scientific</td><td>MS-376-S0</td><td>Clone D33</td></tr><tr><td>Horse serum</td><td>Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>Reagents for primary skin fibroblast isolation</td><td></td><td></td><td></td></tr><tr><td>Transport medium: RPMI 1640 supplemented with 10% fetal bovine serum and 0.2% penicillin/streptomycin</td><td>RPMI medium 1640: GIBCO Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>Human primary fibroblast culture medium: RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin</td><td>Fetal bovine serum: Thermo Scientific</td><td>SH30071.03</td><td></td></tr><tr><td>Collagenase solution: Collagenase type 2 (100 mg) resuspended in 12.5 ml of fibroblast culture medium and filter-sterilized</td><td>Worthington</td><td>4176</td><td></td></tr><tr><td>Sterile 1X Phosphate Buffer Saline (PBS) calcium and magnesium free</td><td>Lonza</td><td>17-516F</td><td></td></tr><tr><td>Materials for isolation of primary myoblasts</td><td></td><td></td><td></td></tr><tr><td>50 ml and 15 ml sterile conical tubes</td><td>GeneMate/Bioexpress</td><td>C-3394-4 (50 ml) ; C3394-1 (15 ml))</td><td></td></tr><tr><td>Sterile scalpels</td><td>Aspen Surgical</td><td>372610</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>1492</td><td></td></tr><tr><td>Sterile 5, 10, and 25 ml pipettes</td><td>Bellco glass</td><td>1226-05010 (5 ml); 1200-10010 (10 ml) ;1228-25050 (25 ml)</td><td>Reusable pipettes are washed, cotton plugged and autoclaved before use</td></tr><tr><td>Sterile tissue culture-treated plastic dishes (10 cm)</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>Sterile nylon cell strainers (100 &amp;micro;m and 40 &amp;micro;m size)</td><td>BD Falcon</td><td>352340 (40&amp;micro;m); 352360 (100&amp;micro;m)</td><td></td></tr><tr><td>&amp;nbsp;Materials for myoblast immortalization</td><td></td><td></td><td></td></tr><tr><td>Sterile 0.45 &amp;micro;m filters</td><td>Millipore</td><td>SLHV013SL</td><td></td></tr><tr><td>Cloning rings</td><td>Corning</td><td>#3166-8</td><td>To be cleaned and autoclaved before and after use</td></tr><tr><td>Materials for fibroblast cell lines&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Sterile 35 mm tissue culture-treated plastic dishes</td><td>Greiner bio-one</td><td>628160</td><td></td></tr><tr><td>Sterile scalpels</td><td>DeRoyal</td><td>D4510A</td><td></td></tr><tr><td>Sterile T25 tissue culture flasks</td><td>Techno Plastic Product, TPP</td><td>90026</td><td>sold in the USA by MIDSCI</td></tr><tr><td>TrypLE express</td><td>GIBCO Life Technologies</td><td>12605-010</td><td></td></tr></tbody></table>

isolation and immortalization of patient-derived cell lines from muscle biopsy for disease modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

图1示出的一些参与了主组织离解的关键步骤:组织的确切量在无菌组织培养培养皿称重（ 图1A，B）。然后将组织细使用无菌手术刀切碎，直到组织浆料得到（ 图1C，D）。下列另外的消化酶，主要肌肉组织离解，通常通过酶消化内45-90分钟达到。组织消化的进展通常监视每15-20分钟，以防止过量酶切和细胞死亡。酶消化可以轻轻吸入混合几次每次15-20分钟。在?...

Watch this Scientific Journal Video about Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling at JoVE.com

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

isolation-immortalization-patient-derived-cell-lines-from-muscle

Research

JoVE Journal

Medicine

16.5K 次观看.  UT Southwestern Medical Center. This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

视频: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

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.

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

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

人成肌细胞的分离，造血分化的评估和存储操作的钙进入测量

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

将人类成纤维细胞直接重新编程到肌细胞中，以研究神经肌肉疾病的治疗方法

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

遗传学

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

生物学

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

医学

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...