这项工作主要是由5-UO1-ES016045支持与1-R21-ES019498和R44-ES021116部分支持。 DMW由NIEHS的训练津贴在环境毒理学T32-ES007020支持。设备由中心环境健康科学P30-ES002109提供。

1，制备芯片<ol style="margin-left: 40px;"><li>从包和地方除去芯片凝胶成一良好的矩形板，凝胶膜的一面朝下。 </li><li>在25ml的1×PBS中进行15分钟洗凝胶，重复两次。 </li><li>对玻璃板与凝胶的相应标签方向设置的凝胶。 </li><li>轻轻按下一个倒置的无底的96孔板上胶;确保无底孔板的所有孔都在凝胶的区域之内。 </li><li>使用在板的每一侧上1.5&#39;长尾夹固定夹紧用玻璃。 </li><li>吸去多余的PBS缓冲液中的各孔中。 </li></ol> 2，细胞装载<ol style="margin-left: 40px;"><li>制备细胞悬液： <ol><li>对于悬浮细胞系，稀释细胞悬液，在媒体上获得10 5 -10 6个细胞/ ml的终浓度。 </li><li>对于贴壁细胞系，按照正常的拆卸/胰蛋白酶协议和Dilute分离的细胞中的介质，以10 5 -10 6个细胞/ ml的终浓度。如果细胞聚集，通过细胞悬浮液中的细胞过滤器，以获得单细胞悬浮液。 </li></ol></li><li>加入100微升细胞悬浮液至96孔板的各孔中。 </li><li>盖板与凝胶膜，以防止介质的蒸发。 </li><li>让细胞装载至少30分钟在37℃培养箱中培养。 </li><li>从孵化器和吸媒体从每个孔中取出芯片。 </li><li>除去粘结剂的剪辑和无底的96孔板;保持与玻璃板的芯片以一角度和用1×PBS轻轻冲洗以除去过量的细胞在琼脂糖。 </li><li>用明视场显微镜4倍物镜检查电池负载。如果负荷不足的观察，从步骤2.1重复。 </li><li>放置在平整的表面（实验台）装芯片。覆盖芯片，用1％低熔点（LMP）琼脂糖即预加热至37℃。使用约2̵需要为每个芯片3毫升LMP琼脂糖; 1。 </li><li>允许覆盖到第一固化在室温下进行3分钟，然后转移到4℃冰箱中5分钟，使完整的凝胶化。 </li></ol> 3，计量和维修注：研究基线DNA损伤程度，跳到步骤4。 <ol style="margin-left: 40px;"><li>小心地将芯片（从前面的夹紧创建）一个新的无底的96孔板的孔中的孔中。再按下无底的96孔板的顶部和可靠的夹紧与粘合剂剪辑。 </li><li>添加100μl的所需剂量的化学物质到96孔板的各孔中;在冰上或4℃冰箱中所需的时间量进行加药/治疗。 </li><li>经过加药，每个吸化学品以及使用1X PBS洗去残留的化学物质。如果直接损害在这里学习，请执行步骤4。 </li><li>学习修动力学，使细胞中的芯片，以修复媒体在37℃。裂解（继续执行步骤4）在特定的时间点。 注：修复可以片上进行了无底的96孔板附，或芯片可以除去无底的96孔板中后切割成单独的部分。 </li></ol> 4，裂解<ol style="margin-left: 40px;"><li>要执行碱性彗星试验： <ol><li>使通过添加1％的Triton X-100至碱性裂解溶液（2.5 M氯化钠，100毫米的Na 2 EDTA，10毫摩尔Tris，pH值为10）的工作碱性裂解缓冲液中。准备大约25毫升工作裂解液为每个芯片。 </li><li>预冷工作碱性裂解缓冲液中于4℃。 </li><li>淹没芯片在阴凉工作碱裂解缓冲液，并允许溶解于4℃冰箱中进行o / N。 </li></ol></li><li>要执行中性彗星实验： <ol><li>使通过添加1％的Triton X-100和10％的DMSO中，中性溶胞溶液（2.5 M氯化钠，100毫米的Na 2 EDTA，10个中性裂解缓冲液毫米的Tris，1％的N -Lauroylsarcosine，pH值9.5）。准备大约25毫升工作裂解液每个芯片。 </li><li>预温工作的空档裂解液在43℃。 </li><li>片浸入温水工作空档裂解缓冲液，并允许溶解于43℃培养箱进行o / N。 </li></ol></li></ol> 5，电泳<ol style="margin-left: 40px;"><li>要执行碱性彗星试验： <ol><li>除去裂解缓冲液和快速冲洗芯片用1×PBS。 </li><li>安全芯片中的电泳室用凝胶膜面朝下使用双面胶带。 </li><li>把室到4℃冷室。 </li><li>填充用冷水碱性电泳缓冲液（0.3 M氢氧化钠，1毫米的Na 2 EDTA），以刚刚覆盖胶的水平室。 </li><li>允许用于碱性开卷40分钟。 </li><li>在1伏/厘米和300毫安在冷室中30分钟，跑电泳。调整了茶的电泳缓冲液的体积MBER以实现期望的运行电流。 </li></ol></li><li>要执行中性彗星实验： <ol><li>除去裂解缓冲液和冲洗芯片与中性电泳缓冲液（2毫米的Na 2 EDTA，90毫摩尔Tris，90mM的硼酸，pH值8.5）为2×15分钟，在4℃下。 </li><li>安全芯片中的电泳室用凝胶膜面朝下使用双面胶带。 </li><li>把室到4℃冷室。 </li><li>室内冷中性电泳缓冲液（2毫米的Na 2 EDTA，90毫米的Tris，90 mM的硼酸，pH值8.5），以填补刚刚覆盖胶的水平。 </li><li>使凝胶坐在冷室中静置60分钟。 </li><li>跑电泳在0.6伏/厘米和6毫安在冷室中60分钟。调整电泳缓冲液的体积，在室内达到预期的运行电流。 </li></ol></li></ol> 6，荧光成像<ol style="margin-left: 40px;"><li>从山坳电泳室中取出芯片ð室。 </li><li>中和中和缓冲液（0.4摩尔Tris，pH7.5）中的凝胶在4℃冰箱中2×15分钟。 </li><li>染色与选择的DNA荧光染色的芯片（ 即 SYBR金，溴化乙锭）在工厂推荐的程序。图片使用荧光显微镜。 </li></ol> 7，数据分析分析用标准彗星试验软件彗星图像，如Komet公司5.5。

