This work was supported by the European Research Council (ERG) and EMBO installation (ERG) and by a PhD fellowship from the Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (MP).

注意:一个鼠标的产量足够的成肌细胞大约两张35毫米的菜肴或两个活成像菜，所以计划mattings，夹层，和涂层（步骤2.6）相应。 10只动物 - 由于肌细胞是通过连续的离心和预镀隔离，协议应在5个批次进行。 所有涉及动物对象的程序是由动物伦理委员会研究所德MEDICINA分子与大学皮埃尔与玛丽·居里批准 1.新生小鼠后肢肌肉解剖<ol><li>提前（ 材料表 ）准备所有的解决方案，并通过过滤（0.22微米过滤器）消毒。确保所有媒体都在37℃下除细胞之前，除了含有基底膜基质（ 例如，基质胶）的制剂。 </li><li>消毒夹层材料（每个之一:剪子弯，直剪刀，镊子定期，和细尖镊子），并通过用70％乙醇擦拭的工作台上。 </li><li>制备100毫米培养皿用5ml Dulbecco氏磷酸盐缓冲盐水（DPBS）的肌肉收集和保持在冰上，直到切碎步骤。 </li><li>斩首P6 - P8小鼠直剪刀和消毒用70％乙醇的皮肤。 </li><li>使背部皮肤切口，然后轻轻向外拉出后肢，直到它被删除，完全暴露后肢肌肉组织。 </li><li>使用钳去除脂肪组织而不损伤肌肉。 </li><li>要卸下背后肢肌肉，保持伸展肢体和弯曲的爪子，露出脚后跟肌腱。骨用剪子弯分离肌肉，从肌腱出发，通过轻轻滑动和切割以上。切除的肌肉，并放置在冰DPBS。 </li><li>由俭用细尖镊子肌肉和周围切割而不会损坏或股骨的膝关节隔离股四头肌。</li><li>解剖所有动物后，继续到无菌层流细胞培养罩，确定应执行以下所有步骤。 </li></ol> 2，成肌细胞分离<ol><li>除去多余的DPBS的。讳言灭菌弯曲剪刀组织，以获得一个均匀的质量。 </li><li>收集在用5毫升消化混合物的50毫升锥形离心管中的组织糜和在37℃搅拌孵育它为90分钟。 </li><li>通过在75 XG加入6毫升夹层介质和离心5分钟的悬浮液，以沉淀剩余的组织停止消化。 </li><li>仔细收集上清。 确保不收集组织碎片 。在350×g离心5分钟离心它;它悬浮在5毫升夹层介质中。 </li><li>通过40微米的细胞滤网过滤细胞悬浮液。添加25夹层介质中的溶液，并preplate它在150毫米的菜用于在细胞培养孵育箱（4小时37℃，5％CO 2的&lt;/子&gt;），以允许成纤维细胞附着。 </li><li>在冷的IMDM 100在RT 1小时:在预镀，外套菜肴基底膜基质的500微升稀释1。用DPBS洗一次，并立即板上的细胞（步骤2.8）或生长培养基，直到电镀离开。 </li><li>预镀后，收集上清液并在350 xg离心离心它为10分钟。 </li><li>重悬在它生长介质和细胞计数的血球。调节音量，使细胞150,000 250,000是每个基底膜基质涂，镀菜。保持所述细胞在细胞培养孵化器。 </li></ol> 3.肌纤维分化注:3天后，将细胞应在约70％汇合（ 图1B）开始保险丝和形式肌管。 <ol><li>在这点上，转染的细胞，如果需要的话，用siRNA或感兴趣的DNA。如果细胞不被转染，直接切换到分化培养基和滑雪p依次3.4。 </li><li>用按照制造商的说明书转染试剂转染。孵育用siRNA脂质复合物5小时的细胞（为20nM + 1微升试剂）或DNA-脂质复合物（1微克试剂+1μL）。如有必要，优化siRNA和DNA的浓度。 </li><li>与分化培养基清洗过一次，然后切换到新的分化培养基。 </li><li>在冰冷的分化培养基2:第二天，稀释基底膜基质1。删除现有的媒体，并添加冰冷的基质200微升到每道菜。 </li><li>孵育在细胞培养孵化30分钟。 </li><li>补充与集聚蛋白的分化培养基（100毫微克/毫升），并小心加入2毫升到细胞中。 </li><li>仔细改一半培养基的每2天，总是与集聚蛋白补充至100毫微克/微升的最终浓度。 </li><li>监测细胞分化和生存力。取决于多种因素（如胎牛血清和chicken胚胎提取物的起源），细胞可能需要5之间- 10分化d可达到完全成熟（ 图2）。 </li></ol> 4.免疫染色玻璃底菜<ol><li>对于免疫染色，在感兴趣的任何时间点，用DPBS洗涤细胞一次，并解决这些问题用4％PFA在RT 10分钟。 </li><li>用DPBS洗两次。在这一点上，可以将细胞储存于4℃。 </li><li>用0.5％的Triton X-100通透它们在RT 5分钟。 </li><li>用PBS洗两次，并用在室温阻断30分钟溶液阻塞。 </li><li>用在阻断溶液O / N在4℃下稀释的初级抗体孵育它们。 </li><li>洗3次用DPBS在RT 5分钟。 </li><li>与二级抗体和DAPI的0.2微克/毫升1小时在室温下孵育它们。 </li><li>洗3次用DPBS在RT 5分钟。 </li><li>添加安装介质的200μL并继续成像。 </li></ol>

