Name: the isolation and culture of primary epicardial cells derived from human adult and fetal heart specimens
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这项研究得到了荷兰科学研究组织 (NWO) (VENI 016.146.079) 的支持, 还有一个 LUMC 研究金与 AMS, LUMC Bontius Stichting (MJG)。

所有人体组织标本的实验均由莱顿大学医学中心伦理委员会批准, 并符合赫尔辛基宣言。所有步骤都用无菌设备在细胞培养流柜中进行。
1. 筹备工作
<ol><li>在1:1 的比例下, 将 Dulbecco 修饰的鹰培养基 (DMEM 低葡萄糖) 和中 199 (M199) 混合, 制备 EPDC 培养基。添加10% 热灭活胎牛血清 (血清, 热灭活25分钟在56摄氏度), 并补充 100 U/毫升青霉素和100毫克/毫升链霉素。预热的 EPDC 介质在37°c 水浴。</li>
	<li>预热胰蛋白酶 0.25%/EDTA (1:1) 在37°c 水浴。</li>
	<li>用明胶涂水井。将0.1% 的明胶/PBS 添加到每一个井中, 在37摄氏度时至少15分钟孵化出板材。在表 1中总结了所需的单元格区域性板的准则。在电镀细胞之前小心地去除所有液体。</li>
	<li>准备一个10毫米 SB431542 (锑) 的库存溶液, 稀释在亚砜 (谨慎)。使50µL 整除数在锥形底部聚丙烯离心管, 如离心管, 并存储在-20 摄氏度。请注意, 某人整除数只能解冻一次。</li>
</ol>2. 成人和胎儿心脏标本的检索和贮存
<ol><li>在50毫升的15毫升高葡萄糖 DMEM 的外科手术中, 直接储存成人耳部, 含10% 只血清, 100 毫升青霉素, 100 毫克/毫升链霉素, 4 摄氏度。标本一般 3-5 厘米的大小, 可以储存最多48小时后解剖。</li>
	<li>储存胎儿心脏从选择性流产材料获得的 EPDC 培养基在4°c, 24 小时后隔离。对于本协议, 请在 12-22 周之间使用妊娠期样本。请注意, 整个心脏可以用来隔离心外膜。</li>
</ol>3. 心外膜层的隔离
<ol><li>在层流柜中, 用 pbs 准备一个100毫米的细胞培养皿, 并在盖子上放置一个单独滴 (~ 200 µL) 的 pbs。填充15毫升管与5毫升预热 EPDC 培养基。</li>
	<li>将组织放在充满 PBS 的细胞培养皿中。在手术过程中要确保组织经常湿润。</li>
	<li>使用显微镜, 从组织样本中取出尽可能多的心外膜层, 用镊子将其剥离。 注意: 心外膜可以被识别为非常薄, 透明的层紧密附着在心脏的外部 (图 1A)。尽量避免与心外膜脂肪组织和血管的污染, 因为这将阻碍隔离。</li>
	<li>在盖上的 PBS 液滴上收集心外膜组织的部分。</li>
	<li>使用手术刀 (图 1B) 将心外膜组织切成小块 (0.5 毫米3), 或者使用小钳的锋利尖。请注意, 这些片断应该能够通过 P1,000 吸管提示 (步骤 3.6)。</li>
	<li>将1毫升的胰蛋白酶加入到心外膜组织中, 用 P1,000 吸管尖端收集切片和胰蛋白酶。将组织转移到1.5 毫升锥形离心管 (图 1C)。</li>
	<li>在37摄氏度的水浴中孵化出10分钟的管子, 同时晃动在60转每分钟。</li>
	<li>从水浴中取出管子, 用70% 乙醇清洗管子的外部。</li>
	<li>允许组织下沉到管的底部 (图 1D), 并小心地将含有心外膜细胞的上清液转移到含有5毫升 EPDC 培养基的15毫升管中, 以使胰蛋白酶失效。</li>
	<li>用1毫升胰蛋白酶补充锥形离心管中剩余的组织, 并通过轻轻吹打上下混合。</li>
	<li>重复步骤3.7 至3.10 两次。在最后一步, 在总共30分钟的胰蛋白酶孵化后, 将上清和其余的心外膜组织转移到含有 EPDC 培养基的管内。</li>
