该项目由加州大学旧金山分校计划突破生物医学研究（由桑德勒基金会提供部分资助），美国国立卫生研究院途径来独立奖（R00HL114738）和爱德华马林克罗特小基金会。 JJ是由来自美国国立卫生研究院（T32HL007731）的博士后奖学金支持。作者在此工作，并不一定代表美国国立卫生研究院的官方意见的内容负责。

这里列出的所有程序已获得批准的机构动物使用及护理委员会在加州大学旧金山分校。 注:简单地说，来自小鼠胸腔中提取的心脏后，冠状动脉逆行灌注被用来有效地消化用胶原酶和蛋白酶XIV细胞外基质。然后心室被隔离，机械分离并过滤成一个单一的细胞悬浮液。重力沉降进行分离的心肌细胞，这是从基质细胞样的成纤维细胞和内皮细胞可以通过高密度分离。纯化的心肌细胞可用于在层粘连蛋白涂层的聚苯乙烯周中培养，并用腺病毒基因载体转导。 1.心肌隔离<ol><li> 准备缓冲器，外科站和灌注设备 <ol><li>按Tab键准备缓冲区和媒体乐1。 </li><li>开启循环水浴，设定输入温度，使得加热的水套的输出达到37℃的温度。 </li><li>通过用70％乙醇冲洗干净灌注系统。然后用无菌水几卷​​冲洗除去从系统中乙醇的所有痕迹。 </li><li>装入灌注缓冲液（ 见表1）灌注系统。一是排水系统从，然后用2体积的灌注缓冲液冲洗。然后填充灌注缓冲液使得热套的内侧列充满灌注缓冲液的死空间，但脱泡腔室是空的。 提示:确保无气泡存在于脱泡室下方的线。要删除顽固的气泡，使所有旋塞，让压力线内建立了几秒钟。然后松开下活塞，使高压灌注缓冲区从出口射流;快速流动和turbulencË将有助于驱逐被抓灌注出口附近的气泡。 </li><li>准备的注射器针头被用于主动脉插管。附加一个18G的钝针到1ml注射器充满灌注缓冲液中。去除气泡和修复注射器组件与实验室磁带解剖miscroscope照明的一只手臂。 提示:若要从斜口针创建一个钝头针，用横切线切割针轴。然后磨练与打磨石头尖，直到提示是平坦的。 注意:为降低插管期间从针主动脉滑移，粗糙化用剃刀刀片的插管针头的轴。由轮毂和上轴固定针头，然后看到剃刀来回垂直于针杆的长轴旋转的同时，直到几个沟槽已1厘米针杆的尖端的内创建。切换到一个新的剃须刀当刀片变钝。 </li><li>将一个洁净解剖显微镜下面的培养皿中插管遏制心脏。所述照射器臂，使得所述套管的尖端靠近视场的边缘定位，但不妨碍清扫。确保套管的前端在培养皿的边缘的下方。 注:要帮助清扫过程中固定的心脏，放置一个小的（〜5 - 6 平方厘米），215微米网孔方形的培养皿。当心脏被放置在该网格，它会产生磨擦，以减少滑动和机械操作过程中心脏的旋转。 </li><li>领带2缝线与周围的套管针的毂松双反手结用于主动脉固定。 </li></ol></li><li> 心脏提取和插管 <ol><li>管理通过腹膜内注射肝素（5 IU / g体重）。等待20分钟，使肝素流通至心脏。 </li><li>麻醉小鼠腹腔注射鼠标20％的氨基甲酸乙酯（2毫克的氨基甲酸乙酯/ g体重）的离子。密切观察，直到鼠标是深麻醉下（大约3 - 5分钟）。 注:深麻醉是由缺乏既脚趾捏和角膜反射的证实。如果鼠标还没有由8达到深度麻醉 - 10分钟，然后施用的氨基甲酸乙酯的额外剂量。虽然麻醉个体反应将小鼠之间有所不同，未能进入在第一次给药麻醉过深可能表明退化的氨基甲酸乙酯，这可能会妨碍深麻醉即使经过多次给药。为了防止退化，准备的氨基甲酸乙酯每次使用前新鲜溶液。 </li><li>通过确保与手术磁带每个肢体固定鼠标的手术平台。用70％乙醇的自由量消毒切口区域。抹干了实验室擦拭。 </li><li>提起用组织钳只是胸骨下方皮肤。请用小剪刀外侧切口，切割救援人员到场唉皮肤和腹部。 </li><li>用止血钳经胸骨轻轻抬起胸腔。使用小的解剖剪刀，在朝向腋窝V字形延伸的切口。通过第一肋切割并穿刺每侧隔膜。 </li><li>继续解除与止血胸腔和使用小光圈剪刀完全横切隔膜。小心不要刺破心脏。 </li><li>缩回吻侧使用连接止血，并迅速肋骨但仔细通过朝向两侧腋下胸腔切割延伸的V形切口。