Name: simultaneous isolation of high quality cardiomyocytes, endothelial cells, and fibroblasts from an adult rat heart
Uploaded: 2017-05-19T14:00:00.000+00:00
Duration: 9 min 38 s
Description: Watch this Scientific Journal Video about Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart at JoVE.com

感谢L. Rinaldi，S.Schäffer，D. Reitz，H. Thomas和A. Weber的技术支持。作者还希望感谢Dr. E. Martinson博士对手稿进行广泛的阅读和语言编辑。该研究由Giessen Anschubsfinanzierung大学授予M. Aslam和D.Gündüz授予的支持。

调查符合美国国家卫生研究院出版的"实验动物护理和使用指南"（NIH出版号85-23 1985），并得到了吉森大学当地伦理委员会的批准。在本研究中使用体重200-250g的成年雄性Wistar大鼠。 高压灭菌<ol><li>在进行手术至少一天之前，将所有手术器械，移液管吸头和巴斯德吸管进行高压灭菌，以在121℃的隔离程序中使用30分钟。 </li></ol> 2.媒体和解决方案的准备注意:步骤2.1-2.4中的解决方案可以在隔离前一周准备并储存在4°C，但在隔离当天的步骤2.5-2.8中制备溶液。缓冲液和介质的完整配方见表1 。将所有培养基和溶液加热至37°C水浴开始准备之前。 <ol><li>通过将表1中列出的化学物质在室温（RT）下溶解在4.5L H 2 O中来制备鲍威尔培养基。用2.0N NaOH将pH调节至7.4，使体积达到5.0L。无菌过滤溶液，并将其储存在4℃。 </li><li>通过将M199粉末培养基溶解在4.5升H 2 O中来制备肌酸，L-肉毒碱和牛磺酸（CCT）培养基。将根据表格的HEPES粉末加入培养基中并混合15分钟。根据表1添加肌酸，肉碱，牛磺酸和Ara-C。混合均匀，用2.0 N NaOH将pH值调节至7.4，使体积达到5.0 L.无菌过滤器并在4℃下储存。 </li><li>通过向培养基M199中加入10％胎牛血清（FCS）和2％青霉素/链霉素（Pen / Strep），为成纤维细胞制备细胞培养基。 DMEM同样适用于M199，也可用于成纤维细胞培养。 </li><li>准备MV2细胞培养e培养基用于内皮细胞，根据制造商的说明书将补充混合物的所有内容物添加到基础MV2培养基中。取5 mL等分试样，并在水浴中温热至37°C。 </li><li>通过将0.05g牛血清白蛋白（BSA）溶解在50mL Hanks平衡盐溶液（HBSS）中并向其中加入0.5mL的200mM乙二胺四乙酸（EDTA）溶液来制备内皮细胞分离缓冲液。将其无菌过滤并将其储存在4°C。 </li><li>通过加入0.1mL 200mM至9.9mL PBS并将其储存在4℃来制备内皮细胞洗涤缓冲液。 </li><li>通过将27毫克胶原酶溶解在5毫升的鲍威尔培养基中并加入12.5μL的100mM CaCl 2溶液来制备胶原酶溶液。在水浴中将其温热至37°C。 </li><li>通过加入12.5μL，25μL和120μL的100mM CaCl 2溶液来制备增加的Ca 2+修复溶液1,2和3用于心肌细胞分别离子至6mL，6mL和12mL的Powell培养基。 </li><li>通过在每个35-mm细胞培养皿中加入1mL含有层粘连蛋白的预包衣培养基，在分离前一天预先涂覆心肌细胞的细胞培养皿。将它们在细胞培养箱中37℃孵育过夜。 </li></ol> 3.磁珠的制备<ol><li>将0.5 mL内皮细胞分离缓冲液置于1.5 mL样品管中。 </li><li>加入5μL（2×10 6 ）的平板小鼠IgG磁珠并充分混合。 </li><li>将管放在磁性架上1分钟。珠将粘附到管的壁朝向磁体。用1 mL移液管小心清除上清液。 </li><li>重复洗涤两次，最后重新悬浮在50μL内皮细胞分离缓冲液中，并保存在4°C直到使用。 </li></ol> Langendorff灌注系统的准备<ol><li>清洁t他Langendorff系统灌注2升蒸馏水。 </li><li>通过系统循环50 mL鲍威尔培养基5 min，弃去培养基。 </li><li>加入80 mL新鲜鲍威尔培养基。通过从气瓶中用玻璃巴斯德吸管鼓泡，持续地将其与"碳霉素"一起灌注。让介质循环通过系统。该系统现在可以灌输心脏（ 图1A ）。 </li></ol> 5.解剖大鼠<ol><li>将大鼠放在通风柜中的5L干燥器中，并通过排气口加入1.5mL的4-5％异氟烷，并关闭盖子。 注意:将动物放在干燥器内的高架多孔板上，以避免动物与液体异氟烷直接接触。 </li><li>等到大鼠失去盖子反射（通常〜5分钟），然后将大鼠从干燥器中取出。通过颈椎脱位安乐死老鼠。 </li><li>切开腹部皮肤和下面的肌肉水平线在中间使用一把锋利的剪刀。此后，将肌肉垂直切割至大鼠胸骨，打开胸部。与肺一起取出心脏，立即将器官置于冰冷的0.9％NaCl溶液中。 </li><li>在一把剪刀的帮助下取出肺，然后将其丢弃。清洁任何结缔组织的心脏，并在头臂主干的分支尾部切开主动脉。 </li></ol>心脏灌注和胶原酶消化<ol><li> 通过 Langendorff灌注系统的主动脉（第4节），使用两对薄的弯曲镊子安装心脏。为了确保以逆行方式灌注心肌，将灌注系统的插管的末端置于第一主动脉分支和主动脉瓣之间。用鳄鱼夹固定主动脉。 </li><li>用手术缝合线固定心脏，取出鳄鱼夹（ 图1B ）。 </li><li>打开套管的阀门，使灌注液（35 mL）逐滴通过心脏（60滴/ min; 5 mL / min），以清洗任何残留的血液。在这个阶段放弃任何流通。 注意:避免在灌注的任何阶段形成气泡，因为这将导致不完全消化和细胞分离过程失败。 </li><li>将悬挂的心脏放置在收集漏斗（心室; 图1B ）中，并启动蠕动泵，这将使灌注介质从收集漏斗再循环到储存器（ 图1A ）。 </li><li>将胶原酶溶液（从步骤2.8）加入到灌注培养基中。用循环胶原酶溶液灌注心脏30分钟。 注意:此步骤对于优化细胞产量非常关键。灌注时间取决于胶原酶的活性，这可能因批次而异。测试3-4批胶原酶以找到具有opti的胶原酶活动不良那么可以购买大量最适合的批次，这些批次足够至少一年可重复的结果。 </li><li>在消化时间结束时，停止灌注，使用一把剪刀从Langendorff系统中取出心脏，并将其放在玻璃培养皿中。 </li><li>切除并丢弃两个心房，并清除任何残留的脂肪组织。借助两对镊子仔细剥离心包膜，以避免上皮细胞的污染。 注意:如果需要，可以用胶原酶进一步消化心包，以获得纯心脏上皮细胞。在本研究中没有给出详细的方案，因为这超出了本议定书的范围。 </li><li>用一把剪刀将心脏放在中间，放入组织切碎机中。使心脏2-3次，并从Langendorff灌注中将切碎的组织重新悬浮在12 mL消化缓冲液中。 注意:不要砍h过量过量，因为这会导致死亡心肌细胞比例较高。 </li><li>将切碎的组织转移到15mL含胶原酶的解离介质中，并使其在水浴中消化5-10分钟，偶尔混合通过使用5mL一次性塑料移液管重新悬浮。 </li><li>在消化期结束时，通过100μm筛子过滤细胞悬浮液。丢弃组织碎屑和未消化的物质。 </li><li>在Langendorff灌注结束时，立即用至少1升蒸馏水冲洗灌注系统。通过将100mL的0.1N NaOH再循环30分钟来对系统进行清洗，最后用2-3L蒸馏水冲洗该系统。通过冲洗空气干燥系统，并用对分膜覆盖开口。 </li></ol> 7.大鼠心脏细胞分离<ol><li> 心肌细胞的分离培养 <ol><li>将细胞悬浮液以25 xg离心1分钟（无Brakes）以沉淀心肌细胞。