	<li>当所有细胞以 EPDC 培养基收集时, 通过10毫升的注射器轻轻地将悬浮液通过一个19口径的针头插入一个新的15毫升管, 机械地分离细胞 (图 1E)。 注: 在通过针头通过溶液前, 一定要融汇吹打的悬浮液, 以防止针头被堵塞。</li>
	<li>要进一步离解细胞, 重复步骤3.12 与21口径针。</li>
	<li>将100µm 细胞过滤器放在50毫升管的顶部, 用10毫升吸管将含有心外膜细胞的培养基转移到滤网上, 以除去所有剩余的团簇 (图 1F)。</li>
	<li>通过吹打5毫升 EPDC 培养基将过滤器清洗到过滤器上以收集残余细胞。</li>
	<li>吸管细胞悬浮从50毫升管变成15毫升管。由于细胞下沉到管底部, 在吹打前轻轻摇动溶液。 注意: 虽然这一步是不必要的, 一个较小的管有助于可视化颗粒后离心。</li>
	<li>离心机在室温下为 200 x 克5分钟 (图 1G)。</li>
	<li>在 EPDC 介质 (表 1) 的所需卷中, 移除上清和并用重悬单元格颗粒(图 1H)。 注: 对于胎儿 EPDCs, 直接使用含有10µM ALK5 激酶抑制剂 (EPDC + 锑) 的 EPDC 培养基。在第一段中, 成人细胞在没有某人的情况下可以被电镀。</li>
	<li>板上的细胞悬浮在明胶涂层培养板 (图 1I)。井的大小取决于心外膜组织样本的大小 (表 1)。注意低融合会诱发急诊的发生。</li>
	<li>将单元格放在孵化器中至少48小时, 在37摄氏度, 5% CO2 , 以允许单元格附加到培养板上。</li>
</ol>4. EPDCs 文化
<ol><li>至少每3天补充一次 EPDC。</li>
	<li>使用显微镜至少每3天检查一次细胞。当单元格达到融合时,即, 当培养板完全覆盖单元格时, 通过1:2 表面比的单元格。请注意, 到达融合可能需要 5-10 天。<ol>
<li>使用吸管或吸入系统 (如, 例如) 将玻璃吸管吸出介质。</li>
		<li>通过将 PBS 添加到细胞中, 仔细冲洗细胞。轻轻地旋转盘子, 吸入 PBS。</li>
		<li>在盘子里加入胰蛋白酶。用胰蛋白酶轻轻旋转盘子覆盖所有细胞, 在37摄氏度时将板材孵化1分钟。 注: 尽可能少使用胰蛋白酶 (指示: 200 µL 每井6井板)</li>
		<li>轻敲盘子, 机械地从盘子的底部分离出细胞。使用显微镜目视检查细胞是否已分离。如果没有, 孵化细胞培养板多一分钟。</li>
		<li>将所需的 EPDC + 锑培养基添加到细胞中, 并用重悬吹打向上和向下, 并将细胞悬浮液转移到新的明胶涂层培养板上。 注意: 一般情况下, 细胞可以保持在鹅卵石形态, 直至8。</li>
	</ol>
</li>
</ol>5. 在 EPDCs 中诱导急诊医师
<ol><li>为了诱导急诊医师, 将细胞与胰蛋白酶分离, 将细胞转移到 EPDC + 锑培养基中1:2 表面比的新明胶涂层井, 如4所述, 在37摄氏度、5% CO2上孵育至少24小时。</li>
	<li>检查融合是否为 50-70%, 如果 EPDCs 有鹅卵石形态。 注意: 融合影响 EPDCs 接受急诊医师的能力。</li>
	<li>从细胞中吸取培养基, 用 PBS 仔细冲洗细胞 (见步骤 4.2.1-4.2.2)。</li>
	<li>在 EPDC 培养基中刺激 1 TGFβ3/毫升的细胞, 并将它们放在孵化器中37摄氏度, 5% CO2 5 天。注意, 在刺激过程中, 不需要补充含有 TGFβ3的 EPDC 培养基。</li>
	<li>每天监视单元格。5天后, 接受急诊的细胞由纺锤形形态学 (图 2A) 识别。</li>
	<li>培养纺锤形 EPDCs 根据4描述的方法, 不添加某人或 TGFβ到 EPDC 培养基。请注意, 一般情况下, 主轴形状的 EPDCs 可以培养到20通道。</li>
	<li>用抗体对间充质标志物αSMA 或波形或罗丹明检测 F 肌动蛋白应激纤维 (图 2B) 的形成, 或使用 qRT PCR 技术对急诊患者相关基因进行免疫荧光染色, 验证其发生情况 (例如, WT1, Periostin, Col1A1, αSMA, n-钙黏蛋白, MMP3, 蜗牛, 蛞蝓) (图 2C和表 2)。</li>
</ol>

