Name: preparation and culture of myogenic precursor cells/primary myoblasts from skeletal muscle of adult and aged humans
Uploaded: 2017-02-16T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans at JoVE.com

This work is supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/L021668/1), the MRC and Arthritis Research UK as part of the MRC - Arthritis Research UK Centre for Integrated Research into Musculoskeletal Ageing (CIMA) and the Wellcome Trust Institutional Strategic Support Fund (097826/Z/11/A). The authors would like to thank Dr Dada Pisconti (University of Liverpool) for her expertise and advice in the isolation of muscle progenitor cells.

所有涉及本文所述人体组织的实验是由英国利物浦大学，大学医院安特里医院和西南威尔士研究伦理委员会的事先批准（批准号:13 / WA / 0374）和实验是按照良好做法指导意见执行。利物浦大学担任道德赞助这项研究。所有捐助者给知情同意这项研究的招生。肌肉是从孤立的人（BMI &lt;25）:成人:30±2.8岁，年龄:69±5岁。 1.准备文化<ol><li> 文化的涂层表面层粘连蛋白 <ol><li>制备10微克/毫升的层粘连蛋白的在1×DPBS（Dulbecco氏磷酸盐缓冲盐水）的工作溶液。 </li><li>吸取的层粘连蛋白溶液的最小量以完全覆盖在其上的细胞将要被镀（ 表1）的表面上。孵育ŧ电镀细胞之前他培养皿至少30分钟，在加湿37℃，5％CO 2培养箱。小心处理层粘连蛋白，避免使用旋涡。在DPBS稀释10微克/毫升的层粘连蛋白的工作溶液可以储存在4℃和再使用数次。 </li><li>使用60毫米（20 cm 2）的培养皿或2个孔在6孔板（2×10 cm 2）的每〜18 - 19毫克骨骼肌到板的细胞（5.50×10 4个细胞的数量）。在任何时候都镀细胞功能研究执行时，细胞计数。 最初从女性患者（30±2.8岁，年龄:69±5岁，BMI &lt;25人）的脚手术（趾短伸肌，胫骨前肌或外展肌halluces）中获得的样品:注意。 </li></ol></li><li> 酶溶液的制备 <ol><li>准备250毫米氯化钙2有效的解决方案:在10毫升1X DPBS 277毫克的股票氯化钙 。过滤等等lution用0.2微米滤膜和储存在4℃。 </li><li>制备1.5 U / ml的胶原酶的D，2.4 U / ml的分散酶II的和2.5mM 氯化钙在无血清的DMEM（Dulbecco氏改良的Eagle氏培养基）（ 表2）的工作溶液。 </li><li>拌匀并过滤通过0.2微米滤膜进行灭菌的酶溶液。制备酶溶液提前，并冷冻向下（-20℃）中的等分试样以供将来使用。 </li></ol></li></ol> 2.组织消化:机械和酶解<ol><li>在DPBS在样本采集后，肌肉保持在4°C，直到消化。制备2毫升胶原酶分散酶- 氯化钙每18溶液的最小体积- 19毫克的肌肉组织和组织分解之前温热至37℃。 </li><li>浸入肌肉活检简要地在70％乙醇，用新鲜的DPBS洗涤并放置在新的培养皿的组织用1mL的酶的解。 </li><li>丢弃尽可能多的纤维化和脂肪组织尽可能快速地而是轻轻撕开肌肉成小，但区分片（约&gt; 0.5， 平方毫米）用无菌剪刀或手术刀（刀片第10号）。 </li><li>将样品转移到50 mL管中与剩余的1毫升胶原酶分散酶- 的 CaCl 2。 </li><li>孵育在37℃下将组织高达30 - 40分钟。 10分钟 - 轻轻搅拌筒，每5移动肌肉组织。 </li><li>外套与媒体的吸移管，以避免释放的细胞到移液器的塑料壁的粘附性。添加2倍体积的无菌生长培养基， 例如，4毫升的DMEM 20％FBS（胎牛血清），1％L-谷氨酰胺和1％P / S（青霉素-链霉素）的，以停止消化。 </li><li>吸管上下数次用5毫升吸管，以帮助从肌纤维细胞的释放。 注:样品通常被完全由于其体积小分离。 HH但是，如果肌肉的片段仍然存在，使用第二孵育肌肉的剩余部分，用新鲜的酶溶液。 </li><li>过滤通过70微米的细胞过滤过的50mL锥形管肌肉溶液。通过过滤洗涤与多个媒体和过滤剩余的细胞。 </li><li>离心在443×g离心5分钟，在室温以沉淀细胞。小心弃上清。 </li><li>溶解沉淀到F-12介质（咸的F-12营养混合），20％FBS，10％HS（马血清），1％P / S，1％α谷氨酰胺和2.5纳克/ FGF-B中的溶液（重组人碱性成纤维细胞生长因子）。这里，用4毫升媒体为60毫米的培养皿。 </li></ol> 3.细胞接种<ol><li>收集预先涂覆有从培养箱（第1.1节）10微克/毫升的层粘连蛋白的培养容器中。 </li><li>小心地从培养皿去除多余的层粘连蛋白，并避免接触到表面（或它会干扰蛋白质结构）。洗培养皿用DPBS（可选）。 </li><li>直接镀在层粘连蛋白涂覆的容器中的细胞，并在湿润的37℃，5％CO 2培养箱培养24小时。 </li><li>可视化明场显微镜（100X总放大倍率）第二天下的细胞。