也可以将心肌细胞在室温下静置10分钟而不进行离心。 </li><li>借助于一次性塑料移液管，在新的50mL管中，小心地去除含有内皮细胞和成纤维细胞的上清液。 </li><li>在Ca 2+ 6mL溶液1中重悬心肌细胞沉淀（步骤2.9）。允许细胞1分钟调节至低Ca 2+浓度环境，并以25 xg再次离心细胞悬浮液1分钟（无制动）。 </li><li>弃去上清液并将细胞沉淀重悬于6mL Ca 2+溶液2中（步骤2.9）。允许细胞1分钟调节至中等浓度的Ca 2+ ，最后加入12 mL的Ca 2+溶液3（步骤2.9）给细胞。在一次性塑料移液器的帮助下混合。 </li><li>以25 xg离心细胞悬浮液1分钟。 </li><li>弃去上清液，并将细胞重悬于20 mL预处理液中受热CCT培养基。将1mL细胞悬浮液加入到用层粘连蛋白预包被的20个无菌35mm细胞培养皿中（步骤2.9）中，并使细胞在无CO 2的细胞培养培养箱中附着2小时。 注意:心肌细胞也可以在二氧化碳环境下培养;然而，在这种情况下，应使用碳酸氢盐缓冲液系统的介质。 </li><li> 2小时后，用新的2 mL CCT培养基更换培养基，以去除任何死细胞。 注意:细胞可以进行任何计划的实验，并且可以在典型的细胞培养箱（不含CO 2 ）中培养长达3天，而不会明显损失活细胞。典型的健康的棒状心肌细胞显示在图2A中 。 </li></ol></li><li> 内皮细胞和成纤维细胞的分离 <ol><li>将含有内皮细胞和成纤维细胞的上清液（步骤7.1.2）以250×g离心10分钟。 </li><li>丢弃supe在2 mL样品管中将细胞沉淀重悬于1.5 mL内皮细胞分离缓冲液中。 </li><li>向细胞悬浮液中加入3μg（1:500）小鼠抗大鼠CD31抗体，并在4℃温育30分钟，并进行端对端旋转。 注意:建议用最佳稀释滴定抗体。该步骤应在4℃下进行，在室温下，抗体经内皮细胞的快速内化将导致低产率。 </li><li>将先前制备的（第3部分）泛抗小鼠IgG珠添加到细胞悬浮液中，并在4℃下以端对端旋转孵育20分钟。 注意:或者，可以将小鼠抗大鼠CD31抗体预连接到磁珠上，然后加入细胞悬液。然而，这将增加珠与细胞悬浮液的孵育时间，并且可能导致非内皮细胞的非特异性结合增加，因此增加其他细胞类型的污染。 </li><li>一个在抗体孵育时间结束时，将含有细胞悬浮液的管放置在磁性架中，并允许2分钟将附着于内皮细胞的磁珠沉积到管侧。 </li><li>用1 mL移液管小心地取出剩余的细胞悬浮液（主要含有成纤维细胞），并加入到含有10mL成纤维细胞培养液的10cm细胞培养皿中（步骤2.3）。将其置于含有5％CO 2的典型细胞培养箱中。步骤7.3继续。 </li><li>使用含有内皮细胞的磁珠，向管中加入1 mL洗涤缓冲液。 </li><li>盖住管子，将其从磁性架上取下，并通过轻轻摇动4〜5次，仔细重新悬挂珠子。将管再次放置在磁性架上，使珠子沉淀在管壁上1分钟，并小心地取出洗涤缓冲液。 </li><li>重复洗涤步骤5 - 7次以摆脱任何污染的n内皮细胞。 </li><li>将附着在珠上的内皮细胞重悬于1mL MV2内皮细胞培养基中，加入12孔板的单孔中，并在CO 2细胞培养箱中孵育。 </li><li>允许细胞附着过夜，并在第二天，然后每2-3天更换培养基，直到细胞变得半融合（通常为5-7天）。 注意:分离7-10天之后3-4天和融合内皮细胞的典型非汇合内皮细胞分别显示在图3A和3B中。然后将细胞进行胰蛋白酶消化（第7.4节）并接种到T25细胞培养瓶中。 </li></ol></li><li> 成纤维细胞的洗涤和培养（从7.2.6步继续） <ol><li>培养1小时后，通过加入适量体积的预热的PBS将培养基吸出并洗涤附着于细胞培养皿的成纤维细胞。吸出PBS并重复step 2-3次，最后加入预热新鲜成纤维细胞培养液。 </li><li>第二天用预先加热的PBS（如上一步）洗涤附着的细胞，并加入预热的新鲜培养基。