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作者没有什么可透露的。

在这里, 我们描述了一个详细的协议, 以隔离和培养从人类成年和胎儿心脏的原发性心外膜细胞。这些单元格的广泛描述以前已发布17。我们已经表明, 两种细胞类型可以保持作为上皮鹅卵石样细胞, 当培养与 ALK5 激酶抑制剂 SB431542。在发育和损伤后反应过程中, 急诊急救是心外膜激活的体内的一个组成部分。通过添加 TGFβ, 可以对急诊医师进行研究。重要的是, 我们以前观察到, 当某人被移除时, 胎儿 EPDCs 迅速接受自发急救, 而成年 EPDCs 只在刺激17时接受急诊医师。研究胎儿 EPDCs 的这些过程有助于了解如何在损伤后最佳激活成人心外膜。
所提出的方法依赖于患者的材料, 这是在手术中得到的。因此, 人们可以期待在材料的状态的一些变化, 这是由于病人的变异性或在操作剧场收集材料的速度。这种变异可以解释过程中的差异: 1) 如何容易从心肌解剖心外膜, 2) 黏附的孤立细胞的板, 3) 的能力, 以预防或接受急诊医师, 4) 增殖速度。一般来说, 快速隔离是更好的细胞生存。主要的关键点是从心肌中剥离心外膜。如果心外膜是强烈坚持的基础组织, 15 分钟前治疗心脏组织与胰蛋白酶可以帮助消除心外膜更容易。此外, 胰蛋白酶治疗的功效取决于多种因素, 包括胰蛋白酶活性和组织组成。因此, 如果观察到低产量, 可以调整胰蛋白酶的潜伏期。此外, 如果在向下旋转后没有可见的单元颗粒, 则在运行筛选器之前, 该解决方案可能没有完全分离。在通过注射器或使用额外的小注射器之前, 将溶液混合在一起, 可能有助于分离细胞。应仔细考虑播种 EPDCs 的井径, 使细胞保持其上皮状态。表 1给出了一个指示, 但如果只有一小部分胎儿心脏可以使用, 并且在较小的井中电镀更方便, 则可以偏离本指南。
由于胎儿细胞将立即接受急诊医师没有某人17, 胎儿 EPDCs 应始终与某人镀。然而, 成年 EPDCs 倾向于保持他们的上皮形态学, 因此可以在第一段的前48小时内没有某人被镀, 以促进细胞黏附。在第一个段落以后, 成人和胎儿 EPDCs 连续培养与某人防止自发急诊医师。此外, 低细胞密度是急诊急救的重要诱因。因此, EPDCs 不应培养在50% 融合以下。另一方面, 如果需要急救, 请确保 EPDCs 的种子不太密, 并且保持在70% 融合以下。虽然这个协议使用 TGFβ3, 刺激与 TGFβ1和-2 可能导致急诊医师在 EPDCs 并且 (未发表的观察)。
在本协议中, 细胞直接分裂而不向下旋转, 因为我们在使用离心机时观察到较低的细胞存活率。因此, 在添加含血清的培养基时, 使用低容量的胰蛋白酶来保证其失活是至关重要的。
经过 6-8 通道, EPDCs 与上皮形态学将停止生长或将接受急诊医师自发。因此, 在实验中, 我们用鹅卵石 EPDCs 在通道3和通道6之间。相比之下, EPDCs 与骨髓间质形态学的脆弱程度较低, 可以培养到20通道。然而, 在实验中, 我们从来没有在 10th通道后使用主轴 EPDCs。
由于心外膜位于心脏的外侧, 很容易与底层组织分离, 因此细胞的真实性很明显, 严重污染的几率很低。虽然脂肪组织或血管有时会粘在单独的心外膜层, 但大多数细胞在隔离过程中不会通过过滤器, 否则在 EPDC 细胞培养中无法存活。此外, 如前所述, 使用人体材料提供了一个独特的模型来调查人的心外膜细胞行为.人们知道, 例如, 心外膜脂肪组织不同物种之间, 强调人类心外膜细胞模型的必要性21。
应注意的是, 心脏耳廓是在病人的心脏手术期间获得的, 因此, 在疾病, 使用药物, 年龄和性别的差异, 捐献者可能影响的重现性实验。因此, 应在几个隔离上进行实验, 以获得有效的结果, 并允许得出坚实的结论。此外, 根据研究问题, 人们可以专门选择只使用心外膜的患者的缺血性疾病或患者的非缺血性瓣膜病, 这是预期的行为不同。
提示心房心外膜可能有不同的特点从脑室心外膜2, 从而质疑, 如果心外膜从心房心脏耳廓提供了一个有效的模型的心外膜行为。在这方面, 必须指出, 我们不能发现胎儿心房和心室衍生 EPDCs (未发表的数据) 之间的差异。然而, 使用心外膜从成人心室将是最终的细胞来源, 以验证这一点。然而, 采集成人心室大样本 EPDC 隔离对患者具有很强的侵入性, 因此目前在本院尚不可行。
我们观察到, 某人并不总是足以防止急救, 主要是在胎儿 EPDCs。当胎儿 EPDCs 在某人面前接受急诊医师的时候, 我们将他们排除在实验之外。因此, 用于实验的细胞被选择以保持上皮表型的能力来回应某人。我们推测胎儿细胞在急救过程中已经超出了一定的阈值, 而与某人的抑制无法阻止。
这个心外膜细胞模型有几个应用, 因为开发和成人心外膜可以被调查。在我们的实验室中, 我们专注于改善心脏损伤后的心外膜再生反应。成人 EPDCs 可用于检测诱发急诊的化合物, 目的是寻找一种潜在的治疗药物, 改善心外膜的再生反应。此外, 还可以测量 EPDCs 分泌的因素, 以理解分泌信号对 (再) 生成心肌的影响。此外, 由于我们观察到胎儿 EPDCs 比成人 EPDCs 更容易接受急诊手术, 我们调查胎儿和成人 EPDCs 之间的差异。确定增加胎儿活化的基础机制, 可以为改善成人心脏的心外膜活化提供线索。