轮小细胞附着在表面，而其余的碎片可以在培养基中可以看出。 </li><li>媒体改变到有20％FBS，10％HS，1％P / S，1％α谷氨酰胺和2.5纳克/ FGF-B中的溶液补充新鲜的F-12培养基。 </li></ol> 4.培养和细胞传代<ol><li>改变介质每2 - 3天，以避免自发尽快分裂细胞作为细胞群是可见的，在显微镜下（100X总放大倍数，例如，在图2中 ，从老年人细胞，通道0，后7天）分化。 </li><li>在第一通道（P1），介质改变到有20％FBS，10％HS，1％补充高糖DMEMP / S，1％α-谷氨酰胺。避免使用FGF-B从这一点，作为FGF是一种有效的有丝分裂原和重要的因素在培养开始，但是，如果在培养使用较长它可以促进成纤维细胞的过度生长。 </li><li>对于细胞传代: <ol><li>删除媒体并用DPBS洗涤细胞两次。 </li><li>添加0.25％的EDTA胰蛋白酶的最小体积以覆盖细胞的表面上。轻轻摇动，以确保所有的细胞被分离液覆盖，孵育在室温下10秒钟，然后将其删除。 </li><li>孵育将细胞在增湿的37℃，5％CO 2培养箱3 - 5分钟。轻轻点击和明亮的光学显微镜（100X总放大倍率），这些细胞是圆的，但是从表面没有完全脱落下检查。如果没有变化在细胞中观察到，孵育5更多分钟。 </li><li>加5生长毫升培养基（高糖DMEM补充有20％FBS，10％HS，1％P / S，1％α-谷氨酰胺）以收集细胞，混合好，转移与新媒体的细胞成T75烧瓶中。洗剩余的细胞重复这一步骤与生长培养基的另外5毫升（10毫升总一个T75烧瓶）中。 </li><li>到preplate（在第一通道优选），孵育所述细胞在加湿37℃，5％CO 2培养箱40分钟。收集与没有在新的T75烧瓶连接，并培育它们的细胞上清液。这应该充实成肌细胞的文化，因为大部分的成纤维细胞，应在第一瓶已连接。 </li></ol></li><li>改变介质每2 - 3天，并尽快分裂细胞1至4，因为它们达到的最大收率70％汇合。 </li><li>对于分化，一旦细胞达到70％ - 80％汇合，改变培养基分化培养基（DM）:用2％HS，1％P / S，1％的α-谷氨酰胺补充高糖DMEM。 7天取决于成肌细胞C的质量和纯度 - 细胞应5内有所区别ulture。 </li></ol> 5.转染协议<ol><li>种子50,000个细胞/孔在MF 20，衰老和存活测定一个12孔板。培养细胞在盖玻片上或涂上层粘连蛋白一盘。对于Ki67的染色，种子25,000个细胞/孔在12孔板中。 </li><li>对于转染，按照使用5微升转染试剂，100nM的控制微小RNA模仿或抑制剂，为100nM微RNA模拟物或每孔为100nM微RNA抑制剂的制造商的程序，用1毫升的总体积。 </li><li>切换到分化培养基6 h后（高糖DMEM补充了2％HS，1％P / S，1％的α-谷氨酰胺）。对于增殖实验，将细胞染色2天转染后;对于衰老，转染后7天;和MF20染色，染后七天。 </li></ol> 6.免疫染色的细胞<ol><li> Ki67的，MyoD的和MF 20染色<ol><li>准备块1（10％HS和在PBS中的0.1％的Triton-X）和块2（10％HS和0.05％的Triton-X的PBS中）的解决方案。使用每孔试剂500μL，对于12孔板中。 注:执行使用用于孵育步骤摇动以下步骤。 </li><li>从细胞中取出介质。 </li><li>用PBS冲洗细胞。 </li><li>固定用冷甲醇将细胞上的摇杆10分钟。 </li><li>从细胞中除去甲醇，并用PBS漂洗3次，每次5分钟。 </li><li>在RT上的摇杆添加块1溶液孵育细胞1小时。 </li><li>新增主要抗体溶液:用于Ki67的染色，用兔单克隆抗体来Ki67的（1:1000稀释块2）;为的MyoD，使用MYOD1（D8G3）XP的兔单克隆抗体（1:100稀释在方框2）;对于MF 20染色，使用MYH1E（MF 20）初级抗体（DSHB，1:1000稀释块2）。 </li><li>孵育细胞1小时（RT）到O / N（4℃）上的摇杆的第一抗体。 </li><li>收集主AB（它可以如STO重复使用几次红色在4°C）。 </li><li>冲洗细胞，用PBS，每次5分钟的3倍。 </li><li>添加适当的二级抗体:对于Ki67表达或MyoD的染色，山羊抗兔IgG（H + L）次级抗体，的Alexa Fluor 488缀合物（1:1,000稀释在PBS中）;为MF 20染色，山羊抗小鼠IgG（H + L）次级抗体，的Alexa Fluor 488缀合物（1:1,000稀释在PBS中）。 </li><li>包裹含有细胞的板孵育二级抗体在黑暗中2小时就在RT摇杆。 </li><li>冲洗细胞，用PBS 3倍，每次5分钟。 </li><li>加的DAPI溶液（1:1,000稀释在PBS中）到细胞上，并培育上在RT摇杆5 - 10分钟。 </li><li>冲洗细胞，用PBS 3倍，每次5分钟。 </li><li>加入1毫升新鲜的PBS。 </li><li>安装在盖玻片细胞在安装解决方案或密封含有细胞的PBS用封口膜，以避免蒸发板。 </li><li>保存在4℃。 </li><li>可视化与细胞荧光显微镜尽快（不迟于2周）。 </li></ol></li><li> 生存试验 <ol><li>从细胞中取出介质。 </li><li>冲洗在PBS中的细胞。 </li><li>添加1:1,000溴化乙锭和1:在PBS中稀释1000吖啶橙。 注意:注意，健康和安全法规中工作 - 溴化乙锭是一种致癌物质。 </li><li>包含在染色溶液中的细胞的板，并在室温孵育在摇臂5分钟。 </li><li>采取与荧光显微镜下细胞的图像:绿色通道，吖啶橙;红色通道为溴化乙锭。 </li></ol></li></ol>