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+alkaline+comet+assay+for+measuring+DNA+repair+capacity+in+human+populations.">A modified alkaline comet assay for measuring DNA repair capacity in human populations.</a> Radiation research. 169 (1), 110-121 (2008).</li><li>Marcon, F., Andreoli, C., Rossi, S., Verdina, A., Galati, R., Crebelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+individual+sensitivity+to+ionizing+radiation+and+DNA+repair+efficiency+in+a+healthy+population.">Assessment of individual sensitivity to ionizing radiation and DNA repair efficiency in a healthy population.</a> Mutation research. 541 (1-2), 1-8 (2003).</li></ol>

The CometChip has been licensed to Trevigen, Inc. based upon a patent submission that includes Wood, Weingeist, Bhatia and Engelward.

在环境中，并在人类细胞中普遍存在的基因毒素产生不可避免的应力和损坏的细胞DNA。事实上，据估计，有存在于人体细胞在稳定状态18的DNA损伤的1000个。了解损伤的敏感性和修复动力学人类不仅带来洞察力的基础研究，也有利于药物的开发。讽刺的是，尽管DNA的损伤，促进癌症的功能，D​​NA损伤剂被用于以非常高的水平，以治疗癌症，使左右的DNA损伤知识和修复相关的个性化药物。该CometChip是利用微细加工的做法彻底改变长期存在的DNA损伤试验了一种新的研究设备。建立在传统彗星测定法的相同的原理，该芯片提供显著更高的吞吐量和更好的再现性。我们在这里描述了采用该芯片的方法。为了明确其实用性和耐用性，我们的结果显示来回米几个实验，其中该芯片已被用于评估诱导的几种不同的DNA损伤剂的损害，并且它已被用于评价DNA修复。总之，这里给出的结果提供有用的信息，使用该芯片，并展示其广泛的适用性研究DNA损伤与修复研究。 一个在该协议中最重要的步骤是将完成有效的小区负载。许多细胞系进行了检验该系统上，包括悬浮和贴壁细胞。中效率取决于许多变量，例如细胞类型，细胞大小，加载浓度和时间。悬浮细胞系，例如淋巴细胞样细胞是最简单的工作。将细胞悬浮液的浓度可以在10 4〜10 6个细胞变化每96孔和装载通常在15-30分钟完成。更高的细胞密度有利于负载，并缩短所需的时间为细胞沉降到微孔的量。 加载参数的贴壁细胞可以不同于悬浮细胞。细胞系，例如中国仓鼠卵巢（CHO）细胞中，被发现有相似的装载效率为悬浮细胞（以下胰蛋白酶消化），因此使用类似的载荷条件。