心外膜, 一个单细胞上皮层, 信封心脏, 是至关重要的心脏发育和修复 (在史密特et 。1). 发育, 心外膜产生于 proepicardial 器官, 一个小结构位于发展的心脏基地。在发展天 E9.5 在老鼠和4星期以后构想在人, 细胞开始从这个花椰菜结构迁移并且包括开发的心肌2。一旦形成单个上皮细胞层, 心外膜细胞的一部分就会进行上皮间质的转移 (急诊室)。在急诊室, 细胞失去了上皮特征, 如细胞粘连, 并获得一个间充质表型, 使他们有能力迁移到发展中的心肌。经形成的心外膜衍生细胞 (EPDCs) 可分化为多种心肌细胞类型, 包括成纤维细胞、平滑肌细胞和潜在心肌细胞和内皮干细胞3, 虽然后两个细胞的分化人口仍需进行辩论 (在史密特等) 中进行审查。4). 此外, 心外膜提供了指导分泌信号的心肌调节其生长和血管化5,6,7,8。多项研究表明, 心外膜形成受损导致心肌发育缺陷9,10, 血管11, 传导系统12, 强调至关重要的心外膜对心脏形成的贡献。
虽然在成人心脏心外膜是存在作为休眠层数, 它在缺血13以后变得激活。心外膜重新激活损伤后概括为心脏发育, 包括增殖和急诊14所描述的几个过程, 尽管效率较低。有趣的是, 虽然确切的机制还没有完全理解, 但通过治疗可以改善心外膜对修复的贡献,例如, 胸腺素β415或修改 VEGF-mRNA16, 从而改善心脏心肌梗死后功能。因此, 心外膜被认为是一个有趣的细胞来源, 以加强内源性修复受伤的心脏。
心脏发育的机制经常概括在损伤期间, 虽然以一种较不有效的方式。为了寻找心外膜激活剂, 我们可以确定和比较胎儿和成人心外膜的全部容量, 这是至关重要的。此外, 从治疗的角度来看, 重要的是, 除了动物实验外, 我们还要对人类心外膜的反应提供知识。在这里, 我们描述了一种方法, 以分离和培养人成体和胎儿心外膜衍生细胞 (EPDCs) 在上皮细胞样的形态学和诱发急诊。本模型旨在探讨和比较成人和胎儿心外膜细胞的行为。
该协议的主要优点是使用了人的心外膜材料, 尚未深入研究。重要的是, 所描述的隔离和细胞培养协议提供了一个统一的方法来获得胎儿和成人鹅卵石 EPDCs, 使这两个细胞来源的直接比较。此外, 由于心外膜是根据其位置隔离的, 因此确保单元格实际上是 epicardially 派生的17。
虽然以前已经建立了人类的 EPDC 隔离方法, 但它们大多依赖于将心脏或心外膜组织镀到细胞培养皿18,19的生长协议。这种方法可以专门为部分丧失其上皮表型的细胞进行移植, 更容易接受自发的急诊急救。在当前的协议中, 心外膜首先被处理成一个单一的细胞溶液, 允许隔离的 EPDCs 保持其上皮状态。因此, 该方法提供了一个固体的体外模型来研究心外膜急诊。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium + GlutaMAX</td><td>Gibco</td><td>21885-025</td><td></td></tr><tr><td>Medium 199&amp;nbsp;</td><td>Gibco</td><td>31150-022</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>Trypsin 0.25%</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>Penicillin G sodium salt</td><td>Roth</td><td>HP48</td><td></td></tr><tr><td>Streptomycin sulphate</td><td>Roth</td><td>HP66</td><td></td></tr><tr><td>Trypsin 1:250 from bovine pancreas</td><td>Serva</td><td>37289</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E4884</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Culture plates 6 well</td><td>Greiner bio-one</td><td>657160</td><td></td></tr><tr><td>Culture plates 12 well</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>Culture plates 24 well</td><td>Greiner