完全缩回胸腔的中间部分和放下所附止血就位持有。 </li><li>删除使用细镊子心包。 </li><li>当使用弯钳轻轻提起心脏，横切连接到心脏的静脉和动脉。立即将心脏在冰冷的灌注缓冲减缓肌肉收缩。 </li><li>放置在心脏下一个培养皿解剖显微镜（地方上的一小块215微米的网格，以帮助清扫过程中固定）。修剪用细虹膜剪任何过量的非心脏组织。主动脉附近切割时要小心。 </li><li>断面附近brachioencephalic动脉主动脉，并注意不要让气泡或组织碎片进入开通。 </li><li>填充含有用冰冷的灌注缓冲液，使得液位上升套管的开口上方的心脏培养皿。鞘主动脉到套管，使得尖端只是远离主动脉瓣。 提示:为了减少空气栓塞的风险，在插管的注射器略微压下插管之前，以除去可能在套管的端部已经开发潜在气泡。此外，保持套管的尖端和主动脉溶液的表面之下，以避免引入气泡而护套主动脉。 </li><li>滑动追平前7-0丝线下来插管针和位置正好针尖上方的轴上。拧紧用细镊子结。重复此步骤，以放置正上方的第一的第二缝合线。 注:确保该针尖仍主动脉瓣上述前和紧后。 </li><li>慢慢地压下注射器通过冠状动脉血管弹出0.5毫升灌注缓冲。 注:如果插管成功，血液得以显现而使冠状动脉血管和汇集了背静脉和动脉。 重要:从胸腔打开的时间（特别是穿刺隔膜）到初始灌注应尽量少，以减少缺氧暴露并最大限度地分离的心肌细胞的存活和健康。理想情况下，这个时间应小于5分钟。 </li></ol></li><li> 灌注 注:逆行灌注装置可在一个清洁的环境中方便地操作一个标准的实验室工作台上或放置在层流罩最大化无菌。然而，灌注完成后，工作应在层流进行，以尽量减少污染。 <ol><li>轻轻地但坚定慢慢扭动来回移除注射器（使用无菌手套）套管座。而拉动插管针从注射器的，慢慢地喷射灌注缓冲液进针毂，以防止在灌注期间的气泡。 </li><li>从灌注出口下方取出循环收集管，并用废抓烧杯代替。附加针毂，其固定于灌注出口端口的路厄氏适配器（ 图1，图2）上的水套，而泵是在最低设置运行（15 RPM，〜6毫升/分钟）。 提示:当针集线器连接到鲁尔适配器，让滴水灌注缓冲精读NECT到在针毂的液体，以避免引入气泡。 </li><li>灌注心脏而不再循环直到8毫升灌注缓冲液已经通过入口软管吸出。 注:灌注缓冲液中在该步骤由入口软管吸气体积假定为7毫升的灌注系统死体积，总共为15ml灌注缓冲液中通过心脏泵送。 </li><li>从灌注缓冲液移除入口管。允许空气通过所述入口为小于系统的死体积被吸出。 注意:在这个步骤中吸入的空气的体积，必须进行调整，以防止空气进入冠状脉管（见步骤1.3.7）。 </li><li>放置入口管进入消化缓冲液（ 表1），只是在管的底部的上方。订阅缓慢并允许进口管抽吸足够体积，以避免过满管。 </li><li>可视化和遵循的气泡在灌注缓冲液并行进通过热夹套的消化缓冲液。观察在加热夹套的中心灌注缓冲液的水平开始下降，一旦气泡到达脱泡室。一旦消化缓冲到达脱泡室的液体中的热量夹套水平将保持稳定。 重要说明:如果灌注缓冲水平降得太低，关闭出口活塞，打开排活塞。允许电平上升到一个舒适的水准（3 - 第5英寸出口段）。然后，停止灌注泵和关闭排活塞。打开出口活塞并重新启动灌注泵。 </li><li>观看解决方案从心脏出现。作为最后的灌注缓冲液中离开心脏和消化缓冲液开始通过心脏循环，从清晰的颜色变化到浅棕色将观察到由于胶原酶的存在。 </li><li>灌注消化缓冲的心脏约10 - 15分钟。心脏就会开始懒散的胶原降解，它失去的机械支撑。 </li></ol></li><li> 机械分离与纯化 <ol><li>从在干燥培养皿镊子和地点插管取出心脏。用细虹膜剪修剪快额外心室组织。转移心室到新鲜培养皿含有培养皿灌注缓冲液洗。移动至培养皿无菌罩和心室转移到新鲜，干燥培养皿。 </li><li>从热护套收集7.5毫升消化缓冲液，并添加1M的氯化钙 2.8微升，颠倒混匀。用氯化钙 3毫升消化缓冲液到心室中的陪替氏培养皿-添加2。 