There is nothing to disclose.

在这里，我们提出从伸digitorium短，胫骨前肌或外展肌halluces成年和老年人类隔离肌肉祖细胞/成肌细胞初级的简单，功能强大，价格低廉，可重复和有效的方法。该协议的目的是允许研究使用人原代成肌细胞从成年和老年人，特别是当更复杂的方法，例如FACS-或MACS分选，是不可能或不实际的。 在这个手稿提交的分离方法需要大约2个小时。期间肌肉隔离，肌肉在70％的乙醇，以避免污染洗涤。在酶促肌肉的解离之前，它向肌肉切成小但可见件，并避免过多切碎细胞损伤是重要的。消化的结果在肌纤维的解离和卫星细胞和成肌细胞前体细胞的释放。在我们的情况下为约20毫克的骨骼肌，一种60毫米（20 cm 2）的培养皿是收获的细胞的最适当的表面积。镀上一更大的表面细胞表现出降低的增殖，而细胞铺板到更小的表面显示出增加的细胞死亡和凝集。 在分离，将细胞培养和层粘连蛋白覆盖的平板展开。使用非涂层表面的倾向减小隔离的成功。出于这个原因，细胞可以优选在分离后直接收获预涂面上。成纤维细胞富集的培养物将起主要作用，而不是成肌细胞来源的细胞，如果细胞被分离后直接在非涂布面收获。除了层粘连蛋白，可以使用用其他的细胞附着的解决方案，如基质胶和基于胶原的试剂。涂料溶液可以包括生长因子和其它化合物，以促进细胞生长，但这些可能会改变细胞行为，因此，实验结果。在我们的经验，10微克/毫升层粘连蛋白是最佳浓度和卫星细胞和成肌细胞附着和增殖适当涂层试剂，因为它缺乏任何生长因子或其他互补的。此外，层粘连蛋白是在基底层，直接链接到肌膜，它通过骨骼肌纤维起着卫星细胞附着和迁移的重要功能自然存在。 所述培养基的补充也可以具有在一次成肌细胞的行为产生不利的影响。例如生长因子基团，如的FGFs或胰岛素样生长因子，对初级成肌培养多效作用，与FGF-2的同时控制促有丝分裂和细胞程序性死亡的响应31。因此，有必要严格地控制培养条件，特别是因为在从成年和老年人的肌肉分离的原代成肌细胞的行为的差异很可能是由于纯度ÒF中的培养物和成纤维细胞中的长期培养物35超越在培养的成肌细胞的可能性。我们已经以降低与成纤维细胞的培养物的污染第一拆分过程中使用的细胞的1小时的预镀到非涂覆表面。 我们描述的方法适合于从成年和老年人的肌肉分离生肌祖细胞。分离的细胞组成的细胞的代表性生肌人口的由肌细胞的高百分比指示（由MF20免疫染色图1可视MyoD的表达和肌性和2），并且可以用作用于处理功能研究的体外模型肌肉平衡有关。 先前的研究已经表征的人原代成肌细胞从隔离和性能的差异，或它们的缺乏，成人和老年人6，20，27，28，29，30，31，35，3 6，37，38。老年和/或非功能性人的MPC的存在已被证实6，20，22。然而，在新鲜分离的人的MPC的行为没有区别也已经显示27。我们的协议允许初级成肌细胞，其至少部分保留其表型，如从老年人的肌肉分离的原代成肌细胞的减少增殖潜能或衰老的分离和允许使用的这些衰老22中细胞的肌肉平衡的分子机制功能的研究。 使用此处描述的方法分离的初级成肌细胞可以不仅用于肌分化研究，但还调查细胞内的变化，如在老化过程中在人肌细胞前体细胞产生的基因表达的变化。然而，需要分析老化过程中发生的表型和基因型变化时，需要考虑延长体外培养期间发生的细胞的变化。我们建议使用新鲜分离细胞用于此目的。 此外，这里所描述的主成肌细胞培养方法允许膨胀和相对人原发性成肌细胞的长期培养，允许鲁棒体外功能研究。我们以前曾表明，用我们的方法分离成肌祖细胞可用于既表达谱和复与肌肉老化22相关的进程nctional研究。这种方法也适用于成人和旧啮齿动物的肌肉，并允许成肌细胞的富集培养可用于分析老化和功能研究22中的遗传和表观遗传变化的隔离。这种方法的局限性包括使用，在一定程度上，将细胞而非卫星信元的纯群体的混合群，其中可使用更复杂的公开的方法如图6所示 ，28，29，39，40，41，42来获得， 43。 我们提出一个简化的，负担得起的，重现性协议的主要成肌细胞从成年和老年隔离人类。根据我们的经验，（排序FACS卫星细胞如MACS-或）可用的，更复杂的隔离和人原代成肌细胞培养的方法，非常适合某些类型的研究，如分析在细胞内转录或蛋白质的变化。然而，这些方法是昂贵的，需要至少一些专业知识水平，并可能证明是困难的，由于纯原成肌细胞培养物和成纤维细胞过度生长成肌细胞的低增殖率。 我们提出了一个可重复的协议，允许在功能研究使用人原代成肌细胞的简单分离和培养。此外，我们提出使用的层粘连蛋白42的和有限的使用的bFGF作为关键因素为成功培养44。我们也建议避免通过离心产生的应力分裂细胞，并在第一通道45 1预镀步骤时。总而言之，我们有优化的分离和从成年和老年人的肌肉也适用于啮齿动物的肌肉，使表达和肌稳态功能研究主要成肌细胞/的MPC的培养的高效的协议。