bio-one</td><td>662160</td><td></td></tr><tr><td>SB 431542</td><td>Tocris</td><td>1614</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Merck</td><td>102931</td><td></td></tr><tr><td>100-1000&amp;micro;L Filtered Pipet Tips</td><td>Corning</td><td>4809</td><td></td></tr><tr><td>10-ml pipet</td><td>Greiner bio-one</td><td>607180</td><td></td></tr><tr><td>5-ml pipet</td><td>Greiner bio-one</td><td>606180</td><td></td></tr><tr><td>Cell culture dish 100/20 mm</td><td>Greiner bio-one</td><td>664160</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010056</td><td>Or home-made and sterilized</td></tr><tr><td>Eppendorf tubes 1.5 mL</td><td>Eppendorf</td><td>0030120086</td><td></td></tr><tr><td>15-ml centrifuge tubes</td><td>Greiner bio-one</td><td>188271</td><td></td></tr><tr><td>50-ml centrifuge tubes</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>10 mL Syringe</td><td>Becton Dickinson</td><td>305959</td><td></td></tr><tr><td>Needles 19 Gauge</td><td>Becton Dickinson</td><td>301700</td><td></td></tr><tr><td>Needles 21 Gauge</td><td>Becton Dickinson</td><td>304432</td><td></td></tr><tr><td>EASYstrainer Cell Sieves, 100 &amp;micro;m</td><td>Greiner bio-one</td><td>542000</td><td></td></tr><tr><td>TGF&amp;beta;3&amp;nbsp;</td><td>R&amp;amp;D systems</td><td>243-B3</td><td></td></tr><tr><td>Monoclonal Anti-Actin, &amp;alpha;-Smooth Muscle</td><td>Sigma</td><td>A2547&amp;nbsp;</td><td></td></tr><tr><td>Anti-Mouse Alexa Fluor 555</td><td>Invitrogen</td><td>A31570</td><td></td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Invitrogen</td><td>A12379</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Pipet P1,000</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>Pipet controller</td><td>Integra</td><td>155 015</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>Inverted Light Microscope</td><td>Olympus</td><td>CK2</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Waterbath</td><td>GFL</td><td>1083</td><td></td></tr></tbody></table>

