这项工作是由欧盟（STREP项目241577“大心脏”，第七届欧洲框架计划，CP），梅纳里尼国际业务卢森堡（AM），马拉松式节目GGP07133（CP）和Gilead Sciences公司（AM）的支持。

对人体组织的实验方案已获大学Careggi酒店，医院（2006/0024713; 2009年续签5月）的伦理委员会。每个患者的书面知情同意书。 1，解决方案和设备的准备解决方案是在表1中说明。细胞分离方法的简化流程图，在图1中找到。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2">解</td><td> CP </td><td> DB </td><td> KB </td><td>结核病</td><td> PS </td><td> EB1 </td><td> EB2 </td></tr><tr><td rowspan="23">试剂（MM） </td><td> KH 2 PO 4 </td><td> 50 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 硫酸镁 </td><td> 8 </td><td> 1.2 </td><td> 5 </td><td></td><td></td><td> 1.2 </td><td> 1.2 </td></tr><tr><td> HEPES </td><td> 10 </td><td></td><td></td><td> 10 </td><td> 10 </td><td></td><td></td></tr><tr><td>腺苷</td><td> 5 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>葡萄糖</td><td> 140 </td><td> 10 </td><td> 20 </td><td> 10 </td><td></td><td> 10 </td><td> 10 </td></tr><tr><td>甘露醇</td><td> 100 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>牛磺酸</td><td> 10 </td><td> 20 </td><td> 5 </td><td></td><td></td><td> 20 </td><td> 20 </td></tr><tr><td>氯化钠</td><td></td><td> 113 </td><td></td><td> 136 </td><td></td><td> 113 </td><td> 113 </td></tr><tr><td>氯化钾</td><td></td><td> 4.7 </td><td> 85 </td><td> 5.4 </td><td> 25 </td><td> 4.7 </td><td> 4.7 </td></tr><tr><td> 氯化镁 </td><td></td><td> &lt;/ TD&gt; <td></td><td> 1.2 </td><td> 5 </td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td></td><td> 0.6 </td><td> 30 </td><td></td><td></td><td> 0.6 </td><td> 0.6 </td></tr><tr><td>的Na 2 HPO 4 </td><td></td><td> 0.6 </td><td></td><td></td><td></td><td> 0.6 </td><td> 0.6 </td></tr><tr><td> 碳酸氢钠 </td><td></td><td> 12 </td><td></td><td></td><td></td><td> 12 </td><td> 12 </td></tr><tr><td> KHCO 3 </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td>丙酮酸钠</td><td></td><td> 4 </td><td></td><td></td><td></td><td> 4 </td><td> 4 </td></tr><tr><td> BDM </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> BHBA </td><td></td><td></td><td> 5 &lt;/ TD&gt; <td></td><td></td><td></td><td></td></tr><tr><td>琥珀酸</td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> EGTA </td><td></td><td></td><td> 0.5 </td><td></td><td></td><td></td><td></td></tr><tr><td> K 2 +-ATP </td><td></td><td></td><td> 2 </td><td></td><td></td><td></td><td></td></tr><tr><td>丙酮酸</td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td>肌酸</td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> KMES </td><td></td><td></td><td></td><td></td><td> 115 </td><td></td><td></td></tr><tr><td rowspan="2">酶（U / ml的） </td><td>胶原酶V型</td><td></td><td></td><td></td><td></td><td></td><td> 250 &lt;/ TD&gt; <td> 250 </td></tr><tr><td>蛋白酶XXIV型</td><td></td><td></td><td></td><td></td><td></td><td> 4 </td><td></td></tr><tr><td> pH值</td><td></td><td> 7.4 KOH </td><td> 7.3氢氧化钠</td><td> 7.1 KOH </td><td> 7.35氢氧化钠</td><td> 7.2 KOH </td><td> 7.3氢氧化钠</td><td> 7.3氢氧化钠</td></tr></tbody></table> 。表1用于标本采集，细胞分离和细胞的功能特性 CP =心脏停搏液解决方案 ; DB =分离缓冲液; KB =卡夫-Bruhe的解决方案; TB =台氏缓冲液; PS =吸管的解决方案; EB1 =酶缓冲液1; EB2 =酶缓冲液2。 <ol><li>准备心脏停搏（CP）的解决方案。 CP溶液可以储存在4℃下长达1周。 </li><li>制备的Ca 2 +的解离缓冲液（DB）。该溶液应在一天内使用。 </li><li>制备牛皮纸Bruhe（KB）溶液。 KB溶液可以储存在4℃下长达1凌晨K表。 </li><li>制备的Ca 2 +游离的Tyrode缓冲液（TB）。该溶液应在一天内使用。 </li><li>使用针头式过滤器在使用前过滤所有的解决方案。 </li><li>制备消化装置（ 图2），由有机硅弹性体的两个相对的电刷，其中一旋转由电机一刮容器。消化设备定制。消化设备上的细节都在图8;该装置的图像是在图2 C和2D。用70％乙醇和水冲洗组织腔。 </li></ol> 2，收集心肌标本和处理<ol><li>倾将40ml cardiplegic（CP）的溶液在50毫升的试管，并将其存储在冰中从手术室内的细胞分离的实验室样品运输。 </li><li>切除后立即收集从手术室内心室心肌标本，用冰冷的洗CP溶液，并将其存储在所述管。使用来自心脏直视手术中的上跨室间隔，权重&gt; 100毫克内膜切除标本。 </li><li>样品迅速转移到实验室区域;内切除标本10分钟开始检体处理。 </li><li>在保持在冰冷的CP缓冲试样，小心地取出使用立体显微镜下精剪心内膜纤维化层;事后，切心肌组织小片（长2-3毫米）。取决于组织样品的大小，切割100毫克和1 g之间心肌的总量为每个隔离。 </li><li>当组织切碎后，转移心肌块放入消解器，用干净的冰冷CP的解决方案。避免与心肌块填充两个硅刷子间的总容​​积（3-4毫升），使用不超过总组织1克。 </li></ol> 3，洗涤和大块心肌消化<ol><li>船尾洱块被转移到消化装置的刮腔，改变腔室中的CP的缓冲用冷的Ca 2 +的解离缓冲液（DB）。 </li><li>将消解装置的恒温槽中，为了使该腔室以与被加热的水在浴（ 图1）接触。将沐浴到37.5℃，打开它，才能慢慢提高组织室的温度。把消化装置的马达上，旋转速度设置为1转/秒。 </li><li>进行3次洗涤用DB中，改变该室中的溶液用干净的数据块，每8分钟。数据库温热（37℃），并获得与心肌块接触之前氧饱和。 </li><li>通过增加胶原酶V和4 U / ml的蛋白酶种类四条250 U / ml到数据库的解决方案准备酶缓冲液1（EB1）。加入250 U / ml的胶原酶V至DB解决方案准备酶缓冲液2（EB2）。热身（37°C）和oxygenatËEB1和EB2。 </li><li>进行消化的2 12分钟的周期在旋转消解器以100％的含氧EB1（在37℃）。在每个周期中，使用〜3毫升EB1的。用移液管抽吸除去该溶液，每个循环后丢弃它。 </li><li>制备6 15ml试管为细胞收集和〜80毫升洗脱缓冲液的冷（4℃）KB溶液中。 </li><li>执行第一15分钟消化循环用3毫升100％氧化EB2在37℃下消化周期后，收集含在一个15毫升管的第一解离细胞的溶液，并稀释细胞悬浮液用12ml冷的KB溶液中。存储管扁在室温下。 </li><li>稀释剩余EB2解决方案与DB等量的，以减半胶原酶Ⅴ的浓度以下的消化周期。 </li><li>执行其他5 12分钟的消化周期，用3毫升EB2在37℃;之后，他们每个人收集含有缓冲在一个15毫升的锥形管心肌细胞和用12ml KB液稀释。存储6含细胞试管在室温下放置30分钟。 </li></ol> 4，细胞再悬浮和Ca 2 +的再改造<ol><li>加入1毫克/毫升的牛血清白蛋白（BSA）至20ml的Ca 2 +游离的Tyrode缓冲液（TB）。过滤该溶液。 </li><li>离心六含心肌细胞锥形管，在100×g离心5分钟，迫使细胞定居。去除上清并将细胞重新悬浮在每个管与BSA的含在RT TB可变数量（1-3毫升，取决于产率）。 </li><li>逐渐加入100毫摩尔/升的CaCl 2溶液的小等份增加Ca 2 +浓度的含细胞的缓冲区。在第一和第二步骤的Ca 2 +浓度升高至50μmol/ L和100μmol/ L的分别。以下的Ca 2 +另外的步骤中进行，每​​5分钟，浓度在每一步吨提高100μmol/ L的0.9毫摩尔/ L OA终浓度</li><li>评估该分离过程的产率。将0.5毫升含有心肌细胞溶液到显微镜的玻璃底室。评估15显微镜领域在10倍物镜放大倍数，计算健康细胞（ 如杆状细胞有明确的条纹和无显著夹杂物， 图2）的百分比。预期收益率是20％左右。 </li></ol>隔离型心肌5。功能评价。 下面的协议是人类心肌功能评估，包括动作电位和细胞内Ca 2 +通量的同时记录的一个例子。 <ol><li>准备电极内液（PS）在穿孔膜片配置膜片钳实验。该溶液可以贮存于-20℃下进行长达3个月。 </li><li>加1.8毫摩尔/升的CaCl 2，以Ca 2 +的无的Tyrode缓冲液（TB）。 ü本身这个解决方案为心肌灌流过程中膜片钳/荧光实验。 </li><li>传送1 ml的细胞悬浮液到1.5ml管中，加入10μmol/ L的Fluoforte和10μl型电力浓缩。孵育30分钟，在室温。此后，设置管垂直位置和离开细胞定居5分钟;悬浮细胞中的Ca 2 +含结核。 </li><li>转让0.25 ml细胞悬液至小（0.5毫升）中，温度控制的显微镜安装记录室中，以0.3毫升/分钟的流速灌流通过重力与加热microperfusor系统（温度：37±0.5℃）。 </li><li>使用微量牵拉，制备片钳吸量管为3至5微米的尖端直径为3〜4.5米时填充PS了阻力。 </li><li>两性霉素B加至一个批次的PS（250微克/毫升），并用它来填充电极。 </li><li>选择一个杆状细胞有明显的条纹，缺乏夹杂物，FORM的千兆密封和等待5到10分钟，直到下访问抵抗力下降20MΩ。 </li><li>使用短脉冲（&lt;3毫秒）刺激在不同频率（0.2赫兹，0.5赫兹和1赫兹，在每个频率1分钟）在电流钳模式引起动作电位。在录制阶段，打开明亮的视场照明在492±3 nm和检测Fluoforte荧光在505-520纳米。获得的荧光，并使用Digidata 1440A和pClamp 10.0软件膜电位信号。重复的记录序列多次，如果需要的;然而，保持低于15分钟对每个单元的总记录时间。 </li></ol>