骨骼肌质量和功能结果疾病 - 与年龄相关的逐渐丧失的脆弱，下降强度和降低老年人的生活质量。骨骼肌占大约40％的体重1。期间衰老和疾病，个体的肌纤维和减少肌肉质量的进行性萎缩，由于脂肪和纤维化的渗透发生1，2，3，4，5，6。 最近，已经提出了在骨骼肌的老化物种特异性差异发生，特别是在啮齿动物中存在的肌纤维损失，可能不会出现在人类7。尽管如此，老年哺乳动物的剩余肌纤维的特征在于增加的易感性损伤和受损再生<sup类="外部参照"&gt; 8。 成年肌肉的修复和维护由卫星细胞9,10介导的。当肌肉损伤及其他相关线索，卫星细胞被激活和增殖。细胞的子集返回到静止状态，而其余的进展为成肌细胞（肌祖细胞 - 的MPC）。这些都有助于修复现有的肌纤维11。卫星细胞的功能性确定肌肉再生的成功和与老化已经证实12，13，14，15中的变化在卫星细胞的可用性。而且，从旧人类和啮齿类动物的肌卫星细胞表现出转录的开关，降低再生潜能16，17， </sup&gt; 18，19。从旧的小鼠和人类的肌肉卫星细胞也被证明经历导致其功能减弱的20衰老。 最成熟的细胞使肌肉平衡的研究是鼠C2C12细胞系21。研究一个显著量也用鼠类主要成肌细胞22。这些培养物已导致鼠和脊椎动物成肌以及肌肉再生，肌管/肌纤维萎缩和肌肉疾病过程中发生和老化23，24，25，26肥大过程的显著理解。最近，一些团体已经使用人类初级成肌细胞研究肌细胞和肌肉衰老描述。然而，缺乏与雷加共识DS从成年和老年人27，28，29，30，31的肌肉分离的原代成肌细胞之间的差异。尽管其特征在发展过程中发生的全身和局部环境，老化和疾病6，32，33，34，差别在体外成肌和肌管培养物仍然是最可获得的工具用于研究肌肉发育，生长和萎缩相关的分子机制。此外，这些研究提供的不仅一个健壮的，但也有相对快速，廉价和高通量的体外工具。此外，随着人类肌肉的相关研究伦理含义意味着对于涉及基因表达的操作功能实验<em&gt;，在体外培养的人成肌细胞和肌管文化的，仍然可以脊椎动物模式生物的唯一选择。 在这里，我们显示了主成肌细胞，或多用途储值卡的功能强大，价格低廉，重现性隔离一个简单的实验方案，从成年和老年人的肌肉，并描述在体外培养（ 图1）的标准条件。由于肌肉小学文化通常含有除成肌细胞成纤维细胞，我们建议预镀步旨在提高纯度和主成肌细胞的质量。总之，我们建立了一个协议，允许高效率和可重复的分离，培养和成年和老年人的骨骼肌肉丰富和功能的MPC /主成肌细胞的功能研究。