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作者什么都没有透露。

使用这种协议的主要成肌细胞的培养产生了一个特殊的利基，极大地培育肌纤维的发展。这部分是由于其也存在于非常小的数字的其它细胞类型。必须实现成肌细胞的浓度和纯度文化之间的平衡。一个良好的细胞培养物也取决于用于所述介质制剂中的产品的质量。动物来源的所有产品应彻底测试。在我们的经验中，消化条件下也应被监控。 像往常一样，小学文化，实验变异可能比孤立纤维或成肌细胞永生化的研究水平。这种变异可以通过介质和消化成分，小鼠的年龄和大小，以及用于培养操作和结果收集时间点的标准化减少。然而，在实时审议的错综复杂的机制的优点必要的肌纤维的发展大大超过了可变性的缺点。 这个协议赋予的体外方法的优点而不损害细胞的分化。肌纤维成熟直至形成打黑收缩偶联钙火花。这些功能性的输出可以在不同的实验条件下进行访问。此外，可存在到协议作出许多技术的变化。成肌细胞可以从与肌肉发育的兴趣突变新生小鼠收获。细胞可被裂解为在不同分化的时间点的生化分析。钙指标可以加到培养遵循其动态。光遗传学的结构可以用来执行一定的信号通路或诱导特异性当地反应。最后，肌纤维可以与其他细胞类型一起共培养，以研究它们的相互作用。

骨骼肌组成到体重1的40％。肌肉相关疾病代表一个巨大的健康和经济负担2。如何形成这种高度复杂的和有组织的组织，维持和再生的构成广泛和很好建立的研究领域。取决于具体的科学兴趣，最适合的方式的范围可以从简单的肌管培养物，以复杂的体内模型3 - 6。 该协议的目的是提供一种体外系统，该系统允许通过实时成像和免疫监视肌形成的。与传统方法相比，该系统提供了一个非常完整的和动态的洞察鼠标生肌过程。细胞可以从成肌细胞阶段横向显示三合会及周边核7成熟，多核肌纤维被遵循。这个成熟水平可使用常规细胞培养设备来实现，而不需要复杂的刺激或机械装置。虽然在体外系统一些成功的报道8,9，据我们所知，这是唯一的协议产生与T管与肌浆网（SR）横向配对鼠标成熟肌纤维。因此，该体外系统可以用来研究三元形成的分子机制，目前仍在知之甚少10。 使用该系统的进一步的优点是有效的鼠标定位资源，诸如抗体，药物，和RNAi工具的可用性。相对简单的协议不需要费力的步骤，技术娴熟的操作，或昂贵的和专用设备。成熟肌纤维开始5天的文化差异7后出现的，显示收缩加之钙火花（未公布数据）。在一个星期内，在不同的发展在哺乳动物体中最复杂的细胞之一的人阶段可以结合研究与各种体外测定法。