每2-3天更换一次。在分离的第2天，成纤维细胞的典型表型如图4A所示 。 注意:成纤维细胞通常在5-7天内半融合，然后准备进行胰蛋白酶消化（第7.4节），并用于计划实验。通过这个程序，我们通常获得95-99％的纯成纤维细胞。分离第7天的成纤维细胞的典型表型如图4B所示 。 </li></ol></li><li> 内皮细胞和成纤维细胞的胰蛋白酶消化 <ol><li>从细胞培养皿吸出培养基，并加入足够的预热PBS以覆盖细胞。 </li><li>吸出PBS并加入足够的胰蛋白酶-EDTA溶液（分别为0.05％和0.02％）以覆盖细胞并置于37℃孵育5分钟。 </li><li>通过用1毫升移液管的帮助，将细胞上下移液重悬细胞，并通过加入10％FCS停止胰蛋白酶作用。 </li><li>通过在室温下250xg离心10分钟来沉淀细胞悬液。 </li><li>将细胞重悬于适量体积的预热的细胞培养基中。 </li></ol></li></ol> 8.内皮细胞表征注意:血管内皮细胞的纯度通过Vil Willebrand因子（vWF）的Dil-Ac-LDL摄取和免疫染色测定。通过荧光或共焦显微镜观察细胞（ 图3C-3E ）。 <ol><li> Dil-Ac-LDL摄取内皮细胞 <ol><li>对内皮细胞进行胰蛋白酶消化（步骤7.4），并在12孔板（约10×10 4个细胞/ cm 2 ）的玻璃盖玻片上培养它们过夜。 </li><li>吸出培养基，并用1 mL的pre-w冲洗细胞武装HBSS。 </li><li>吸入HBSS并加入含有10μg/ mL Dil-Ac-LDL的1mL内皮细胞培养基，并将细胞置于37℃培养箱中4小时。 </li><li>吸出培养基并用预热的PBS冲洗细胞三次，并用1mL的4％多聚甲醛（PFA;溶解在PBS中）固定。用PFA在37℃下孵育20分钟。 注意:PFA具有腐蚀性;在处理PFA溶液时，始终使用耐酸手套。 </li><li>吸出PFA，用1 mL PBS将12孔板上的盖玻片上的细胞冲洗3次，并在室温下用核染色溶液TO-PRO（10μM）孵育1小时。 注意:如果适用于显微镜的过滤器，也可以使用4&#39;，6-二脒基-2-苯基吲哚（DAPI;1μg/ mL）。在这种情况下孵育时间应减少到10分钟。 </li><li>用1 mL PBS在12孔板上的盖玻片上冲洗细胞，抽出PBS，重复3次。 </li><li>拿c超过12孔板，并将其安装在带有一滴安装介质的玻璃滑块上。细胞准备在荧光/共焦显微镜下显现。对于Dil-Ac-LDL，对于TO-PRO使用530-560nm的激发滤光片，对于TO-PRO使用640nm，对于DAPI使用340-380nm。 </li></ol></li><li> Von Willebrand（vWF）染色 <ol><li>如步骤8.1.1所述培养内皮细胞。 </li><li>吸出培养基并用1mL预热的HBSS冲洗细胞;然后如步骤8.1.4所述用4％PFA固定。 </li><li>吸出PFA，用12mL平板的盖板上的细胞用1mL PBS冲洗两次，并用1mL的0.1％Triton X-100（在PBS中）将细胞在室温下透化20分钟。 </li><li>吸出透化溶液，并用封闭溶液（PBS中5％BSA / 5％FCS）孵育细胞1小时。 </li><li>用兔抗vWF抗体（1:100）在4℃下在潮湿室中孵育细胞过夜。添加一个盖玻片没有the一抗作为阴性对照。 </li><li>用1 mL PBS冲洗12孔板盖玻片上的细胞3次，并用驴抗兔Alexa Fluor-488抗体（1:500）和核染色溶液TO-PRO（10μM）孵育细胞1小时在RT。 </li><li>用1 mL PBS冲洗12孔板盖玻片上的细胞，抽出PBS，重复3次。 </li><li>将盖玻片从12孔板中取出，并将其安装在带有一滴安装介质的玻璃载玻片上。细胞准备在荧光/共焦显微镜下显现。对于vWF使用490nm的激发滤光片，对于核染色使用640nm的激发滤光片。 </li></ol></li></ol>

