Name: isolation and culture of neonatal mouse cardiomyocytes
Uploaded: 2013-09-06T12:00:00.000+00:00
Duration: 7 min 50 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Neonatal Mouse Cardiomyocytes at JoVE.com

我们感谢名誉教授让 - 克洛德·Perriard和伊夫林Perriard（瑞士联邦理工学院，瑞士）的引入隔离技术的新生大鼠和小鼠心肌细胞。我们要感谢朱教授和陈教授西尔维娅·埃文斯（加州大学圣地亚哥分校，美国）的支持。在EE的实验室工作是由一个MRC事业建立格兰特。 SL是由K99/R00途径独立性奖从NIH / NHLBI（HL107744）的支持。 TMM是由美国心脏协会（11POST7310066）的博士后奖学金支持。

下面的过程描述了为期两天的协议16,17对新生小鼠心肌细胞的分离和培养。所有溶液是无菌的或无菌过滤。所有的工具都通过表面杀菌消毒用75％的乙醇。除了最初的组织提取，所有步骤都在无菌层流细胞培养罩进行。此协议的目的是为新生小鼠心脏从一个个垃圾（次）的隔离 - 约5-14幼崽，但可以适用于较大的窝产仔数和新生大鼠心肌细胞。规模媒体/酶用量（如适用）。 对于新生儿啮齿动物的工作，是指规定由立法机关和/或动物护理方案您当地的大学指引和规则，并坚持你的制度上批准的动物协议。在本协议中所述的所有方法已获得加州大学圣地亚哥分校机构动物护理和使用委员会（IACUC），并坚持FEDE拉尔和国家有关规定。 每日1次。 1。从新生小鼠心肌组织中分离<ol><li>制备50毫升的1X PBS（无Ca 2 +的，Mg 2 +的），补充有20mM BDM 6-8，并分散到置于冰上2无菌细菌菜肴。 </li><li>准备10毫升分离培养基的50ml的无菌锥形Falcon管中。置于冰上的所有解决方案。消毒剪刀（一个弯，一条直线），和钳（弯，杜蒙第7号）。 </li><li> 1-3日龄新生小鼠在表面杀菌75％的乙醇溶液快速冲洗。幼仔用无菌剪刀（直）断头，胸部沿着胸骨打开，以允许访问胸腔和心脏（ 图1A中 ， 追加电影S1）。 技术评论:年长超过3天新生小鼠可以使用，但导致更少的活细胞2。 </li><li>心是摘录自主体具有弯曲剪刀和用20mM BDM立即转移到细菌培养皿含有1x PBS（不含Ca 2 +，Mg 2 +的），在冰上。所有以下步骤在无菌的细胞培养罩进行。 </li><li>去除肺组织，较大的血管（和心房，如果需要的话）。洗心在1X PBS液（不含Ca 2 +，Mg 2 +的）与20毫米BDM（冰），以清除血液。转让洗涤从第一盘心进入第二细菌培养皿含有1x PBS（不含Ca 2 +，Mg 2 +的）与使用镊子的20mM BDM（在冰上）或穿孔的勺子（ 图1B）。 </li><li>转让清洗/清洗的心，一滴分离培养基（约250微升; 图1C）在第三细菌培养皿（冰），并使用剪刀弯剁碎的心切成小块（约0.5-1毫米3，或更小; 图1D，1E）。 </li><li>转心剁碎成圆锥形含有10毫升分离培养基（在冰上）的管中，并轻轻摇动孵育在4℃下过夜。 </li></ol>第2天。 2。酶消化组织细胞及电镀<ol><li>称重在15毫克胶原酶/分散酶混合物（罗氏公司），并溶解酶混合物在10ml L15-5介质补充有20mM的BDM（消化培养基）。无菌过滤的消化培养基在细胞培养罩到一个新的无菌50毫升Falcon管中。 </li><li>准备30毫升的L-15培养基中添加20mM的BDM和镀敷介质。 </li><li>外套细胞培养板的胶原蛋白溶液（Sigma C-8919）最少1小时的。除去胶原溶液（可重复使用）和干胶原包被细胞培养皿中无菌层流细胞培养罩。 </li><li>取出锥形管含有从4℃的简化的心组织碎片应合并计算（ 图1G）。