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M., Rudy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+IKr+and+IKs+heterogeneity+on+action+potential+duration+and+its+rate+dependence:+a+simulation+study.">Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence: a simulation study.</a> Circulation. 99, 2466-2474 (1999).</li><li>Volders, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+contribution+of+IKs+to+canine+ventricular+repolarization:+key+role+for+beta-adrenergic+receptor+stimulation.">Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation.</a> Circulation. 107, 2753-2760 (2003).</li><li>Sanguinetti, M. C., Jurkiewicz, N. K., Scott, A., Siegl, P. 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Mechanism of action.</a> Circulation Research. 68, 77-84 (1991).</li><li>Coppini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+translational+approach+to+treatment+of+hypertrophic+cardiomyopathy:+pre-clinical+rationale+and+design+of+a+prospective+randomized+pilot+trial+with+ranolazine.">A translational approach to treatment of hypertrophic cardiomyopathy: pre-clinical rationale and design of a prospective randomized pilot trial with ranolazine.</a> Circulation. 125, 1 (2012).</li></ol>

作者宣称，他们有没有竞争的财务权益。

我们已经描述和验证的方法从人心肌的手术标本分离出可行的心肌细胞。从前面描述的协议已被成功地用于分离的细胞从心房手术样品，该技术允许从患病心室肌单个活细胞分离的开发和微调开始。早期的报道表明，从心房和心室组织块选择性受损复极钾电流，导致改变的电生理特性和响应于生理刺激8,24，单个心肌细胞的分离，而输送通过冠状动脉灌注含有缓冲液的酶没有损害延迟整流?...

的心肌的电生理特性解剖技术的发展为单个心肌细胞分离后已取得进展显着。在心肌兴奋收缩偶联（EC-耦合）的认识的最新发展，也已成为可能，可行的隔离单个心肌细胞可在保留完整的组织的所有生理特性的能力。膜片钳方法被常规使用，研究心肌细胞膜离子电流的功能和药理学调制。细胞内钙动力学与钙离子敏感染料的录音也定期进行单心肌细胞从不同的健康和疾病模型，提供对EC-耦合的生理以及对病理改变细胞内Ca 2 +的重要数据动态平衡，导致机械损伤和心脏疾病的增加心律失常的负担。信息tion从这些研究对于了解药物在临床上的电和机械作用的关键。但是，也有物种的跨膜电流和在该占心脏动作电位和心脏力学的具体特征对EC-偶联蛋白特异性差异。因此，当细胞从非人类哺乳动物中分离的研究已阐明的生物物理属性和特定的跨膜离子通道和EC-偶联蛋白的生理功能，它们不一定提供人类心肌细胞的相关模型。因此，从人类心肌存活心肌细胞的隔离是必要的，充分了解心脏疾病的病理生理学和验证新的治疗方法。 人类心房组织是现成的作为心耳在手术过程中通常被丢弃。成人人心肌动作电位和离子立方米初步定量研究rrents采用酶解分离心房细胞1-4。动作电位或从孤立的成人心室肌细胞电流记录已随后报告3,5-10。大部分这些研究使用了从外植心脏获得和利用胶原酶或冠状动脉段或数量较大的切除的组织暴露于胶原酶，得到分离的细胞的灌注细胞。这些研究使一些来自健康人的心脏心室肌细胞，并从患者的终端心脏衰竭跨膜离子电流的详细特征。 L型的录音的Ca 2 +电流（I CA-L）5-7，瞬时外向钾电流（I 至 ）8，内向整流钾电流（Iκ1）8，延迟整流钾电流的不同组成部分（我κ ）9已有报道。进步和精炼分离过程10，允许在终端心脏衰竭，包括动作电位延长11增加的潜在致心律失常的离子基础的明确表征，去极化12后延迟并增加有趣电流13到舒张期去极化和早搏。 成年心肌细胞通常是从小型动物的整个心脏与各种酶的混合物，一种能够产生的Ca 2 +的容错电池14的高产量技术的逆行灌注隔离。从组织碎片心肌细胞的分离是可能是因为酶的有限访问单个肌细胞与由冠状动脉灌注取得了比较本来就不太成功。由于未使用捐助者的心非常有限的，唯一可行的方法，以获得正常的人心肌细胞定期是通过酶digestio不适用不择期外科手术的切除往往非常小的组织碎片。已经彻底的特点在细胞水平上的唯一人类疾病模型是终端心脏衰竭，因无障碍移植后的心脏。然而，终端心脏衰竭发生在少数患者并且经常涉及心肌细胞的严重重塑，这是相对独立的根本原因15的共同通路。以评估患者的单个心肌细胞的功能在疾病的早期非故障段的能力是至关重要的理解不同遗传或后天条件的特定病理生理学。肥厚型心肌病（HCM）是一个生动的例子。 HCM是一种常见的（1/500的个体）的特征在于心脏肥大可继承的心脏状况，由于流出道梗阻和舒张功能障碍16增加心律失常风险和收缩变化。从HCM心心肌ündergo参与细胞结构的变化（肥大，肌原纤维混乱）和EC-17耦合复杂的重塑过程。然而，在胡志明市心肌功能障碍多数信息都来自转基因动物模型。由于HCM患者，只有少数演变对终端心脏衰竭，需要心脏移植，HCM的心中都很少适用于细胞的分离与标准方法。然而，HCM患者中至少有30％的收缩（HCM）18发展过程中由于大量室间隔肥厚改变流出道血流梗阻症状。最有效的可用的治疗选择梗阻的HCM的救济是手术室间隔心肌切除术：这种手术过程中，上部隔一个大小可变的部分是由反式主动脉瓣的方法去除。肥大隔膜的这一部分，因此可用于从新鲜组织细胞的分离。 一种用于人类ventricula的分离方法r是单一的，小静脉心内膜心肌活检标本细胞先前已经制定和公布19。我们实现了一个方法，从接受心脏手术，包括HCM患者接受室间隔心肌切除术和心脏瓣膜置换手术的患者患者分离单个心肌细胞室间隔从心室心肌标本。除了 ​​隔离协议，有代表性的电生理学和Ca 2 +的荧光的详细描述测量呈现，显示了分离的人心肌细胞的生存能力和膜片钳和细胞内Ca 2 +的研究的可行性。