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在本文中，描述了用于分离和培养心肌细胞，内皮细胞和成纤维细胞的可重复方案。该方案描述了与仅仅一种细胞类型相反的心脏组织， 即心肌细胞，内皮细胞和成纤维细胞的主要细胞类型的同时和高质量分离。分离过程中的关键步骤是适当消化心脏组织。如果消化不完全，仍然可以获得大量的肌细胞，但其质量非常差，并且它们大多是超收缩和圆形的（ 图2B ），并且在实验中没有价值。此外，内皮细胞和成纤维细胞的产量也将减少。这个问题可以通过控制消化时间来解决。在从灌注系统中取出心脏之前，可以借助镊子和指尖检查心脏的柔软度。如果组织仍然不明显心脏可能会再灌注5-10分钟。健康心肌细胞分离的第二个关键步骤是分离缓冲液中Ca 2+增量的恢复。培养基中高浓度Ca 2+的快速恢复将导致圆形心肌细胞。用于制备缓冲液的水质是影响心肌细胞质量的另一个重要因素。因此，建议使用优质水。 内皮细胞的纯度高度依赖于洗涤步骤。 EDTA必须存在于洗涤缓冲液中以防止非内皮细胞与磁珠的非特异性结合。细胞悬浮液中血细胞的存在将大大降低内皮细胞的产量;因此，在将心脏装入灌注系统之后和加入胶原酶之前，心脏的适当洗涤是非常关键的。残留血液成分也会减少t他的胶原酶活动，从而阻碍了心脏的消化。 成纤维细胞培养物中最常见的污染物是上皮细胞的卫星菌落的出现（ 图4C ）。这种污染可以通过两步培养成纤维细胞来避免。在第一步中，将成纤维细胞培养基中的FCS的浓度降低至5％，并将细胞悬浮液在培养箱中附着于细胞培养皿30分钟。上皮细胞迅速粘附，但在低浓度血清存在下，成纤维细胞粘附时间增加。孵育30分钟后，将含有非粘附成纤维细胞的细胞悬浮液小心地移至新的细胞培养皿中。以这种方式，可以降低成纤维细胞的产量，但其纯度大大提高。