让组织碎片沉入在管的底部，去除上清（确保不丢失任何组织片段;通常，约1ml所隔离介质的可保留在管）。加入5 ml消化介质和5毫升的L-15的补充有20mM的BDM到组织片段，并使用氧气或空气为1分钟含氧化合物的悬浮液。 </li><li>密封锥形管中含有消化培养基的心脏组织片段转移到37℃水浴中约2分钟，以调节消化溶液的温度。 </li><li>孵育心肌组织片段在37℃下缓慢搅拌20-30分钟（ 例如摇动器在37℃下，设定为不超过60转以上）。消化时间可能大大依赖于酶的混合物，并批号注意:较长的潜伏期或更高的酶浓度可能会降低细胞活力。 </li></ol> 技术注释:我们建议胶原酶/分散酶混合物的使用量从罗氏（编号:10269638001），有非常少很多到很多变化。经典的酶的小鼠心肌细胞的分离的胰蛋白酶和/或胶原酶II型3,16-20可从Worthington（目录号CLS-2）。然而，胶原酶Ⅱ的性能可能从很多到很多差别很大。沃辛顿通常允许几个胶原酶大量的测试，优化消化时间和酶用量。另外，沃辛顿生产销售心肌细胞分离试剂盒与预测试的酶混合物，包括可适应对小鼠心肌细胞17（目录号:NCIS）的隔离。 <ol start="7"><li>将无菌细胞过滤器（40-100微米尼龙网）的新鲜无菌的50ml锥形猎鹰管。预湿的细胞过滤，用5ml的L-15补充有20mM的BDM。使用预润湿10毫升细胞培养移液器约10-20倍轻轻磨碎的组织碎片。组织碎片应该在这个步骤大多分散，释放出细胞进入悬索桥N（ 图1H）。 </li><li>让更大的组织碎片沉淀和含悬浮细胞到新鲜的锥形管通过细胞过滤器（ 图1I）转移上清。 </li><li>重悬未消化的组织碎片在5ml消化培养基孵育额外的5-10分钟，在37℃下缓慢搅拌。 </li><li>其余的组织碎片继续消化后，轻轻磨碎组织的10-20倍，并加入到锥形管含有细胞从第一通过消化细胞过滤。漂洗细胞器 - 网式过滤，用5ml的L-15添加20mM BDM允许所有消化细胞的通道。 </li><li>离心锥形管含有悬浮的心肌细胞在300转5分钟（约50-100 XG）。除去上清液（主要含有成纤维细胞和内皮细胞）和在10ml平板培养基重悬细胞沉淀。 </li><li>板细胞进入10厘米细胞培养皿（ 图2A）并孵育1-3小时在细胞培养孵化器。此预镀步骤除去成纤维细胞和内皮细胞（ 图2B），这将附着在未涂覆的细胞培养皿中（ 图2C）。 </li><li>温育后，（通过反复吹打在盘镀介质重新悬浮细胞）洗涤非贴壁心肌将从10cm培养皿中，并传输重悬细胞于无菌锥形猎鹰离心管（15毫升或50毫升）中。 可选:重复镀前一步，从细胞悬液中删除额外的成纤维细胞。如果需要的话，粘合成纤维细胞和内皮细胞可进一步培养。 </li><li>细胞计数（ 如使用的Neubauer血细胞计数，台盼蓝拒染21）。 </li><li>板的细胞在胶原包被细胞培养皿以每厘米2（ 图2D约1.5×10 5个细胞的密度，也可参见评论于表1上镀和涂层）。 将餐具放入细胞培养孵化器，离开静置12-18小时，以便坚持和心肌细胞的扩散。 </li></ol>第3天 - 乳鼠心肌细胞的培养<ol><li>准备维修中，prewarm在37℃水浴。参照表1的除了增殖抑制剂或变时性剂，或程序的新生小鼠的心肌细胞的脂质体转染。 </li><li>有一天电镀后，心肌细胞应该坚持的细胞培养皿，最佳开始自发地收缩（ 图2E，2F，补充电影S2，参考表2为隔离/养殖过程中常见的问题）。更换电镀液与维持液和培养更多的1-5天。改变介质是必要的。 </li></ol>

利用动物模型来研究心脏疾病在心血管研究已经成为标准。这些模型的建立更紧密的生化特性（ 即研究心肌细胞的直接反应，生物化学或生物力学刺激）通常需要心脏组织或心肌细胞的分离。调查研究心脏的生理反应体外 （ 如对乙酰胆碱40，或在缺血再灌注场景41）一般采用的Langendorff灌注整个心灵42,43，而植体培养心肌组织碎片44,45允许对心?...