<table border="0" cellpadding="0" cellspacing="0" width="1059">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:139px;"><a href="http://www.sigmaaldrich.com/catalog/product/sigma/p9791?lang=it&amp;region=IT">P9791&#160;</a></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium sulfate heptahydrate(MgSO4 * 7H2O)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m1880?lang=it&amp;region=IT">M1880&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/h3375?lang=it&amp;region=IT">H3375&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9251?lang=it&amp;region=IT">A9251&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/g8270?lang=it&amp;region=IT">G8270&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m4125?lang=it&amp;region=IT">M4125&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7653?lang=it&amp;region=IT">S7653</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p9333?lang=it&amp;region=IT">P9333&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7907?lang=it&amp;region=IT">S7907&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s6297?lang=it&amp;region=IT">S6297&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium bicarbonate (KHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/237205?lang=it&amp;region=IT">237205</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p2256?lang=it&amp;region=IT">P2256&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/b0753?lang=it&amp;region=IT">B0753&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide(NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s8045?lang=it&amp;region=IT">S8045&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamic acid monopotassium salt monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/fluka/49601?lang=it&amp;region=IT">49601</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvic acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/107360?lang=it&amp;region=IT">107360</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-Hydroxybutyric acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/166898?lang=it&amp;region=IT">166898</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#8242;-triphosphate dipotassium salt dihydrate (K2-ATP)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a8937?lang=it&amp;region=IT">A8937</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatine</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c0780?lang=it&amp;region=IT">C0780&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Succinic Acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s3674?lang=it&amp;region=IT">S3674&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/e0396?lang=it&amp;region=IT">E0396&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a0281?lang=it&amp;region=IT">A0281</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m8266?lang=it&amp;region=IT">M8266&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from Clostridium histolyticum, Type V</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c9263?lang=it&amp;region=IT">C9263&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase, Bacterial, Type XXIV</td>
			<td>Sigma-Aldrich</td>
			<td>P8038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride solution, ~1 M in H2O</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/21115?lang=it&amp;region=IT">21115</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride 0.1 M solution</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/53704?lang=it&amp;region=IT">53704</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/83000?lang=it&amp;region=IT">83000</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluoForte Reagent</td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-52015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerload concentrate, 100X</td>
			<td>Life Technologies</td>
			<td>P10020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusion Fast-Step System</td>
			<td>Warner Instruments</td>
			<td>VC-77SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B solubilized</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9528?lang=it&amp;region=IT">A9528&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digidata 1440A</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pClamp10.0&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="108">
			<td height="108" style="height:108px;">Digestion Device</td>
			<td>CUSTOM</td>
			<td>CUSTOM</td>
			<td style="width:255px;">The device is custome made in our laboratory using plastic tubes, cast Sylgard and a motor; it is described in detail in Fig 1 C-D and in Fig.7. We can provide further details if requested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone elastomer for the digestion device&#39;s brushes</td>
			<td>Dow Corning</td>
			<td>SYLGARD&#174; 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Variable speed rotating motor for the digestion device</td>
			<td>Crouzet</td>
			<td>Crouzet 178-4765&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mold for brushes casting</td>
			<td>N.A.</td>
			<td>N.A.</td>
			<td style="width:255px;">The mold is custom made from standard PTFE 2.5 cm diameter rods</td>
		</tr>
	</tbody>
</table>