长期以来，啮齿动物模型被用作扩大对健康和疾病心血管生理学理解的工具。 1虽然这些动物模型可以让我们了解器官水平的疾病的病理生理学，并且分析用于治疗心血管疾病的各种药理学药物的药代动力学和药效学，了解心血管疾病发展的分子机制以及特定的细胞类型需要使用体外细胞培养模型。为此，已经开发了来自心血管系统的不同永生细胞系;然而，新鲜分离的原代细胞在生理和功能上与活组织和生物体更相关。 心脏是含有所有主要类型的心血管细胞的通用器官ystem，大鼠心脏仍然是一种常用的心血管生理学理解模型。在过去几十年中，已经描述了将各种细胞类型与心脏组织隔离的不同方法。然而，这些方法仅关注一种特定细胞类型的分离，导致不能再用于细胞分析的其他类型细胞的丧失。这里描述了优化的方案，其能够同时且高质量地分离主要细胞类型的心脏组织，即心肌细胞，内皮细胞和成纤维细胞。所有这些细胞类型可用于不同的实验装置8,9,10以及来自相同动物的细胞 - 细胞相互作用的分析。

<table><tbody><tr><td>anti-vWF</td><td>Santa Cruz Biotech.</td><td>SC-14014</td><td></td></tr><tr><td>Calcium chloride</td><td>Merck</td><td>102378</td><td></td></tr><tr><td>Carnitin</td><td>Sigma-Aldrich</td><td>C0283</td><td></td></tr><tr><td>Collagenase type II</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>Creatin</td><td>Sigma-Aldrich</td><td>C0780</td><td></td></tr><tr><td>D-Glucose</td><td>Merck</td><td>108342</td><td></td></tr><tr><td>Dil-Ac-LDL</td><td>Thermo Scientific</td><td>L3484</td><td></td></tr><tr><td>EDTA Solution (0.2 M)</td><td>Biochrome AG</td><td>L2113</td><td></td></tr><tr><td>Embeding solution</td><td>Citiflour</td><td>AF1-25</td><td></td></tr><tr><td>Endothelial cell medium MV2</td><td>PromoCell</td><td>C-22022</td><td></td></tr><tr><td>Foetal calf serum (FCS)</td><td>Biochrome AG</td><td>S0615</td><td></td></tr><tr><td>Gentamicin</td><td>Serva Chemicals</td><td>47991</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td>TU 061219</td><td></td></tr><tr><td>Laminin&amp;nbsp;</td><td>Roche/Sigma</td><td>11243217001</td><td></td></tr><tr><td>M199 medium</td><td>Thermo Scientific</td><td>11150059</td><td></td></tr><tr><td>M199 medium (Powder)</td><td>Biochrome AG</td><td>T061</td><td></td></tr><tr><td>Magnesium sulphate</td><td>Sigma-Aldrich</td><td>63138</td><td></td></tr><tr><td>Mouse anti-rat CD31 antibody (TLD-3A12)</td><td>Thermo Scientific</td><td>MA1-81051</td><td></td></tr><tr><td>NaCl solution (0.9%), Sterile</td><td>B. Braun</td><td>30820080</td><td></td></tr><tr><td>Pan mouse IgG beads (Dynabeads)</td><td>Thermo Scientific</td><td>11041</td><td></td></tr><tr><td>Paraformaldehyde (PFA) 4% Solution</td><td>Santa Cruz Biotech.</td><td>sc-281692</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Scientific</td><td>15070-063</td><td></td></tr><tr><td>Phosphate buffer saline (PBS) 1x&amp;nbsp;</td><td>PAN-Biotech</td><td>P04-36500</td><td></td></tr><tr><td>Plastic consumables</td><td>Greiner Bio-One</td><td></td><td></td></tr><tr><td>Potassium Chloride</td><td>Merck</td><td>4933</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Merck</td><td>7873</td><td></td></tr><tr><td>Sodium Chloride</td><td>Merck</td><td>6404</td><td></td></tr><tr><td>Sodium Hydroxide Solution (2 N)</td><td>Merck</td><td>109136</td><td></td></tr><tr><td>Sterile filtration system</td><td>Thermo Scientific</td><td>5660020</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T8691</td><td></td></tr><tr><td>TO-PRO</td><td>Thermo Scientific</td><td>T3605</td><td></td></tr><tr><td>Trypsin-EDTA Solution (10x)</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>Water, Sterile</td><td>B. Braun</td><td></td><td></td></tr></tbody></table>

