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 border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>BDM (2,3-Butanedione monoxime) 6-8</td>
			<td>Sigma</td>
			<td>B-0753</td>
			<td>prepare 0.2M stock solution in HBSS (without Ca2+, Mg2+), filter sterilize, can be kept at 4 &deg;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 culture46,47.</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 II3</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>e.g. cellgro</td>
			<td>25-053-CI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1x Penicillin/ Streptomycin solution with EDTA in HBSS</td>
			<td>e.g. cellgro</td>
			<td>30-002-Cl</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1x PBS (without Ca2+, Mg2+)</td>
			<td>e,g, cellgro</td>
			<td>21-040-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HBSS (Hank's balanced salt solution; without Ca2+, Mg2+) 4</td>
			<td>e.g. cellgro</td>
			<td>21-022-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>e.g. cellgro</td>
			<td>10-013-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>M-199</td>
			<td>e.g. cellgro</td>
			<td>10-060-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>e.g. cellgro</td>
			<td>35-011-CV</td>
			<td>cell-culture grade</td>
		</tr>
		<tr>
			<td>horse serum</td>
			<td>e.g. cellgro</td>
			<td>35-030-CV</td>
			<td>cell-culture grade</td>
		</tr>
		<tr>
			<td>Leibovitz L-155</td>
			<td>e.g. cellgro</td>
			<td>10-045-CV</td>
			<td>&nbsp;</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 H2O, filter sterilize, store at 4 &deg;C</td>
		</tr>
		<tr>
			<td>phenylephrine</td>
			<td>Sigma</td>
			<td>P-6126</td>
			<td>chronotropic agent, prepare 100 mM stock solution in H2O, filter sterilize, store at 4 &deg;C or -20 &deg;C</td>
		</tr>
		<tr>
			<td>isoproterenol hydrochloride</td>
			<td>Sigma</td>
			<td>I-6501</td>
			<td>chronotropic agent, prepare 1 mM stock solution in H2O, filter sterilize, store at 4 &deg;C or -20 &deg;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>e.g. Fisherbrand</td>
			<td>22363548</td>
			<td>appropriate filter size:40 &mu;m-100 &mu;m</td>
		</tr>
		<tr>
			<td>syringe filter 0.2 &mu;m</td>
			<td>e.g. Fisherbrand</td>
			<td>09-719C</td>
			<td>for sterile filtration of digestion medium</td>
		</tr>
		<tr>
			<td>straight scissors</td>
			<td>e.g. Fine Sciences Tools</td>
			<td>91460-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>curved scissors</td>
			<td>e.g. Fine Science Tools</td>
			<td>91461-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont No. 7 forceps</td>
			<td>e.g. Fine Science Tools</td>
			<td>91197-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>perforated spoon</td>
			<td>e.g. Fine Science Tools</td>
			<td>10370-19</td>
			<td>optional, for transfer of heart tissue</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>e.g. Gibco</td>
			<td>15250-061</td>
			<td>live cell staining</td>
		</tr>
		<tr>
			<td>Neubauer hemocytometer</td>
			<td>e.g. 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>e.g. Fisherbrand</td>
			<td>14-432-23</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml Falcon tubes</td>
			<td>e.g. Fisherbrand</td>
			<td>05-527-90</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20 ml syringe</td>
			<td>e.g. BD Medical</td>
			<td>14-820-19</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml serological pipette</td>
			<td>e.g. Falcon</td>
			<td>357551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 mm cell culture dish</td>
			<td>e.g. 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>e.g. 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>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Buffers and media: 
			<ul>
				<li>Isolation medium (filter sterilize) 
				20 mM BDM 
				0.0125% trypsin 
				in HBSS4 (without Ca2+, Mg2+)</li>
				<li>Digestion medium (filter sterilize) 
				20 mM BDM 
				1.5 mg/ml Roche Collagenase/Dispase enzyme mix 
				in L15 medium</li>
				<li>Plating medium 
				65% DMEM high glucose 
				19% M-199 
				10% horse serum 
				5% fetal calf serum 
				1% penicillin/streptomycin</li>
				<li>Maintenance medium 
				78% DMEM high glucose 
				17% M-199 
				4% horse serum 
				1% penicillin/streptomycin 
				optional: 
				1 &mu;M AraC 
				1 &mu;M isoproterenol or 0.1 mM phenylephrine</li>
			</ul>
			The plating and maintenance medium can be stored at 4 &deg;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

62.3K 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 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 ...

科研

生物学

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

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 Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

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 of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

新生儿肝外胆管的隔离

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

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 ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...