注:钙离子的浓度是从300至700微米凸起</li><li>很快磨碎用细镊子心室组织，使大部分作品是小于1mm 3。添加保持从步骤1.3.2至在培养皿中解离的心室ING 钙补充消化缓冲液并通过温和搅拌混合。 </li><li>放置培养皿在37℃培养箱，用5％ 的 CO 2。摇动培养皿每2分钟，直到有足够的心肌细胞（见下面的注释）已经从组织释放。 注:孵化的长度可能需要根据实验的需要进行调整，以微调消化的程度。通常，较长的温育时间增加产率和纯度，但可能减少细胞附着和存活率。 5 - 10分钟通常足以获得高的产率（〜5 -每个心脏10×10 5个细胞）。 </li><li>传送培养皿回一个层流罩。使用以45°角切割移液管，轻轻吸取的组织中以进一步分离。 </li><li>使用无菌手套到215微米的网折叠成锥形并容纳超过一个50ml锥形。吸管离解的组织悬浮液到网和让滤液排到50ml锥形。使用7.5毫升预温热终止缓冲液（ 见表1）的通过筛网从培养皿洗心肌细胞，与第一滤液组合。 注: 钙升高到最终的1.1毫米，并在终止缓冲液最终2.5％FBS的中和蛋白酶活性。 </li><li>细胞悬液转移到15毫升锥形，并允许通过重力15分钟以沉淀。 </li><li>小心地取出用移液管这样的上清液，只有50 - 100微升的解决方案仍然是细胞之上。加入10 mL预热，平衡的培养基（ 表1），并轻轻地倒转混合。让心肌细胞通过重力15分钟解决。 提示:为了提高纯度，重复步骤1.4.8应有的作用。 </li><li> 100微升Ø - 小心地用移液管等，只有50取出上清液˚F解决方案仍然是细胞之上。 </li></ol></li></ol> 2.心肌文化<ol><li>预涂层的组织电镀细胞之前培养处理与层粘连蛋白（10微克/毫升），并存储培养皿在37℃下2小时。之前，为了电镀，吸从组织培养板层粘连蛋白的解决方案。用MEM或PBS洗一次，然后吸残余液体以除去未吸附的层粘连蛋白。 </li><li>加入足够的预热的，平衡的培养基以达到所需的细胞密度，轻轻翻转混匀。板所需的细胞浓度到培养容器（一般为5 - 20×10 4个细胞/ cm 2）。为了评估心肌隔离的产率，通过检查在显微镜下密度计通过血细胞计数器或测试板上的细胞。成年小鼠心脏通常会提供0.5 - 1.0×10 6个细胞在这个阶段的协议。 提示:如果使用96孔板，使用1000微升micropipETTE带以45°角切割的前端，以尽量减少由于剪切力的细胞损伤。放置枪头使得其接触孔的底部的细胞悬浮液的喷射过程中，以最小化引入气泡。 </li><li>迅速移动至细胞培养皿于37℃培养箱，用5％ 的 CO 2和95％的湿度。根据需要，以防止酸化改变媒体:每1 - 取决于细胞密度5天。在第一个3最小化培养皿的处理 - 13天以促进附着于细胞培养表面。 注:要在媒体的变化减少细胞的干扰时，细胞还没有完全连接，进行一个半媒体的变化。慢慢吸媒体，同时保持枪头只是媒体的表面之下。然后慢慢地通过保持枪头只是对培养皿的壁的介质的表面上方添加其他介质。 </li><li>为了评估心肌的生存能力，检查下明场显微术的形态。新鲜分离的心肌细胞保持与在光照下尖锐，鲜明边缘的大致棒状的形态（ 图2）。 注意:用台盼蓝染色细胞活力确认。 </li><li>为了评估心肌隔离的纯度，检查细胞形态，以确定心肌细胞。与染色赫司特33342细胞核，以确定细胞总数。 注:心肌可以从非心肌通过特性的形态（棒状）和大小来识别（大约100 - 200微米长）。或者，新鲜分离的心肌细胞将染色阳性特定心肌标记物，如α-辅肌动蛋白26（ 图3C，D）或心脏肌钙蛋白T. 27 </li></ol> 3.转基因腺病毒<ol><li>通过开办的制备腺病毒细胞裂解液编制方法21转导使用来自腺病毒生产细胞10细胞裂解物的心肌细胞。在隔离在分别为0白天或每日1次，小心地将细胞裂解液加入到含有心肌媒体。 注:高滴度的腺病毒制备物将在大约48实现强劲表现- 72小时。调整感染复数以适应实验设计。 </li></ol>