其它类型的细胞，如肿瘤细胞，很容易形成细胞聚集体悬浮液中，需要额外的处理。通过一个单一的细胞过滤器传递的细胞悬浮液，并加入胰蛋白酶的悬浮介质可在装载期间抑制细胞簇的形成。最后，在加载时间上，需要用于捕获粘附细胞系在微孔中可变化从20分钟至1小时的时间。其结果是，它建议用户进行试验实验进行到进一步调查之前确定最佳负载条件。加载功效能明视场显微镜下可视化或更准确地揭示了染色的细胞封装用DAPI或其它DNA在荧光显微镜下评估之前的污渍。 此方法与芯片的一个主要优点是灵活性。首先，研究人员不局限于一个特定的工作细胞浓度，因为过量的细胞洗去，和微阵列可以确保均匀的彗星密度。第二，微孔可制成具有可变的尺寸，以适应不同大小的细胞类型。在多个细胞被捕获在一个单一的微孔的情况下，多细胞彗星是自校准，并产生一致的结果相比，单细胞彗星6,7。在微阵列图案化细胞的另一个优点是，所有的彗星，然后在固定的位置相同的焦平面中，从而降低噪声由于聚焦的彗星和促进自动成像和分析。由CometChip提供的吞吐量和灵敏度的显著改进使该测定的应用对于大规模的研究。两者合计，该芯片提供SA有价值的工具，DNA损伤评估研究，毒理学，临床医生和流行病学家。 当彗星试验是著名的检测范围广泛的DNA损伤，其出色的灵敏度，此法还患有特异性低。使用该协议大大提高了测定的重现性和吞吐量，但并不表现出进步特异性。然而，复用其它方法测定已被报道为有效地实现更高的特异性。一个例子是加入纯化病变特异性的酶。这些酶可以转换，否则无法检测的基础病变为检测链断裂和碱基网站1,19-21。一种广泛使用的例子是修内切酶formamidopyrimidine-DNA糖基化酶（FPG），这表明DNA氧化修饰19,22。因为酶消化步骤是细胞裂解之后施加，该系统可以处理的相同方式无需改动协议SA传统的琼脂糖幻灯片。虽然详细的协议不在此描述，这种方法已经适应了很多传统彗星试验用户，在过去的协议，可以很容易地找到。在以前的出版物中，我们用这种方法来鉴别荧光光损伤22。 该方法的主要优点是它提供了可打开的门，以前几乎是不可能的研究吞吐量。处理96个样品在相同的平台上的能力，不仅减少了劳力和时间，而且还降低了实验的噪声，并大大提高了重现性。除样本的变化比传统的滑动到滑动变化，它可以是2倍，比与该芯片6,7中观察到的变异更高显著低。降低了噪音也会导致更高的灵敏度，能够检测的样品中更细微的差别。由于该设备采用了STAndard 96孔格式中，它是用一般的科研设备兼容和HTS技术。考虑的一项研究，需要100个样品的评价，假定一个平均研究者可以处理〜30的载玻片上的数天的过程中。由于每个样品需一式三份进行，大约需要一个月的时间完成这项研究，这也将不可避免地遭受样品来样变化。相反，处理100的条件下，各一式三份，用该芯片只需要几个板块，可以在三天内完成。吞吐量这一重大改善将打开大门，这在以前是不可能实现的研究。 这里介绍的协议描述了与基本程序，用于执行标准彗星试验使用CometChip平台所需的改性步骤。以同时测量固有的DNA损伤水平和随后的修复反应的能力，该测定是高度相关各种生物及临床应用。在对基因组的特定化学品的接触所产生的影响有利于疾病的预防和治疗。此外，还存在在确定变体DNA损伤的敏感性和个人23,24间修复能力显著兴趣。此技术提供了所需的这种大规模的研究的吞吐量。知识的个体间差异是识别易感人群和设计个性化治疗或防治策略的关键。总之，这里介绍的技术提供了高吞吐量，客观和定量DNA损伤分析平台，有可能成为在不久的将来，一个标准方法的潜力。