<table><tbody><tr><td>Dispase II</td><td>Gibco</td><td>17105041</td><td></td></tr><tr><td>Collagenase&amp;nbsp;type V</td><td>Sigma-Aldrich-Aldrich</td><td>C9263</td><td></td></tr><tr><td>IMDM, Glutamax supplemented</td><td>Gibco</td><td>31980022</td><td></td></tr><tr><td>Matrigel Growth reduced factor</td><td>Corning</td><td>354230</td><td>protein concentration of the lot should be around 10 mg/mL and endotoxin result should be &amp;lt;1.5</td></tr><tr><td>Chicken Embryo Extract</td><td>MP biomedical</td><td>2850145</td><td>it is also possible to prepare in the lab (Danoviz ME, Yablonka-Reuveni Z. Methods Mol Biol (2012))</td></tr><tr><td>Recombinant rat agrin</td><td>R&amp;amp;D systems</td><td>550-AG-100</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Horse Serum</td><td>GE Healthcare&amp;nbsp;</td><td>11581831</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen</td><td>13778-150</td><td>used to transfect siRNA</td></tr><tr><td>Lipofectamine LTX</td><td>Invitrogen</td><td>15338-100</td><td>used to transfect DNA</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668030</td><td>used to transfect siRNA plus DNA</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190094&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Eurobio</td><td>CVFSVF0001</td><td></td></tr><tr><td>Cell strainer</td><td>Corning</td><td>21008-949</td><td></td></tr><tr><td>Fluorodishes</td><td>World precision instruments</td><td>FD35-100</td><td>dishes used to cultivate cells for live imaging</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>16% PFA (Paraformaldehyde)</td><td>Science Services GmbH</td><td>E15710</td><td></td></tr><tr><td>Goat Serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>BSA (Bovine Serum Albumine)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Saponine</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>use at 200 ng/&amp;micro;L</td></tr><tr><td>Fluoromount-G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions and Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Digestion Mix</td><td></td><td></td><td>in DPBS&lt;br/&gt; 5 mg/mL collagenase&lt;br/&gt; 3.5 mg/mL dispase&lt;br/&gt; sterile filtered, can be stored in working aliquotes for 2 weeks at -20 &amp;deg;C</td></tr><tr><td>Dissection Medium</td><td></td><td></td><td>in IMDM Glutamax supplemented&lt;br/&gt; 10% FBS&lt;br/&gt; 1% Penicillin/Streptomycin&lt;br/&gt; sterile filtered</td></tr><tr><td>Growth Medium</td><td></td><td></td><td>in IMDM Glutamax supplemented&lt;br/&gt; 20% FBS&lt;br/&gt; 1% Chicken Embryo Extract&lt;br/&gt; 1% Penicillin-Streptomycin&lt;br/&gt; sterile filtered</td></tr><tr><td>Differentiation Medium</td><td></td><td></td><td>in IMDM Glutamax supplemented&lt;br/&gt; 2% Horse Serum&lt;br/&gt; 1% Penicillin-Streptomycin&lt;br/&gt; sterile filtered</td></tr><tr><td>Blocking Solution</td><td></td><td></td><td>in DPBS&lt;br/&gt; 10% Goat Serum&lt;br/&gt; 5% BSA&lt;br/&gt; add 0.1% saponine when incubating with primary and secondary antibodies</td></tr></tbody></table>

in vitro differentiation of mature myofibers for live imaging

骨骼肌是由肌纤维，在哺乳动物体内最大的细胞和少数的合胞体中的一个。如何，组成它的复杂性和进化上保守的结构组装仍在调查之中。它们的大小和生理特征往往限制操纵和成像应用。永生化细胞系的培养被广泛使用，但它只能复制分化的早期步骤。 在这里，我们描述了一个协议，使从鼠标主成肌细胞起源的肌纤维容易遗传操作。分化的一周后，将肌纤维显示收缩性，对齐肌节和三单元组，以及外围核。整个分化过程可以跟随实时成像或免疫荧光。该系统结合了现有离体和体外协议的优点。容易和有效转染的可能性，以及四通八达的交通网络的所有分化阶段扩大了潜在的应用。肌纤维随后可不仅用来解决相关发育和细胞生物学的问题，但也再现肌肉疾病表型为临床应用。