isolation and functional characterization of human ventricular cardiomyocytes from fresh surgical samples

从患病心脏的心肌细胞受到涉及改变细胞结构，激发收缩偶联和膜离子流复杂重塑过程。这些变化很可能是负责增加心律失常的风险和收缩变化导致的收缩和舒张功能不全的心脏病患者。然而，在心脏疾病心肌细胞功能的改变大部分信息都来自动物模型。 在这里，我们描述和验证协议从心室肌从接受心脏外科手术的病人手术小样本分离出可行的心肌细胞。的协议进行详​​细说明。电生理学和细胞内钙的测量报告来演示了一些在用这种方法获得的人左心室心肌细胞的单细胞测量的可​​行性。 该协议的报道，他再可用于功能性改变人的心脏在不同心脏疾病的存在的细胞和分子基础的未来的调查非常有用。另外，该方法可用于鉴定在细胞水平上新的治疗靶点，并测试新化合物的效力对人心肌细胞，用直接平移值。

上面描述的方法进行表征的心肌细胞从患者的肥厚型心肌病（HCM）谁接受切除术手术室间隔分离的功能异常，如与非故障非增生性手术的患者21进行比较。在包含在本节中的结果是来自于该工件21，在这里被示为如何使用此技术可用于表征在心脏疾病状况的心肌细胞功能的改变的例子。 从与HCM患者的代表性手术样品如图2A所示。手术样本的大小和?...

Watch this Scientific Journal Video about Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples at JoVE.com

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

心脏疾病的细胞基础上的现有知识大多依赖于对动物模型的研究。在这里，我们描述和验证了一种新的方法从人心肌小型手术样品得到单一可行的心肌细胞。人心肌细胞可用于电生理学研究和药物测试。

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

isolation-functional-characterization-human-ventricular

Research

JoVE Journal

Medicine

17.1K 次观看.  University of Florence. 心脏疾病的细胞基础上的现有知识大多依赖于对动物模型的研究。在这里，我们描述和验证了一种新的方法从人心肌小型手术样品得到单一可行的心肌细胞。人心肌细胞可用于电生理学研究和药物测试。 

视频: Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

科研

医学

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

慢性血栓栓塞性肺动脉高压和右心室功能的仔猪评估

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

肌网钙储备与细胞内舒张钙在心室肌细胞中的分离评价

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

成年大鼠心肌细胞的分离与培养

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

小鼠致耐受性树突状细胞的发育及功能表征

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

生成心室样HiPSC衍生的心肌细胞和用于钙处理特征的高质量细胞制剂

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

内源性表达人RYR1变异体的功能表征

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

朗根多夫方法的修改，用于从成年小鼠中同时分离心房和心室肌细胞

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...