单细胞凝胶电泳（SCGE）试验（又名彗星试验）是一种广泛使用的技术为DNA损伤，包括基本病变，无碱基位点，单链断裂，双链断裂，交联和碱敏感的广谱的量化网站。该法的基本原则是，受损的DNA比未受损的DNA因碎片和超螺旋结构的丧失迁移更容易。迁移的程度是正比于DNA损伤的量，并且可以使用荧光显微镜观察。图像捕获和个人彗星状DNA的强度分布分析，然后传送参数的测量，如尾长，尾矩，并％的DNA在尾巴露出一个小区1-4内的DNA损伤的程度。 目前，有彗星试验的两种常用的版本：1）碱性彗星试验和B）中性彗星试验。碱性彗星试验是应用最广使用的版本，它采用了高pH缓冲液电泳之前放松的DNA，从而检测出所有类型链断裂，碱敏感的部位。中性彗星实验，在另一方面，是不太老练的，因为它使用中性缓冲液，以保持双螺旋和检测只双链断裂2,5。化学和物理因素诱导DNA双链断裂，办学团体在音乐会中创建，并且被形成在高得多的水平（ 例如 ，γIR诱导办学团体是要比双链断裂更常见的顺序）。对大多数DNA损伤风险，DNA双链断裂是比其他的类的DNA损伤更频繁，并且因此更难以检测。我们已经证明，在以前的出版物两个版本的检测窗。6,7 虽然彗星试验已常规用于DNA损伤与修复和遗传毒性试验1,2,8-15基础研究，该试验有报道其重现性差，由于样本用于─样，人对人及实验室到实验室的变异性。另外，该测定是低的吞吐量，这部分是因为图像采集和数据分析是费力的。一起，这些限制有助于其在大规模的研究相对低的接受。通过集成工程技术和原理，在CometChip带来了解决方案的吞吐量是与传统的彗星试验6,7相关的问题和矛盾。简单地说，打造芯片，模具使用光刻制造microposts直径小到一个单细胞的微细加工。模具然后用于盖印在熔融琼脂糖，导致微孔的凝胶固化后的数组。芯片正在开发为研究人员提供可变大小的孔，以不同类型的细胞相容。加载该芯片，将细胞在介质静置入井由重力;过量的细胞被冲走。然后被困细胞封装我们荷兰国际集团的薄层低熔点琼脂糖，和一个无底的96孔板中，然后夹紧在凝胶表面以产生96微孔，每个都有几百个微孔的在其底表面上。 这个协议描述了用于与该芯片的实验，其包括凝胶制剂，小区负载，计量和修复，细胞溶解，电泳和荧光成像的一般程序。我们证明的剂量响应实验以暴露已知DNA损伤剂，使用碱性和测定的分别中性版本的淋巴母细胞的两个例子。两个修复实验还示出以说明如何修复反应后曝光可以使用该系统来评估。