<table><tbody><tr><td>60 mm Petri dishes</td><td>Greiner Bio One</td><td>628160</td><td>Cellstar Cell culture dish, PS, 60/15 MM, VENTS.</td></tr><tr><td>Cell culture plates (6-well)</td><td>Sigma-Aldrich</td><td>CLS3516</td><td>Corning Costar cell culture plates. 6-well, flat bottom (Individually wrapped) .&amp;nbsp;</td></tr><tr><td>Cell culture plates (12-well)</td><td>Greiner bio-one</td><td>657 160</td><td>Cellstar Cell culture Multiwell Plates.&amp;nbsp;</td></tr><tr><td>Culture flasks</td><td>Greiner Bio One</td><td>&amp;nbsp;690175 (25 cm&lt;sup&gt;2&lt;/sup&gt;); 658175 (75 cm&lt;sup&gt;2&lt;/sup&gt;).</td><td>Cellstar Filter Cap Cell Culture Flasks.</td></tr><tr><td>Standard Disposable Scalpel</td><td>Granton</td><td>91310</td><td>Sterile stainless steel blade, pattern: 10.&amp;nbsp;</td></tr><tr><td>Pipettes</td><td>Greiner bio-one</td><td>606 180 (5 mL); 607 180 (10 mL); 760 180 (25 mL)</td><td>Cellstar Serological Pipettes.&amp;nbsp;</td></tr><tr><td>Pasteur plastic pipettes</td><td>Starlab</td><td>E1414-0311</td><td>3.0 mL Graduated Pasteur Pipette (Sterile), Ind. Wrapped.</td></tr><tr><td>Syringe</td><td>BD</td><td>300613</td><td>20 mL BD eccentric tip syringe.&amp;nbsp;</td></tr><tr><td>0.2 &amp;micro;m filters</td><td>Gilson</td><td>ALG422A</td><td>Sterile Syringe Filters CA 0.2 &amp;micro;m 33 mm Pk50.</td></tr><tr><td>Cell strainers</td><td>Fisher Scientific</td><td>11597522</td><td>Cell culture strainer sterile individually packed 70 &amp;micro;m polypropylene.</td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088882001</td><td>Collagenase D; Activity: &amp;ge;0.15 U/mg&amp;nbsp;</td></tr><tr><td>Dispase II</td><td>Sigma-Aldrich</td><td>D4693</td><td>Dispase II Protease from Bacillus polymyx. Activity: &amp;ge;0.5 U/mg solid.</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>449709</td><td>Calcium chloride, anhydrous, beads, &amp;minus;10 mesh, &amp;ge;99.9% trace metals basis&amp;nbsp;</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>114956-81-9&amp;nbsp;</td><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane. 1 mg/mL.</td></tr><tr><td>DMEM-high glucose</td><td>Sigma-Aldrich</td><td>D5671</td><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium - high glucose. With 4,500 mg/L glucose and sodium bicarbonate, without L-glutamine and sodium pyruvate.</td></tr><tr><td>F-12 media</td><td>Gibco</td><td>21765029</td><td>Ham&#039;s F-12 Nutrient Mix. 1x + L-glutamine.</td></tr><tr><td>FGF-b</td><td>PetroTech</td><td>100-18B&amp;nbsp;</td><td>Recombinant human basic Fibroblast Growth Factor.</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270-106</td><td>Fetal Bovine Serum.</td></tr><tr><td>Horse serum (HS)</td><td>Sigma-Aldrich</td><td>H1270</td><td>Horse Serum. Donor herd, USA origin, sterile-filtered.</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td>Penicillin-Streptomycin with 10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, sterile-filtered.</td></tr><tr><td>L-Glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td>L-Glutamine solution. 200 mM, solution, sterile-filtered.</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4049</td><td>Trypsin-EDTA solution. 0.25%, sterile-filtered.</td></tr><tr><td>TrypLE Express&amp;nbsp;</td><td>Gibco</td><td>12604-013</td><td>TrypLE Express Enzyme (1x), no phenol red.</td></tr><tr><td>DPBS (cell culture)</td><td>Sigma-Aldrich</td><td>D8537</td><td>Dulbecco&amp;rsquo;s Phosphate-buffered Saline. Modified, without calcium chloride and magnesium chloride.