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 border="0" cellpadding="0" cellspacing="0" width="1197">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"><a name="RANGE!A1:D52">Name of Material/ Equipment</a></td>
			<td style="width:187px;">Company</td>
			<td style="width:207px;">Catalog Number</td>
			<td style="width:484px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Equipment</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Tissue&#160;culture biosafety hood</td>
			<td style="width:187px;">Baker Company, Inc.</td>
			<td style="width:207px;">Model SterilGuard Hood</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:320px;">Benchtop centrifuge, such as Beckman Model&#160;</td>
			<td style="width:187px;">Beckman Coulter</td>
			<td style="width:207px;">Model Allegra 6R</td>
			<td>If cell sorting is performed, a centrifuge with refrigeration is preferred</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Microscope</td>
			<td style="width:187px;">Nikon</td>
			<td style="width:207px;">Model Eclipse TS100</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:320px;">Tissue culture incubator, connected to a CO2 source</td>
			<td style="width:187px;">Forma Scientific&#160;</td>
			<td style="width:207px;">Series II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Fluorescence-activated cell sorter (FACS)</td>
			<td style="width:187px;">Becton Dickinson</td>
			<td style="width:207px;">Model Aria</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:320px;">Reagents for isolation of primary myoblasts from tissue</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispase II</td>
			<td style="width:187px;">Roche Applied Science</td>
			<td style="width:207px;">#04942078001</td>
			<td>Prepare a sock solution of 2.4U/ml in DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase D&#160;</td>
			<td style="width:187px;">Roche Applied Science</td>
			<td style="width:207px;">#088882001</td>
			<td>Prepare a stock solution of 10 mg/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1x Sterile Hank&#8217;s Balanced Saline Solution (HBSS), calcium and magnesium free</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">#14185-052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin, fraction V</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">#05470</td>
			<td>Prepare a sterile&#160; solution of 1X HBSS 0.5% BSA for FACS sorting</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Sterile growth medium for myoblasts: Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) with high glucose (4.5g) supplemented with 30% fetal bovine serum</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">11965-092</td>
			<td>Contains L- glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RBC lysis solution</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:207px;">158904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Propidium Iodide stock 10mg/mL</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">P4170</td>
			<td>Prepare the stock solution by diluting the powder in sterile distilled water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Anti-human CD56 antibody for flow cytometry</td>
			<td style="width:187px;">Biolegend</td>
			<td>318310</td>
			<td>APC-conjugated antibody, other labels are avaialable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Reagents for immortalization of primary myoblasts</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ecotropic packaging cell line PE</td>
			<td style="width:187px;">Cell Biolabs</td>
			<td style="width:207px;">RV-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Amphotropic packaging cell line PA3174</td>
			<td style="width:187px;">Cell Biolabs</td>
			<td style="width:207px;">RV-102</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Pig skin gelatin</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">G1890-500g</td>
			<td style="width:484px;">Prepare a stock solution of 0.1% gelatin in water. Coat the dish with the solution at 37&#176;C for one hour. Remove the solution and add medium.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">PolyJet&#160;</td>
			<td style="width:187px;">Signagen</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">G418</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:207px;">345812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hygromycin</td>
			<td style="width:187px;">EMD Biosciences</td>
			<td style="width:207px;">400051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">pBabe plasmids containing mCDK4 and hTERT</td>
			<td style="width:187px;">not commercially available</td>
			<td style="width:207px;"></td>
			<td>Stadler et al, 2013</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Qiagen plasmid midiprep kit</td>
			<td style="width:187px;">Qiagen</td>
			<td>12143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Medium 199</td>
			<td style="width:187px;">Life Technologies&#160;</td>
			<td>Medium 199 (31150022)</td>
			<td style="width:484px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td style="width:187px;">Life Technologies&#160;</td>
			<td>DMEM (11965-092)</td>