<table border="0" cellpadding="0" cellspacing="0" width="797">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Name of Reagent/Material</td>
			<td style="width:129px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td style="width:373px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">UltraPure LMP Agarose (low melting point)</td>
			<td style="width:129px;">Invitrogen</td>
			<td style="width:101px;">16520</td>
			<td style="width:373px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Dulbecco&#39;s phosphate buffered saline (PBS) 1x</td>
			<td style="width:129px;">Gibco by life technologies</td>
			<td style="width:101px;">14190</td>
			<td style="width:373px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Crystalline NaCl</td>
			<td style="width:129px;">Macron Chemicals</td>
			<td style="width:101px;">7581-06</td>
			<td style="width:373px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Crystalline Na2EDTA</td>
			<td style="width:129px;">Macron Chemicals</td>
			<td style="width:101px;">4931-04</td>
			<td style="width:373px;">Multiple suppliers, Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Crystalline Tris (base)</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:101px;">T1503</td>
			<td style="width:373px;">Multiple suppliers, Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Crystalline Tris (acid)</td>
			<td style="width:129px;">J.T.Baker</td>
			<td style="width:101px;">4106</td>
			<td style="width:373px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Crystalline N-lauroylsarcosine</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:101px;">L9150</td>
			<td style="width:373px;">Multiple suppliers, Highly toxic by inhalation, Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Triton X-100</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:101px;">X100</td>
			<td style="width:373px;">Multiple suppliers, Harmful by ingestion., Irritant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:101px;">D4540</td>
			<td style="width:373px;">Multiple suppliers, Combustible Liquid, Target Organ Effect</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Crystalline NaOH</td>
			<td style="width:129px;">Macron Chemicals</td>
			<td style="width:101px;">7708-06</td>
			<td style="width:373px;">Multiple suppliers, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Hydrochloric acid</td>
			<td style="width:129px;">VWR</td>
			<td style="width:101px;">BDH3871</td>
			<td style="width:373px;">Multiple suppliers, Corrosive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Crystalline boric acid</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:101px;">B6768</td>
			<td style="width:373px;">Multiple suppliers, Target Organ Effect, Teratogen, Reproductive hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:129px;"></td>
			<td style="width:101px;"></td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Name of Equipments</td>
			<td style="width:129px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td style="width:373px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">CometChip</td>
			<td style="width:129px;">Trevigen</td>
			<td style="width:101px;"></td>
			<td style="width:373px;">Under development for distribution, contact Trevigen directly to obtain materials necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">1-well dish</td>
			<td style="width:129px;">Thomas Scientific</td>
			<td style="width:101px;">73521-420</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Bottomless 96-wel plate</td>
			<td style="width:129px;">VWR</td>
			<td style="width:101px;">655000-06</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">1.5&#8243; binder clips</td>
			<td style="width:129px;">Staples</td>
			<td style="width:101px;"></td>
			<td style="width:373px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Glass plate</td>
			<td style="width:129px;">Tag Hardware</td>
			<td style="width:101px;"></td>
			<td style="width:373px;">Customized to size of 96-well plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Owl Horizontal Electrophoresis System</td>
			<td style="width:129px;">VWR</td>
			<td style="width:101px;">27372-131</td>
			<td style="width:373px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">PowerPac Basic Power Supply</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:101px;">164-5050</td>
			<td style="width:373px;">Multiple suppliers</td>
		</tr>
	</tbody>
</table>

cometchip: a high-throughput 96-well platform for measuring dna damage in microarrayed human cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