对啮齿动物的心脏细胞的成功分离并培养了最早报道可以追溯到1960年的1,2。即使这样，Harary和法利注意到，培养的心肌细胞"可提供的周期性收缩的要求，研究一个独特的系统[，并可能]提供确定的[跳动]过程中的各种代谢途径的贡献的一种手段"。虽然Harary和法利从幼鼠，和原来的协议分离培养的心肌细胞已被改编，被许多科学家修改多年来，一般隔离和培养过程并没有很大的变化。然而，更好的酶3，标准化解决方案4,5和加法可逆信道和肌球蛋白ATP酶抑制剂BDM分离过程6-9期间保护细胞的有显著改善细胞产量和活力。 成人与新生大鼠心肌核细胞心肌细胞的分离，培养新生小鼠或大鼠有超过成人的心肌细胞培养几个优点。最重要的，在分离过程中对新生小鼠或大鼠心脏更容易且成本更低，相对于从成年小鼠或大鼠心肌细胞10的隔离。新生心肌远远不敏感，再引入含钙介质离解后，极大地提高细胞产量。另一大优势是，新生小鼠心肌细胞进行更快速的分化 - 再分化周期，通常会产生电镀后自发跳动的细胞20小时，而成人的心肌细胞通常需要起搏诱导收缩。乳鼠心肌细胞也更容易转染的脂质体转染方法，而成人的心肌细胞需要病毒载体成功传递转基因DNA的。相反，新​​生心肌细胞秒，成年啮齿动物心肌11-13文化允许的肌原纤维的降解和收缩设备的最终重建的调查。在成人的心肌细胞，这些特征的形态学变化发生在1-2周时间。去分化-再分化周期是伴随胎儿基因程序的再表达，从而模拟在人心肌病14观察到的病理变化。成年大鼠心肌细胞在新生大鼠心肌文化的另一个优点是能够培养这些细胞很长一段时间。 大鼠与小鼠的心肌细胞 新生大鼠心肌细胞的分离和培养有一定的好处超过了小鼠乳鼠心肌细胞，包括活细胞的产量更高，增加了转染率。然而，转基因小鼠模型心脏疾病的广泛使用（ 如&lt;/ em&gt;的肌肉LIM蛋白基因敲除小鼠为模型，扩张型心肌病15），导致了分离过程的适应从新生小鼠的心肌。虽然用于分离新生大鼠和小鼠心肌的协议几乎是相同的，更大的关怀必须采取适当的酶混合物，后者的选择。事实上，新生小鼠心肌细胞一般都比较容易过量，造成降低的细胞产量和活力。此外，铺板密度应调整，因为相比于从新生大鼠心脏的细胞从新生小鼠衍生的心肌细胞是要小一些。 与许多用途的形态，电生理，生物化学，细胞生物学和生物力学参数以及为肌丝的过程进行调查，培养的乳鼠心肌细胞已成为最通用的系统的研究之一心肌细胞功能的体外 。第一步，一个成功的实验然而，依赖于一个简单而可靠的方法来隔离乳鼠心肌细胞。我们的协议借鉴其方法有很多，是为再现性和鲁棒性优化。我们讨论了影响心肌的产量和活力的因素，以及对分离和培养条件的优化提供了多种选择。