			<td style="width:484px;">Mix 4:1 DMEM:199</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:320px;">Myoblast growth medium: 4:1 Dulbecco&#8217;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 &#956;g/l hepatocyte growth factor and 10 &#956;g/l beta fibroblast growth factor.</td>
			<td style="width:187px;">Life technologie (DMEM, F199); Atlanta Biological (FBS); Invitrogen (Hepes); Fisher (ZincSulfate); Sigma (Vit.B12, Dexamethasone); Chemicon international (HGF); Biopioneer (betaFGF)&#160;</td>
			<td style="width:207px;">#15630-080 (Hepes); #Z68-500 (ZnSO); #V2876.#D4902 (Vit.B12, Dex); GF116 (HGF); HRP-0011 (bFGF).&#160;</td>
			<td style="width:484px;">Prepare media and stock solution Vit. B12 (20mg/ml); ZincSulfate (60&#181;g/ml); Dex (55&#181;g/&#181;l) separately for easier use. HGF stock solution (5&#181;g/ml) and FGF (20&#181;g/ml) should be added freshly every week at the final working concentration.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">TrypLE express</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">12605-010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Myosin heavy chain antibody for immunostaining</td>
			<td style="width:187px;">Developmental Hybridoma Bank</td>
			<td style="width:207px;">MF20</td>
			<td>Clone MF20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Desmin Antibody for immunostaining</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>MS-376-S0</td>
			<td>Clone D33</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Horse serum</td>
			<td style="width:187px;">Invitrogen</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Reagents for primary skin fibroblast isolation</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Transport medium: RPMI 1640 supplemented with 10% fetal bovine serum and 0.2% penicillin/streptomycin</td>
			<td style="width:187px;">RPMI medium 1640: GIBCO Life Technologies</td>
			<td style="width:207px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Human primary fibroblast culture medium: RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin</td>
			<td style="width:187px;">Fetal bovine serum: Thermo Scientific</td>
			<td style="width:207px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Collagenase solution: Collagenase type 2 (100mg) resuspended in 12.5 ml of fibroblast culture medium and filter-sterilized</td>
			<td style="width:187px;">Worthington</td>
			<td style="width:207px;">4176</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Sterile 1X Phosphate Buffer Saline (PBS) calcium and magnesium free</td>
			<td style="width:187px;">Lonza</td>
			<td style="width:207px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for isolation of primary myoblasts</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">50ml and 15ml sterile conical tubes</td>
			<td style="width:187px;">GeneMate/Bioexpress</td>
			<td style="width:207px;">C-3394-4 (50ml) ; C3394-1 (15ml))</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile scalpels</td>
			<td style="width:187px;">Aspen Surgical</td>
			<td style="width:207px;">372610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:187px;">Hausser Scientific</td>
			<td style="width:207px;">1492</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 5, 10 and 25 ml pipettes</td>
			<td>Bellco glass</td>
			<td>1226-05010 (5ml); 1200-10010 (10ml) ;1228-25050 (25ml)</td>
			<td>Reusable pipettes are washed, cotton plugged and autoclaved before use</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile tissue culture-treated plastic dishes (10cm)</td>
			<td style="width:187px;">BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile nylon cell strainers (100&#181;m and 40&#181;m size)</td>
			<td style="width:187px;">BD Falcon</td>
			<td colspan="2">352340 (40&#181;m); 352360 (100&#181;m)</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">&#160;Materials for myoblast immortalization</td>
			<td style="width:187px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 0.45&#181;m filters</td>
			<td>Millipore</td>
			<td>SLHV013SL</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Cloning rings</td>
			<td style="width:187px;">Corning</td>
			<td>#3166-8</td>
			<td>To be cleaned and autoclaved before and after use</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Materials for fibroblast cell lines&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 35mm tissue culture-treated plastic dishes</td>
			<td>Greiner bio-one</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile scalpels</td>
			<td>DeRoyal</td>
			<td>D4510A</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">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 height="21">
			<td height="21" style="height:21px;width:320px;">TrypLE express</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">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

16.4K 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

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

科研

医学

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

骨骼肌蛋白质组学性别异形

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

人骨骼肌活检手术使用修改伯格斯坦技术

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨骼肌疾病的定量磁共振成像

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...