the isolation and culture of primary epicardial cells derived from human adult and fetal heart specimens

心外膜是一种覆盖心肌的上皮细胞层, 在心脏发育过程中, 以及在缺血性损伤后心脏的修复反应中具有重要作用。当激活时, 心外膜细胞经历了称为上皮间质转移的过程, 为再生心肌提供细胞。此外, 心外膜通过分泌基本的分泌因子而做出贡献。为了充分了解心外膜的再生潜能, 需要一个人类细胞模型。在这里, 我们概述了一个新的细胞培养模型, 从人类成年和胎儿心脏组织中提取原发性心外膜衍生细胞 (EPDCs)。为了分离 EPDCs, 心外膜从心脏标本的外部进行解剖, 并处理成单个细胞悬浮。其次, EPDCs 被镀和培养在 EPDC 培养基含有5激酶抑制剂 SB431542 保持其上皮表型。TGFβ刺激诱发急诊。这种方法首次能够在控制的环境中对人心外膜急诊的过程进行研究, 并有助于在 EPDCs 的 secretome 中获得更多的洞察力, 从而有助于心脏再生。此外, 这种统一的方法可以直接比较成人和胎儿的心外膜行为。

在这里, 我们概述了一个简单的协议, 以隔离 EPDCs 从人类成人和胎儿心脏组织 (图 1)。此协议利用心外膜在心脏外部的易于访问的位置 (图 1A)。解剖后的心耳廓染色表明, WT1+ 心外膜被删除, 而基础心外膜下细胞外基质和心肌组织保持不变 (图1J)。以前已经进行了广泛的描述, 表明 EPDCs 表达的心外膜标记,例如, ALDH1A2, TBX18, KRT8, KRT19 和缺乏其他心脏常驻细胞类型的表达,例如, PECAM1, ISL1, CD34 和 TNNT2。17
成人 (图 1K) 和胎儿 (图 1L) EPDCs 在 ALK5 激酶抑制剂的存在下培养 SB431542 显示鹅卵石形态学。然而, 根据捐赠者和文化条件, 胎儿 EPDCs 可以接受急诊医师, 尽管有某人在场。这可能导致鹅卵石单元格中的纺锤形细胞 (参见图 1L中的箭头)。请注意, 从胎儿组织中分离出鹅卵石状细胞并不总是可行的, 可能导致大部分纺锤形细胞的衍生。由于这些细胞生长更快, 他们将迅速 overgrow 其他细胞类型。
在成人和胎儿细胞中, 可通过 TGFβ320孵化5天诱发急诊医师, 这是由纺锤形细胞的明显形态学转变 (图 2A) 所证明的。与未经治疗的控制 ("空") 显示, 胎儿 EPDCs 将接受自发的急诊手术后, 移除某人, 而成年 EPDCs 将只接受急诊医师的刺激与 TGFβ3。为验证急诊医师, EPDCs 是 immunostained 与阿尔法平滑肌肌动蛋白 (αSMA), 一个间充质标记 (图 2B)。此外, 在成人 EPDCs 中证实了急诊医师使用 qRT PCR 显示下调的心外膜标记 WT1 和上调的间充质标记 POSTN, αSMA, 胶原蛋白 1A1, MMP3 和 n-钙黏蛋白 (图 2C)。关于成人和胎儿心外膜急诊的综合实验发表在17之前。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组织</td>
			<td>井型</td>
			<td>细胞生长区 (cm2)</td>
			<td>介质容积 (毫升)</td>
			<td>增加某人</td>
		</tr>
<tr>
<td>胎儿</td>
			<td>24</td>
			<td>1。9</td>
			<td>0。5</td>
			<td>直接</td>
		</tr>
<tr>
<td>成人小 (< 4 厘米)</td>
			<td>12</td>
			<td>3。8</td>
			<td>1</td>
			<td>1st通道后</td>
		</tr>
<tr>
<td>成人大</td>
			<td rowspan="2">6</td>
			<td rowspan="2">9。6</td>
			<td rowspan="2">2</td>
			<td rowspan="2">1st通道后</td>
		</tr>
<tr>
<td>(> 4 厘米)</td>
		</tr>
</tbody></table>表 1: 选择适当的细胞培养板的指南.所需的井径取决于所分离的心外膜细胞的数量。这些指南可以作为一个经验法则来选择合适的细胞培养板。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>基因</td>
			<td>序列</td>
		</tr>
<tr>
<td>WT1 转发</td>
			<td>GAA TGC ATG ACC TG</td>
		</tr>
<tr>
<td>WT1 反转</td>
			<td>GGC 的 GC</td>
		</tr>
<tr>
<td>n-钙黏素向前</td>
			<td>ACC 和瑞声 AGC</td>
		</tr>
<tr>
<td>n--钙黏蛋白反转</td>
			<td>AGG 的抗体</td>
		</tr>
<tr>
<td>POSTN 转发</td>
			<td>GGA GGC AAA 级 GT</td>
		</tr>
<tr>
<td>POSTN 反转</td>
			<td>GGC TGA GGA AGG TGC TAA 股份公司</td>
		</tr>
<tr>
<td>SMA 向前</td>
			<td>CCG GGA GAA</td>
		</tr>
<tr>
<td>SMA 反转</td>
			<td>GAA GGA 海湾合作委员会动漫委员会 AG</td>
		</tr>
<tr>
<td>MMP3 转发</td>
			<td>TGG ATG CCG 猫 ATG AAG</td>
		</tr>
<tr>
<td>MMP3 反转</td>
			<td>TGG AAA 级</td>
		</tr>
<tr>
<td>COL1A1 转发</td>
			<td>GAA GAA 猫 CAG</td>
		</tr>
<tr>
<td>COL1A1 反转</td>