<table><tbody><tr><td>BDM (2,3-Butanedione monoxime) &lt;sup&gt;6-8&lt;/sup&gt;</td><td>Sigma</td><td>B-0753</td><td>prepare 0.2M stock solution in HBSS (without Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;), filter sterilize, can be kept at 4 &amp;#176;C up to 6 months; Caution: Prolonged usage of BDM other than during isolation procedure may result in non-beating cells, decreased cell viability and/or significantly altered gene-expression during cardiomyocyte culture&lt;sup&gt;46,47&lt;/sup&gt;.</td></tr><tr><td>Collagenase/Dispase</td><td>Roche</td><td>10269638001</td><td>can be substituted with collagenase type II from Worthington</td></tr><tr><td>Collagenase type II&lt;sup&gt;3&lt;/sup&gt;</td><td>Worthington</td><td>CLS-2</td><td>substitute for Collagenase/Dispase mix from Roche</td></tr><tr><td>1x trypsin solution (0.25%) with EDTA</td><td>cellgro</td><td>25-053-CI</td><td></td></tr><tr><td>1x Penicillin/ Streptomycin solution with EDTA in HBSS</td><td>cellgro</td><td>30-002-Cl</td><td></td></tr><tr><td>1x PBS (without Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>e,g, cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>HBSS (Hank&amp;#8217;s balanced salt solution; without Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;) &lt;sup&gt;4&lt;/sup&gt;</td><td>cellgro</td><td>21-022-CV</td><td></td></tr><tr><td>DMEM high glucose</td><td>cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>M-199</td><td>cellgro</td><td>10-060-CV</td><td></td></tr><tr><td>fetal bovine serum</td><td>cellgro</td><td>35-011-CV</td><td>cell-culture grade</td></tr><tr><td>horse serum</td><td>cellgro</td><td>35-030-CV</td><td>cell-culture grade</td></tr><tr><td>Leibovitz L-15&lt;sup&gt;5&lt;/sup&gt;</td><td>cellgro</td><td>10-045-CV</td><td></td></tr><tr><td>AraC (Cytosine-B-D-arabino-furanoside hydrochloride)</td><td>Sigma</td><td>C-6645</td><td>proliferation inhibitor, prepare 1 mM stock solution in H&lt;sub&gt;2&lt;/sub&gt;O, filter sterilize, store at 4 &amp;#176;C</td></tr><tr><td>phenylephrine</td><td>Sigma</td><td>P-6126</td><td>chronotropic agent, prepare 100 mM stock solution in H&lt;sub&gt;2&lt;/sub&gt;O, filter sterilize, store at 4 &amp;#176;C or -20 &amp;#176;C</td></tr><tr><td>isoproterenol hydrochloride</td><td>Sigma</td><td>I-6501</td><td>chronotropic agent, prepare 1 mM stock solution in H&lt;sub&gt;2&lt;/sub&gt;O, filter sterilize, store at 4 &amp;#176;C or -20 &amp;#176;C</td></tr><tr><td>0.1% collagen solution</td><td>Sigma</td><td>C-8919</td><td>extracellular matrix for coating</td></tr><tr><td>3 mg/ml collagen type 1 solution</td><td>Advanced BioMatrix</td><td>5005-B</td><td>alternative to Sigma collagen solution</td></tr><tr><td>cell strainer</td><td>Fisherbrand</td><td>22363548</td><td>appropriate filter size:40 &amp;#956;m-100 &amp;#956;m</td></tr><tr><td>syringe filter 0.2 &amp;#956;m</td><td>Fisherbrand</td><td>09-719C</td><td>for sterile filtration of digestion medium</td></tr><tr><td>straight scissors</td><td>Fine Sciences Tools</td><td>91460-11</td><td></td></tr><tr><td>curved scissors</td><td>Fine Science Tools</td><td>91461-11</td><td></td></tr><tr><td>Dumont No. 7 forceps</td><td>Fine Science Tools</td><td>91197-00</td><td></td></tr><tr><td>perforated spoon</td><td>Fine Science Tools</td><td>10370-19</td><td>optional, for transfer of heart tissue</td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250-061</td><td>live cell staining</td></tr><tr><td>Neubauer hemocytometer</td><td>Prosource Scientific</td><td>3500</td><td>alternatively use: disposable hemocytometer C-chip or automated cell counting systems</td></tr><tr><td>50 ml Falcon tubes</td><td>Fisherbrand</td><td>14-432-23</td><td></td></tr><tr><td>15 ml Falcon tubes</td><td>Fisherbrand</td><td>05-527-90</td><td></td></tr><tr><td>20 ml syringe</td><td>BD Medical</td><td>14-820-19</td><td></td></tr><tr><td>10 ml serological pipette</td><td>Falcon</td><td>357551</td><td></td></tr><tr><td>30 mm cell culture dish</td><td>Nunc</td><td>153066</td><td>for standard culture of cardiomyocytes</td></tr><tr><td>30 mm cell culture dish, glass bottom</td><td>MatTek</td><td>P35G-0-10-C</td><td>for live cell imaging with inverted microscope</td></tr><tr><td>10 cm cell culture dish</td><td>Nunc</td><td>172958</td><td>for preplating</td></tr><tr><td>Escort III</td><td>Sigma</td><td>L3037</td><td>for liposomal transfection, alternatively use lipofectamin 2000</td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies, Invitrogen</td><td>52887</td><td>substitute for Escort III</td></tr><tr><td colspan="4">&lt;strong&gt;Buffers and media: &lt;/strong&gt; &lt;ul&gt; &lt;li&gt;&lt;strong&gt;Isolation medium (filter sterilize)&lt;/strong&gt;&lt;br/&gt; 20 mM BDM&lt;br/&gt; 0.0125% trypsin&lt;br/&gt; in HBSS&lt;sup&gt;4&lt;/sup&gt; (without Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;)&lt;/li&gt; &lt;li&gt;&lt;strong&gt;Digestion medium (filter sterilize)&lt;/strong&gt;&lt;br/&gt; 20 mM BDM&lt;br/&gt; 1.5 mg/ml Roche Collagenase/Dispase enzyme mix&lt;br/&gt; in L15 medium&lt;/li&gt; &lt;li&gt;&lt;strong&gt;Plating medium&lt;/strong&gt;&lt;br/&gt; 65% DMEM high glucose&lt;br/&gt; 19% M-199&lt;br/&gt; 10% horse serum&lt;br/&gt; 5% fetal calf serum&lt;br/&gt; 1% penicillin/streptomycin&lt;/li&gt; &lt;li&gt;&lt;strong&gt;Maintenance medium&lt;/strong&gt;&lt;br/&gt; 78% DMEM high glucose&lt;br/&gt; 17% M-199&lt;br/&gt; 4% horse serum&lt;br/&gt; 1% penicillin/streptomycin&lt;br/&gt; &lt;em&gt;optional:&lt;/em&gt;&lt;br/&gt; 1 &amp;#956;M AraC&lt;br/&gt; 1 &amp;#956;M isoproterenol or 0.1 mM phenylephrine&lt;/li&gt; &lt;/ul&gt; The plating and maintenance medium can be stored at 4 &amp;#176;C for up to 6 months.</td></tr></tbody></table>