在这第一个例子中，我们描述了使用H 2 O 2暴露后量化在人淋巴母细胞中的DNA损伤的初始水平的氧化损伤的方法。为了评估H 2 O 2诱导的基础性病变和单链断裂，碱彗星条件被使用。加载完成后和细胞已封装的琼脂糖覆盖，无底的96孔板中被按压到表面上，以创建该芯片上的96的室中。每个96孔的可服用不同类型或化学物质的浓度。这里四种不同剂量的H 2 O 2被用于和1x PBS用作阴性对照。排列彗星的代表性图像示于图1A，其中未处理的（顶部），50微米（中间）和100μM的（底部）的H 2 O 2 -exposed样品表现出不同程度的彗星尾巴。彗星图像分析可以perfor使用市售的软件，该软件一般量化基础上的每个彗尾的总荧光强度和迁移距离的损伤程度MED。测量是在50至100彗星每条件和中值（无论％尾部DNA或尾长度）通常进行的计算。 图1B示出的比例尾DNA的响应曲线，从暴露于四种不同浓度的H TK6淋巴母细胞彗星分析2 O 2。间独立实验的一致性表明在评估来自不同水平的细胞中的化学处理DNA损伤反应了这种方法的有效性。 以了解样品中的变化（ 例如 ，微孔中），并且在同一实验的重复中，跨样品和间试验变异分别绘制在图2A和图2B。的96瓦特范围内的每个实验中，每孔埃尔芯片相当于一个不同的样品，因此油井到井的变化揭示了该装置的跨样本的变化。这里，在12个孔的96孔芯片的装载TK6细胞用相同剂量的H 2 O 2和处理后立即溶解。在并行进行所有的其他实验步骤和从各微孔至少50彗星的位数分别绘于图2A中 。中位数的平均值是在尾部77.53％的DNA，标准偏差是3.99％。从这些数据，我们计算该样品间变异系数为5.2％，这是几个比传统的测定倍低，这表明该测定6,16的改进的健壮性。要了解该法的可重复性，我们暴露TK6细胞在75微米H 2 O 2芯片，并分析DNA损伤的直接程度。该实验重复了每个实验的6倍和数据被绘制在F中igure 2B。在相同的实验条件下，我们得到的结果范围从41.76％到57.87％，显示为图中的灰色条。六个重复的平均值是49.57％，而只有2.04％，平均值的标准误差。重复中，低的变化意味着，这种新型装置上执行的彗星试验是高度重复性。 来评价修复动力学，允许细胞治疗后修复其DNA在新鲜培养基中。在不同时间点裂解微孔揭示了在DNA损伤随时间的变化。一个例子示于图3，在该实验中，TK6细胞首先封装在芯片上，以50μM的H 2 O 2处理后，使之修复多达60分钟后曝光。分别将细胞溶解，在0，20，45和60分钟。代表彗星图像在不同时间点示于图3A中 ，并在DNA损伤水平随时间的变化绘制在图3B。这些数据表明，几乎所有的伤害是在45分钟后ħ修复2 O 2处理。许多变量会影响修复的速率，包括使用的细胞株和化学试剂的类型。因此，研究人员可以进行修改的协议（ 即延长维修时间），以适应他们的调查更多的需求。这里我们提供了修复实验使用这种芯片，其特征在于，TK6细胞用不同的基因毒性剂N-甲基N&#39;-Nitro-Ñ-Nitrosoguanidine（MNNG）挑战另一个例子是已知的诱导基病变包括7-甲基鸟嘌呤烷基化剂和3 - 甲基腺嘌呤。这些病变被转换为脱碱基位点或者通过自发脱嘌呤或通过酶促去除DNA糖基化酶。所得的脱碱基位点可以在碱性条件下被裂解，从而在芯片上检测。初次接触会导致显著增加的伤害，从长远明显尾巴，随着时间的推移这些尾巴减少为细胞修复过程中恢复完整的DNA。虽然烷基化和氧化损伤是由碱基切除修复途径都主要修复，产生病变的数量和参与修复的蛋白质而变化。这些变化可导致转化率的不同的速率脱碱基位点，以及维修所产生的脱碱基位点的速率不同。在图4中，TK6细胞的修复动力学暴露于0.1，1和MNNG 3微克/毫升被示出。在这个实验中，我们扩展了实验时间为2小时的暴露后，因为MNNG引起的损伤似乎持续时间更长，清零以较慢的速率相比，H 2 O 2诱导的损伤。 使用中性条件DNA双链断裂的分析是非常相似的协议，用于单链病变使用碱性条件下进行分析。以显示初始损伤程度，TK6细胞暴露于GIR的可变水平<html>将得到的细胞溶解并进行电泳，在中性pH下（ 图5A）。值得注意的是，彗星尾部的形态是从碱测定条件不同。要使用此法达到可观察的DNA片断，用红外剂量显著高（0-100戈瑞）比使用碱性条件下，看损坏所需的剂量。此外，我们发现，线尾长度的是在使用中性彗星实验7时反映DNA损伤的程度是最敏感的参数。所分析的剂量响应曲线示于图5B。修复在细胞中的DNA双链断裂的也可以评估，并且已显示通过Weingeist 等。人 7。综合来看，中性CometChip提供了DNA的双链断裂的高通量分析的重要工具。 1highres.jpg“WIDTH =”500“/&gt; 图1。H 2 O 2的剂量使用碱性彗星试验TK6细胞的反应。（一）未经处理的TK6淋巴母细胞（上图）和TK6细胞暴露在50微米（中）和100微米（下）H 2 O 2摆着微孔彗星为20分钟，在4℃下。比例尺为100微米。 （B）H暴露于不同水平的H 2 O 2 TK6细胞2 O 2的剂量响应。每个数据点是三次独立实验，其中在每次实验中得到的中位数百分比尾DNA的至少50个个体彗星的平均值。