simultaneous isolation of high quality cardiomyocytes, endothelial cells, and fibroblasts from an adult rat heart

大鼠是用于心血管研究的重要动物模型，大鼠心脏细胞常规用于体外分析心血管疾病进展的分子机制，如心脏肥大，纤维化和动脉粥样硬化。尽管已经对从心血管系统开发永生化细胞系以了解这些细胞机制进行了多次成功的尝试，但是对于这些研究，原代细胞提供了更加自然和接近于体内环境。因此，在特定细胞类型上工作的不同实验室已经制定了分离感兴趣的大鼠心脏细胞的各种类型的方案。然而，允许隔离多个单元格类型的协议丢失。这里描述了优化的方案，其允许从单一制剂中分离高质量的主要心脏细胞类型（心肌细胞，内皮细胞和成纤维细胞），并使得该r用于细胞分析。这样可以最有效地利用可用资源，这可以节省时间并降低研究成本。

<html>分离程序导致可用于计划实验的70-80％可行的棒状条纹心肌细胞（ 图2A和2C ）的产量。在我们的实验室中，心肌细胞通常用于分析Ca 2+信号。 图5A显示了在加载Fura-AM（5μM）的心肌细胞中对缺血/再灌注反应的细胞内钙[Ca 2+ ] i振荡。 图5B显示加入ATP（100μM）后内皮细胞和成纤维细胞中[Ca 2+ ] i的变化。如前所述分析不同心脏细胞类型中[Ca 2+ ] i的变化。 6，11，12 re 1"src ="/ files / ftp_upload / 55601 / 55601fig1.jpg"/&gt; 图1:改良Langendorff灌注系统。 （ A ）用于分离心脏细胞的整个Langendorff灌注系统（ B ）"心室"中悬挂心脏的放大视图也用于收集灌注缓冲液进行再循环。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55601/55601fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55601/55601fig2.jpg" /> 图2:成年鼠心肌细胞。 （ A ）在层粘连蛋白包被的细胞培养皿中电镀2小时后典型的棒状健康心肌细胞（ B ）由于不完全组织消化而导致的不良制剂的典型实例。电镀后2 h，在层层蛋白包被的细胞培养皿中，细胞是超收缩并且看起来是圆的，并且几乎没有棒形心肌细胞。比例尺=10μm（ C ）如前所述，用于肌动蛋白可视化染色的单个心肌细胞的共聚焦显微照片。 13比例尺=50μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55601/55601fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55601/55601fig3.jpg" /> 图3:大鼠心脏内皮细胞。 （ A ）分离3天后非汇合内皮细胞单层的相差显微照片。黑斑是仍然附着于母体内皮细胞的磁珠。比例尺=10μm（ B ）分离7天后融合内皮细胞单层的相差显微照片（ C）显示Dil-Ac-LDL摄取（红色）和核染色（蓝色）的内皮细胞的免疫染色（ D ）显示vWF染色（红色）和核染色（蓝色）的内皮细胞的免疫染色（ E ）仅与二抗孵育的细胞和核染色（蓝色）（比例尺=50μm） <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55601/55601fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55601/55601fig4.jpg" /> 图4:大鼠心脏成纤维细胞。 （ A ）隔离第二天的典型纺锤形心脏成纤维细胞。 （ B ）培养7天后融合纯的成纤维细胞。 （ C ）被上皮细胞污染的不良制剂的典型例子。箭头指示的细胞是心脏上皮细胞<html> 。刻度棒=10μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55601/55601fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" src="/files/ftp_upload/55601/55601fig5.jpg" /> 图5:心肌细胞中[Ca 2+ ] i的变化。 （ A ）暴露于化学缺血的大鼠心肌细胞（氰酸钠; 2mM）后再灌注的Fura-2比值测定的细胞内钙[Ca 2+ ] i变化的代表性追踪（ B ）细胞内钙变化的代表性追踪[Ca 2+ ] i在大鼠心脏内皮细胞和成纤维细胞中通过Fura-2比率测量，用所指示的ATP（100μM）处理。 &gt;请点击此处查看此图的较大版本。

Watch this Scientific Journal Video about Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart at JoVE.com

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

已经开发并描述了用于从大鼠心脏分离不同心脏细胞类型的几种方案。在这里，描述了优化的方案，其允许从单一制剂分离高质量的主要心脏细胞类型（心肌细胞，内皮细胞和成纤维细胞），降低了实验成本。

从成年大鼠心脏同时分离高质量心肌细胞，内皮细胞和成纤维细胞

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

simultaneous-isolation-high-quality-cardiomyocytes-endothelial-cells

Universidad San Sebastian

Research

JoVE Journal

Biology

13.3K 次观看.  University Hospital Giessen. 已经开发并描述了用于从大鼠心脏分离不同心脏细胞类型的几种方案。在这里，描述了优化的方案，其允许从单一制剂分离高质量的主要心脏细胞类型（心肌细胞，内皮细胞和成纤维细胞），降低了实验成本。 

视频: Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

科研

生物学

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

从鼠心心室自由壁分离内膜细胞和冠状内皮细胞

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

发育生物学

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 ...

医学

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

代谢综合征心力衰竭模型大鼠心房心肌细胞的分离与保留射血分数的关系

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

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 ...

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. ...