</td></tr><tr><td>PBS (immunostaining)</td><td>Sigma-Aldrich</td><td>P4417-50TAB</td><td>Phosphate-buffered saline tablet. One tablet per 200 mL of deionized water (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4).&amp;nbsp;</td></tr><tr><td>Methanol</td><td>Fisher</td><td>M/4000/PC17</td><td>Methanol Analytical Reagent Grade</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Triton X-100 for molecular biology.</td></tr><tr><td>anti-MF 20 antibody</td><td>DSHB</td><td>MF20-c 2ea 211 &amp;micro;g/ml.</td><td>MYH1E (MF 20) Mouse mAb.</td></tr><tr><td>anti-MyoD antibody</td><td>Cell Signaling Technology</td><td>13812P</td><td>MyoD1 (D8G3) XP Rabbit mAb.</td></tr><tr><td>anti-Ki67 antibody</td><td>Abcam</td><td>ab16667</td><td>Rabbit mAb to Ki67 [SP6].</td></tr><tr><td>Anti-mouse 488 secondary antibody&amp;nbsp;</td><td>Invitrogen</td><td>A-11029</td><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate.</td></tr><tr><td>Anti-rabbit 488 secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11034</td><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate.</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>Sigma-Aldrich</td><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td></tr><tr><td>Senescence &amp;beta;-Galactosidase Staining Kit</td><td>Cell Signaling Technology</td><td>9860</td><td>Senescence &amp;beta;-Galactosidase Staining kit.</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>41639</td><td>Dimethyl sulfoxide. BioUltra, for molecular biology, &amp;ge;99.5% (GC).&amp;nbsp;</td></tr><tr><td>Acridine Orange</td><td>Sigma-Aldrich</td><td>A8097</td><td>Acridine Orange hydrochloride solution, 10 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Ethidium bromide</td><td>Sigma-Aldrich</td><td>E1510</td><td>Ethidium bromide solution. BioReagent, for molecular biology, 10 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Lipofectamine 2000</td><td>ThermoFisher Scientific</td><td>11668019</td><td>Lipofectamine 2000 Transfection Reagent</td></tr><tr><td>Scramble control for transfections</td><td>Qiagen</td><td>1027271</td><td>miScript Inhibitor Neg. Control (5 nmol)</td></tr><tr><td>Hsa-miR-378a-3p miScript Primer Assay</td><td>Qiagen</td><td>218300</td><td>Hs_miR-422b_1 miScript Primer Assay (targets mature miRNA: hsa-miR-378a-3p). MIMAT0000732: 5&#039;-ACUGGACUUGGAGUCAGAAGGC</td></tr><tr><td>Anti-hsa-miR-378a-3p miScript miRNA Inhibitor</td><td>Qiagen</td><td>219300</td><td>Anti-hsa-miR-378a-3p miScript miRNA Inhibitor (targets mature miRNA: hsa-miR-378a-3p). MIMAT0000732: 5&#039;-ACUGGACUUGGAGUCAGAAGGC</td></tr><tr><td>Megafuge 2.0 R Centrifuge</td><td>Heraeus</td><td>75003085</td><td>n/a</td></tr><tr><td>Centrifuge rotor</td><td>Heraeus</td><td>3360</td><td>Heraeus Sepatech Megafuge Centrifuge Rotor BS4402/A. Max. radius: 15.5 cm.</td></tr><tr><td>Eclipse Ti-E Inverted Microscope System</td><td>Nikon</td><td>n/a</td><td>Eyepieces: CFI 10X/22; Total magnification: 100X (MF20, Live/dead and Ki67).</td></tr><tr><td>Axiovert 200 inverted microscope</td><td>Carl Zeiss</td><td>n/a</td><td>Eyepieces: Carl Zeiss 1016-758 W-PI 10X/25; Total magnification: 100X (Senescence &amp;beta;-Galactosidase Staining).</td></tr><tr><td>Axiovert 25 inverted microscope</td><td>Carl Zeiss</td><td>n/a</td><td>Eyepieces: E-PL 10X/20. Total magnification: 100X (bright field).</td></tr><tr><td>Diaphot Inverted Tissue Culture Microscope</td><td>Nikon</td><td>n/a</td><td>Eyepiece: CFWN 10X/20. Total magnification: 100X (bright field).</td></tr><tr><td>Hydromount</td><td>National Diagnostics</td><td>HS-106</td><td>Hydromount</td></tr></tbody></table>