The authors have nothing to disclose.

分离出心肌细胞的整体健康取决于该协议的几个重要方面。首先，从心脏提取到灌注的时间是非常关键的，应在5分钟以下进行。钙的除去有助于解离细胞-细胞相互作用，但能细胞健康长期负面影响。29-32。因此，我们发现它足以通过EGTA灌注的最初几分钟期间去除钙（乙二醇四乙酸）螯合， 22但很快消化和随后的步骤中恢复钙。 利用蛋白酶十四大大提高比单?...

培养的心肌细胞经常用于监测在体外良好控制的环境中细胞的行为。例如，形态，电气，生化或机械细胞性质可在工程衬底进行研究，在确定的培养基1,2，以及响应于小分子药物，肽，基因调控，3或电刺激。4蜂窝含量可以还使用定义共培养物来控制。5 这些体外实验是在大的药物或遗传筛选有用和体内方法各种类型涉及心肌生物学调查补充。 长期的文化使那些需要长的时间内，实现表型改变的实验途径。适时地例子是成年哺乳动物心肌细胞增殖，其中去分化，细胞周期再入，和细胞分裂典型地研究过本身的veral天至数周，6,7这里，延长培养时间有利于遗传操作，7,8分化的功能（ 例如，肌节拆卸）9和潜在的转录分化。6随后细胞周期重新进入和细胞分裂，需要更长的时间培养观察，特别是如果多轮师都是实验性的目标。心肌细胞周期的重要性是中央在心脏再生，其中，预先存在的心肌细胞的分化和增殖已显示负责在斑马鱼和新生小鼠心脏再生几个最近的关键科学著作。10-12。因此，有可能刺激分化和细胞周期重新进入哺乳动物成年心肌留在人的心脏再生13-15的一个关键问题 I N体外实验研究哺乳动物心脏的细胞周期肌细胞已主要使用鼠源，由于其相对容易长期培养的相比小鼠模型16然而，鼠系统提供的充分表征的基因的工具和疾病模型是在体外 和体内有用资源丰富协议。例如，基于Cre的谱系追踪，使预先存在的心肌细胞的鉴定如在体内的新生小鼠心脏再生心肌的一个来源。12 体外谱系追踪新生小鼠的心肌细胞的研究已启用与基质相互作用的检查细胞通过共培养成纤维细胞。5然而，由于它的挑战，17报道很少成年小鼠心肌细胞的分离和长期培养的存在。18,19 单独短期培养存活成年小鼠心肌细胞的分离被称为是一个具有挑战性的任务。这protocOL提供了有关如何实现从可用于短期和长期的调查成年小鼠心肌细胞存活一步一步的指导。使用此协议分离的心肌细胞可以与腺病毒载体20,21和培养周有效地转。这些方法提供了强大的系统来研究生物学心肌体外 。 本文所描述的方法是基于从使用的Langendorff逆行灌注的变化之前的作品几个元素。18,22虽然几个协议已发表 ​​关于短期培养和研究中，23-25 ​​的优点的成年小鼠心肌细胞的隔离该协议是能够培养分离的心肌细胞长期的。这将是在涉及异位基因表达并且需要延长的时间段，例如在细胞的初始化策略细胞过程的研究是有用的。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Heated water jacket</td><td>Radnoti</td><td>158831</td><td></td></tr><tr><td>Circulating heated water bath, Isotemp</td><td>Fisher Scientific</td><td>3013</td><td></td></tr><tr><td>Laboratory pump</td><td>Watson-Marlow</td><td>323</td><td></td></tr><tr><td>Hemostats</td><td>Exelta</td><td>63042-090</td><td></td></tr><tr><td>Tissue forceps</td><td>VWR</td><td>470128034</td><td></td></tr><tr><td>Dumont #7 curved forceps</td><td>FST</td><td>91197-00</td><td></td></tr><tr><td>Dumont #5 fine forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>Small dissection scissors</td><td>VWR</td><td>470128034</td><td></td></tr><tr><td>Extra fine bonn scissors</td><td>FST</td><td>14084-08</td><td></td></tr><tr><td>Fine spring scissors</td><td>FST</td><td>91500-09</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;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>S25837</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>S25414</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>86329</td><td></td></tr><tr><td>Butane dione monoxime (BDM)</td><td>Sigma</td><td>B0753</td><td></td></tr><tr><td>HEPES</td><td>Fisher&amp;nbsp;</td><td>BP310100</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G-7021</td><td></td></tr><tr><td>Insulin</td><td>Novo Nordisk</td><td>393153</td><td></td></tr><tr><td>EGTA</td><td>Amresco</td><td>0732-288</td><td></td></tr><tr><td>Protease, type XIV</td><td>Sigma</td><td>P5147</td><td></td></tr><tr><td>Collagenase II</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>MEM</td><td>Corning</td><td>15-010-CV</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>JRS</td><td>43613</td><td></td></tr><tr><td>Primocin</td><td>Invitrogen</td><td>NC9141851</td><td></td></tr><tr><td>Ethyl Carbamate</td><td>Alfa Aesar</td><td>AAA44804-18</td><td></td></tr><tr><td>215 micron mesh</td><td>Component supply</td><td>U-CMN-215-A</td><td></td></tr><tr><td>20 G blunt ended needle</td><td>Becton Dickinson</td><td>305183</td><td></td></tr><tr><td>20 G beveled needle</td><td>Becton Dickinson</td><td>305176</td><td></td></tr><tr><td>Lab tape</td><td>VWR</td><td>89097-990</td><td></td></tr><tr><td>Surgical tape</td><td>3M</td><td>1527-0</td><td></td></tr><tr><td>Silk suture, 7-0</td><td>Teleflex</td><td>15B051000</td><td></td></tr><tr><td>Mouse anti-alpha-actinin antibody</td><td>Sigma</td><td>A7811</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG1 antibody</td><td>Thermo Fisher</td><td>A21121</td><td></td></tr></tbody></table>