isolation and culture of neonatal mouse cardiomyocytes

培养的乳鼠心肌细胞长期被用来研究肌丝和肌原纤维的功能。培养的心肌细胞可以很容易调查的生化途径和操作，以及它们对自发跳动的心肌细胞的生物力学特性的影响。 下面2日协议描述的新生小鼠心肌细胞的分离和培养。我们将展示如何轻松地从新生儿心脏解剖，游离心脏组织，并从心脏细胞群丰富的心肌细胞。我们讨论了不同酶混合的用法细胞解离，及其对细胞活力的影响。分离的心肌细胞随后可以用于各种形态，电生理，生化，细胞生物或生物力学分析。我们优化了协议鲁棒性和再现性，通过仅使用市售溶液和酶混合了的shOW小批与批之间变化。我们还应对与心肌细胞的分离和培养相关的常见问题，并提供了多种用于分离和培养条件的优化选项。

使用此协议，我们分离的心从8一日龄新生小鼠（ 图1A，1B，追加电影S1）。洗涤和切碎的心，用剪刀（ 图1C-1F）后，组织碎片在预消化分离培养基过夜，4℃，轻轻摇动。以下简化（ 图1G），我们将其组织碎片进入新鲜的消化培养基，并温育的组织碎片20分钟，在37℃下缓慢搅拌。将所得细胞悬浮液（ 图1H），是通过细胞过滤网（ 图1G）</strong...

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

Isolation and Culture of Neonatal Mouse Cardiomyocytes

原代小鼠心肌细胞培养物的肌原纤维组织和功能的研究的关键工具之一。以下协议描述了从新生小鼠心脏原发性心肌细胞的分离和培养。所得到的心肌细胞培养物随后可用于各种生物力学，生物化学和细胞生物学测定。

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-and-culture-of-neonatal-mouse-cardiomyocytes

Research

JoVE Journal

Biology

62.5K 次观看.  King’s College London. 原代小鼠心肌细胞培养物的肌原纤维组织和功能的研究的关键工具之一。以下协议描述了从新生小鼠心脏原发性心肌细胞的分离和培养。所得到的心肌细胞培养物随后可用于各种生物力学，生物化学和细胞生物学测定。 

视频: Isolation and Culture of Neonatal Mouse Cardiomyocytes

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

隔离和乳鼠心肌细胞冷冻保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

科研

发育生物学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

生物学

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

神经科学

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 of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

肺动脉平滑肌细胞从新生小鼠隔离

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

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 ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

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 ...

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. ...