肌纤维发育的程度主要由隔离成肌细胞的纯度和可行性决定。的粘附，增殖和融合的能力可以用来凭经验访问这些参数（ 图1的A，B）。在增殖D2，成肌细胞应该坚持并应该显示典型的梭形。增殖预计在此阶段广泛地发生，导致自发肌管形成翌日（ 图1B）。 细胞汇合可能需要稍作调整。如果成肌细胞需要超过3天的增殖和熔断器它应该增加。如果肌纤维不能生长和细长相对直由于它们的密度应当减少。汇合典型地从中心向盘的外周降低，所以最好肌纤维应朝向外区域被发现。 肌管将快速伸长和显示多个中心对准的细胞核（ 图1C）。由D5，部分细胞开始采集条纹和移动他们的原子核外围。成熟特征的肌纤维的数量将随时间以及与单元厚度（ 图1D）增加。 分化程度可通过免疫荧光来进一步观察。固定在分化D8目前横向黑社会肌纤维。这可以通过T管（DPHR）和SR（triadin），预计在三单元组（ 图2）到共定位的成像元件来确认。 肌纤维的功能性可以通过实时成像来解决。从分化D3起，单元格显示自发抽搐。通过转染钙传感器（ 如:，GCaMP6f 11）中 ，有可能观察到收缩联接与钙的峰（ 图3）。 使用该系统，我们能够以确定在中央核肌病和肌强直性营养不良打乱一个新的分子途径，因此其可以是对创新分子疗法7的新靶点。我们也适于此方法研究了神经肌肉接头（NMJ）12的开发。通过与大鼠脊髓外植体的共培养，我们已在NMJ形成13描述了用于动力蛋白的作用。 <img alt="图1" src="/files/ftp_upload/55141/55141fig1.jpg" /> 图 1: 成肌细胞文化发展阶段。 A）在扩散D2，成肌细胞一直坚守并开始激增。 B）在prolif关合作D3，60铺满 - 80％达到和成肌细胞开始自发地融合。 c）在分化D3，包含位于中央的细胞核肌管占主导地位。 D）从分化D5起（ 例如8天），肌纤维开始呈现出条纹和外设核并开始变厚。比例尺:50微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55141/55141fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55141/55141fig2.jpg" /> 图2:D8肌纤维免疫显的代表共焦成像。 A）免疫染色二氢吡啶受体（DHPR，上图）和triadin（TRDN，中间面板）。在DHPR，TRDN和DAPI通道的重叠图显示了黑社会组件共存。 B）<html>在答Ç绘制的黄线）染色α辅肌动蛋白（绿色）和DAPI（蓝色）肌纤维的容积再现的三维图像的强度分布。比例尺和网格宽度:5微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55141/55141fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55141/55141fig3.jpg" /> 图 3: 自发性抽搐肌纤维钙水平的实时成像。在抽动肌纤维钙火花的 ）高速长延时（20ms的帧）显微镜。通过GCaMP6f的表达（Addgene质粒＃40755）中检测到的钙。 B）的荧光强度的定量随时间在面板A中的钙传感器_blank"&gt;请点击此处查看该图的放大版本。

Watch this Scientific Journal Video about In Vitro Differentiation of Mature Myofibers for Live Imaging at JoVE.com

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

在成熟的肌纤维诱导分化实时成像

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

in-vitro-differentiation-of-mature-myofibers-for-live-imaging

Research

JoVE Journal

Developmental Biology

In Vitro Differentiation of Mature Myofibers for Live Imaging

10.2K 次观看.  Universidade de Lisboa. Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

视频: In Vitro Differentiation of Mature Myofibers for Live Imaging

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

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

生物学

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.

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 ...

医学

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 ...

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 ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

在成细胞分化和融合过程中利用活成像跟踪核

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

单一肌纤维培养测定法用于评估体外成人肌肉干细胞功能

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

使用基于光学的系统对水凝胶嵌入的完整小鼠肌纤维进行高通量收缩测量

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...