			<td>CGC 猫法 GGG</td>
		</tr>
<tr>
<td>GAPDH 转发</td>
			<td>GCT ATC C</td>
		</tr>
<tr>
<td>GAPDH 反转</td>
			<td>海合会民航局</td>
		</tr>
<tr>
<td>B2M 转发</td>
			<td>CCC CT</td>
		</tr>
<tr>
<td>B2M 反转</td>
			<td>GCT 交 ATG</td>
		</tr>
</tbody></table>表 2: 用于验证 EPDCs 中的急诊医师的底漆序列.qRT PCR 的正向和反向引物序列, 用于测定成人 EPDCs 中几种急诊相关基因的表达。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57370/57370fig1.jpg"> 图 1: 对心外膜派生单元的隔离 (EPDCs).(A) 描述的是从人的心脏去除的成人耳廓与一个稀薄的外层膜层, 心外膜, 被去除。黑色箭头指向心外膜。(B I)EPDCs 隔离方法的可视化表示。黑色箭头指向单元格颗粒。(J) 对心外膜的无解剖的心耳廓进行免疫荧光染色。缩放栏:50 µm. (K) 两个不同的成人 EPDC 隔离的代表性图片 (L) 两个不同的胎儿 EPDC 隔离培养的代表图片黑色箭头表示胎儿间充质样细胞EPDC 文化。刻度条: 200 µm.<a href="https://cloudflare.jove.com/files/ftp_upload/57370/57370fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57370/57370fig2.jpg"> 图 2: 验证成人和胎儿心外膜衍生细胞 (EPDCs) 中的急诊医师.成人和胎儿 EPDCs 培养与某人, 不治疗 (空) 或刺激与 TGFβ3 5 天。(A) 代表明亮的字段图片。缩放栏: 200 µm (B) 染色 DAPI 和αSMA。刻度条: 100 µm (C) 与急诊医师相关基因的 mRNA 水平, 用 qRT PCR 法测定成人 EPDCs。测量值被规范化为 GAPDH 和 B2M 表达式。值被描述为平均值 + SD 2 ^-ΔΔct (n = 2)。缩写: WT1:Wilms ' 肿瘤 1, MMP3: 基质金属蛋白酶 3, POSTN: Periostin, αSMA: 阿尔法平滑肌肌动蛋白, Col1A1:Collagen 1A1。<a href="https://cloudflare.jove.com/files/ftp_upload/57370/57370fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens at JoVE.com

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

心外膜在心脏的发育和修复中起着至关重要的作用, 它为心肌壁提供细胞和生长因子。在这里, 我们描述了一种培养人原发性心外膜细胞的方法, 能够对它们的发育和成人特征进行研究和比较。

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

the-isolation-culture-primary-epicardial-cells-derived-from-human

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.7K 次观看.  Leiden University Medical Center. 心外膜在心脏的发育和修复中起着至关重要的作用, 它为心肌壁提供细胞和生长因子。在这里, 我们描述了一种培养人原发性心外膜细胞的方法, 能够对它们的发育和成人特征进行研究和比较。

视频: The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

心外膜生长培养测定和<em&gt;离体</em&gt;心外膜源性细胞迁移的评估

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

科研

发育生物学

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

心外膜细胞小鼠胚胎心脏的体外培养

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

工程学

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

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

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 ...

医学

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. ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

从小鼠心脏中分离主黑素细胞样细胞

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

生物学

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

从固定心脏分离心肌细胞进行免疫细胞化学和倍体分析

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

人体心室心肌细胞与颤音-切切心肌切片分离

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

人成体和胎心标本中原发性心外膜细胞的分离培养

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心外膜生长培养测定和