preparation and culture of myogenic precursor cells/primary myoblasts from skeletal muscle of adult and aged humans

骨骼肌稳态依赖于肌肉的生长（肥大），萎缩和再生。在衰老和几种疾病，发生肌肉萎缩。肌肉质量和功能的丧失与肌纤维类型萎缩，纤维型开关，具有卫星细胞，肌干细胞，和其它病理生理过程的功能障碍有关的缺陷的肌肉再生相关联。这些变化与细胞内的变化以及局部和全身壁龛有关。除了肌肉老化的最常用的啮齿动物模型中，有必要研究肌肉稳态和使用人的模型，由于伦理影响，包括主要在体外培养物的浪费。尽管广泛使用人体肌祖细胞器（MPC），并在成肌原成肌细胞，对使用人原代成肌细胞和肌管培养物研究的分子机制调节年龄相关亩的不同方面有限的数据CLE虚损，在对啮齿动物的肌肉衰老提出的机制验证帮助。利用人的MPC，主要的成肌细胞和肌管成人和老年人中分离，提供了的肌肉生长，萎缩和再生过程相关的分子机制生理相关的模型。在这里，我们详细描述用于分离和人类的MPC和它们的后代的维护一个健壮的，廉价的，可再现的和高效的协议 - 成肌细胞和肌管从用酶消化人的肌肉样品。此外，我们已经确定在从成年和老年人初级成肌细胞在体外培养经历衰老的通道数量。最后，我们要展示这些转染成肌细胞的能力，并表征其增殖和分化能力，并提出其适用于执行的成肌和肌肉在体外浪费分子机制的功能性研究的能力。 </p&gt;