误差棒代表平均值由三个独立实验的标准误差。 <a href="https://www.jove.com/files/ftp_upload/50607/50607fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50607/50607fig2highres.jpg" width="500" /> 图2。间采样和间变性实验（A）样品到样品的变化。 TK6人类淋巴母细胞，装入12孔的96孔的芯片。在各孔中暴露于100μl的50μM的H 2 O 2的 为20分钟，在4℃下。细胞立即溶解并测定％尾部DNA。每个孔中的数据如下所示。每个方块代表中位数，从每个孔中的至少50个个体的彗星。的12个孔的平均值是77.53％，标准偏差为3.99％，变异系数被计算为5.2％。 （ 二）实验对实验的变化。 TK6人类淋巴母细胞，装入96孔的芯片，在4℃下暴露于100μl的75μM的H 2 O 2 20分钟。细胞立即溶解并测定％尾部DNA。相同的实验重复6次，每次重复的数据被显示为灰色条。每个框代表中位数％尾部DNA从每个重复了至少100个人彗星。 6重复的平均值为49.57％，表现为这里的回吧。 6重复的标准偏差是4.99％，而平均值的标准误差被计算为2.04％，表示为图中的误差棒。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/50607/50607fig3highres.jpg" width="500" /> 图3。修复的H 2 O 2诱导的损伤淋巴母细胞TK6（A）修复的研究具有代表性彗星示意图。 TK6细胞用损伤剂，并使其在37℃下裂解之前修复中的介质。 （ 二）TK6细胞的修复动力学为50微米H 2 O 2处理20分钟后，在4＃176℃。每个数据点是三次独立实验，其中在每次实验中得到的中位数百分比尾DNA的至少50个个体彗星的平均值。误差棒代表平均值的重复实验的标准误差。 <a href="https://www.jove.com/files/ftp_upload/50607/50607fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/50607/50607fig4highres.jpg" width="500" /> 图4。修复中的淋巴母细胞TK6 MNNG引起的损伤。TK6细胞在4℃下以0.1，1和3微克/毫升MNNG处理后30分钟，并使其在37℃下以修复在媒体多达120分钟后曝光。对于每个三次独立实验中获得的平均百分比尾DNA的至少50个个体的彗星。误差棒表示标准误差的平均值由三个独立的实验。 <img alt="图5" fo:content-width="3in" src="/files/ftp_upload/50607/50607fig5highres.jpg" width="300" /> 图5。使用中性彗星试验TK6细胞的红外剂量反应（A）微孔摆着彗星未经处理TK6淋巴母细胞（上图）和TK6细胞暴露在40戈瑞（中）和80戈瑞（下）伽玛射线。比例尺为100微米。 （B）的红外剂量暴露于不同水平的γ辐照TK6细胞的响应。每个数据点代表至少300彗星从芯片六个微孔池平均尾长（微米）。 <a href="https://www.jove.com/files/ftp_upload/50607/50607fig5highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells at JoVE.com

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

我们在这里描述了一个平台，使彗星试验检测DNA损伤了前所未有的吞吐量。该器件的图案哺乳动物细胞成芯片，使96个样品进行并行处理。这种方法有利于基础水平的DNA损伤，暴露诱导的DNA损伤和DNA修复的动力学分析。

CometChip：一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

cometchip-high-throughput-96-well-platform-for-measuring-dna-damage

Research

JoVE Journal

Bioengineering

17.2K 次观看.  Massachusetts Institute of Technology. 我们在这里描述了一个平台，使彗星试验检测DNA损伤了前所未有的吞吐量。该器件的图案哺乳动物细胞成芯片，使96个样品进行并行处理。这种方法有利于基础水平的DNA损伤，暴露诱导的DNA损伤和DNA修复的动力学分析。 

视频: CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

科研

生物工程

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto：一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...