isolation, culture and transduction of adult mouse cardiomyocytes

培养的心肌细胞可用于研究是否可使用那些对体内系统补充技术心肌生物学。例如， 在体外培养的纯度和可访问性使得在生化分析，实时成像，和电精细控制。心肌细胞长期培养提供了访问，不能在短期内培养进行额外的实验方法。例如，去分化，细胞周期再入，和细胞分裂的体外调查迄今基本上被限制在大鼠心肌细胞，这似乎是在长期培养更加健壮。然而，转基因小鼠线和发达的疾病模型的丰富的工具集的可用性使鼠标系统的心脏研究的吸引力。尽管一些报道成年小鼠心肌细胞分离存在的，一些研究展示自己的长期培养。这里介绍的，是为一步一步的方法成年小鼠心肌细胞的分离和长期培养。首先，逆行的Langendorff灌注被用来有效地消化用蛋白酶的心脏，接着通过重力沉降纯化。一段去分化下述分离后，将细胞逐渐附着到培养，并且可以是数周培养。腺病毒的细胞裂解物，用于有效地转导的分离的心肌细胞。这些方法提供了一种简单，但功能强大的模型系统来研究生物学的心脏。

野生型成人ICR（CD1）小鼠心脏通常产生从一个成功的隔离500​​,000到1,000,000心肌细胞。分离后立即将细胞保持大多棒状外观（ 图3A）与完整肌节和可用于涉及心肌收缩功能研究。棒状心肌（90％以上）的高百分比是有效灌注和消化的指示。可行的心肌细胞将是大的（〜100 - 200微米长）和出现有在光照下（ 图3A）的急剧外膜。免疫染色特定心肌肌节...

Watch this Scientific Journal Video about Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes at JoVE.com

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

分离培养和成年小鼠心肌细胞转导

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

isolation-culture-and-transduction-of-adult-mouse-cardiomyocytes

Research

JoVE Journal

Biology

26.6K 次观看.  University of California, San Francisco. This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability. 

视频: Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

科研

生物学

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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

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

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

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

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

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

一种持续分离小鼠心肌细胞的简单有效的方法

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

医学

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

成年小鼠和大鼠心肌细胞的隔离、转染和长期培养

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

来自成人鼠心脏的心外肌细胞、心室肌细胞和非肌细胞的同步隔离和文化

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...