的MPC /初级成肌细胞应该是可见的24小时后接种到层粘连蛋白涂层的表面（ 图2）。细胞应采取的纺锤状的形状，并应在通道4仍然表达MyoD的（ 图1A，B）。成纤维细胞可以通过他们的明星般的形态，缺乏MyoD基因（ 图1B，C）的表达来区分。一旦细胞附着在第二天，媒体应该用新鲜的bFGF介质代替。文化传媒应每隔48小时更换。 这里示出和来自我们实验室22目的公布的数据支持我们的分离和培养协议，并证明可用于人类初级成肌细胞的功能性研究的不同的技术代表性结果。成肌细胞增殖可以用Ki67的免疫和生存能力使用stainin进行研究克细胞活力测定（ 图3）。对于分化培养基应改为分化培养基。肌管应该形成在5 - 7天，并肌球蛋白重链阳性（ 图3）。注意，肌管形成可能是效率较低时成肌细胞从老年人（ 图3）的肌肉中分离。衰老（SA-β半乳糖苷酶）染色，也可以建立衰老细胞的培养液中的百分比（ 图3）进行的。我们观察到具有较长培养物（ 图3，通道4），从老年人的肌肉分离更多的成肌细胞显示衰老。 用于功能研究，基因和微RNA表达可使用的表达载体，的siRNA，微RNA模拟物和antimiRs（ 图4）的亲脂转染试剂介导的递送来操纵。这允许40 -70％的转染效率与基因/微小RNA水平被上调或生理范围（ 图4; 22）内下调。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 培养容器 </td><td> 约。面积（每孔） </td><td> 10微克/毫升的层粘连蛋白的体积 </td></tr><tr><td> 35mm培养皿</td><td> 10cm 2的</td><td> 1毫升</td></tr><tr><td> 60mm培养皿</td><td> 20厘米2 </td><td> 2毫升</td></tr><tr><td> 100mm皿</td><td> 60厘米2 </td><td> 4毫升</td></tr><tr><td> 24孔板</td><td> 2 平方厘米</td><td> 200微升/孔</td></tr><tr><td> 12孔板</td><td> 4厘米2 </td><td> 500微升/孔</td></tr><tr><td> 6孔板</td><td> 10cm 2的</td><td> 1毫升/孔</td></tr><tr><td> T25 </td><td>为25cm 2 </td><td> 3毫升</td></tr><tr><td> T75 </td><td> 75cm 2的</td><td> 5毫升</td></tr></tbody></table> 表1。 层粘连蛋白DPBS溶液（10微克/毫升）涂层培养表面的推荐最低卷。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> 具体活动/摩尔质量 </td><td> 终浓度 </td><td> 质量或体积需要10毫升溶液 </td></tr><tr><td> 胶原酶ð </td><td> 0.15单位/毫克</td><td> 10毫克/毫升（1.5单位/ mL）的</td><td> 100毫克</td></tr><tr><td> 分散酶II </td><td> 0.5单位/毫克</td><td> 4.8毫克/毫升（2.4 U /毫升） </td><td> 48毫克</td></tr><tr><td> 250毫米氯化钙2 </td><td> 1100.98克/摩尔</td><td> 2.5毫米</td><td> 100μL </td></tr></tbody></table> 表2酶制剂肌肉消化。 <img alt="图1" src="/files/ftp_upload/55047/55047fig1.jpg" /> 图1.图形抽象总结协议的步骤。人肌肉活检用剪刀或外科手术刀（I）的离解。孵育在37℃，30酶溶液- 40分钟（II）。通过加入生长培养基并过滤通过70微米的膜过滤该溶液到离心管中的消化的端部（ 三 ）。离心443×g离心5分钟（IV）。弃去上清，在含有2.5毫微克/毫升的FGF（V）的生长培养基中重悬。电镀细胞上涂覆有10菜81克/层粘连蛋白和24小时（VI）后更改媒体毫升。 <a href="http://ecsource.jove.com/files/ftp_upload/55047/55047fig1large.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/55047/55047fig2.jpg" /> 图2.在成肌细胞不同代的成年和老年人类肌肉隔离。 一 。图像代表从趾短伸肌，胫骨前肌或外展halluces女性患者的肌肉组织中分离成肌细胞（成人:30±2.8岁，年龄:69±5岁，BMI &lt;25）。在通道0之后被镀5天，细胞仍然是圆而小，但可见明亮的光学显微镜（A）下。然后成肌细胞将采用细长形状，如示出在通道2（A）中。 MyoD的是在成肌细胞中表达，但不在成纤维细胞<html> （B）。 MyoD的阳性细胞的定量示出;误差条显示标准偏差; n = 3的（B）中。代表图像展现成肌细胞和成纤维细胞形态（C）之间的差异。 <a href="http://ecsource.jove.com/files/ftp_upload/55047/55047fig2large.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/55047/55047fig3.jpg" /> 图3.人原发性肌细胞的特点可以用不同的染色技术。细胞的生存力可以通过对细胞生存力测定法染色进行可视化，增殖可以用Ki67的免疫染色来评价，分化可使用MF20进行评估（肌球蛋白重链）的免疫染色和衰老可使用衰老相关β-半乳糖苷酶被可视化（SA-β半乳糖苷酶）染色。<html> ecsource.jove.com/files/ftp_upload/55047/55047fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55047/55047fig4.jpg" /> 图4.人原发性肌细胞，可用于肌肉动态平衡体外的功能性研究。 答 MF20（肌球蛋白重链）从成年人表示的miR-378的过表达或抑制对肌管大小和数量的影响分化初级肌管的免疫染色。 B.定量PCR显示的miR-378和IGF1R的，经过验证的miR-378靶基因的相对表达，在之后人类初级成肌细胞的miR-378表达或抑制。相对于RNU-6和β-2-微球蛋白的表达，分别被示出。误差棒显示SEM; N = 3，* - P &lt;0.05，t检验。JPG"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans at JoVE.com

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

本协议描述的隔离和成肌细胞祖细胞培养的成年和老年人的骨骼肌肉健壮，重复性好，简单的方法。这里使用的肌肉包括脚和腿部的肌肉。这种方法使对于功能研究初级成肌细胞的富集群体的分离。

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

preparation-culture-myogenic-precursor-cellsprimary-myoblasts-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

21.8K 次观看.  University of Liverpool. 本协议描述的隔离和成肌细胞祖细胞培养的成年和老年人的骨骼肌肉健壮，重复性好，简单的方法。这里使用的肌肉包括脚和腿部的肌肉。这种方法使对于功能研究初级成肌细胞的富集群体的分离。 

视频: Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

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

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

生物工程

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 ...

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

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

评估人体骨骼肌微组织骨骼肌健康的功能指标

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

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

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 ...