这项研究部分由美国国立卫生研究院资助，奖励编号为R43GM137651、R61HL154249、R16GM146687和NSF授予CBET 2150798。作者感谢RIT机械车间的铝模具制造。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。

该设计可以根据实验要求和最终用户的偏好在各种模式下使用。在每次实验之前，请查阅 图2 所示的决策流程图，以确定方案的必要步骤和模块。例如，如果用户打算在整个实验过程中保持开孔格式，以直接将其与Transwell型系统进行比较，则细胞接种不需要图案模板。核心模块是市售的（见 材料表），超薄纳米膜可以从具有不同孔隙率和孔径的材料库中进行选择，以满足实验需求。
1. 图案模板的制作
注意：图案化模板用于将电池专门放置在膜芯片的多孔区域上，防止电池沉降到周围的硅层上，在添加流量模块后，它们可能会受到损坏16 （参见 图3）。单层的损坏会对屏障完整性产生不利影响，并影响实验结果。在开放、静态的培养中，模板是不必要的，因为没有损坏的风险。
<ol><li>使用 补充编码文件 1 （图 4A）中提供的设计购买、加工或 3D 打印模具。 注意：与高纵横比直壁特征相比，模板壁呈锥形，以增加介质容量并最大限度地减少气泡捕获。</li>
	<li>使用冷辊将 130 μm 压敏胶 （PSA） 薄膜连接到 3.2 mm 亚克力板上（参见 材料表）。</li>
	<li>根据 补充编码文件 2 中提供的设计激光切割亚克力板，以创建空腔阵列（图 4B）。</li>
	<li>从PSA薄膜上剥下保护层，将亚克力板连接到模具上。 注意：确保激光切割的板材与模具对齐，使模具特征在型腔内居中（图 4C）。细胞在膜上定位的准确性取决于此步骤。</li>
	<li>充分混合PDMS预聚物（使用10：1的碱与催化剂比例）（参见 材料表）并在真空室中脱气，直到没有可见的气泡残留（5-15分钟）。</li>
	<li>将PDMS慢慢倒入模具型腔中，用平坦的边缘将其调平。 注意： 为防止气泡滞留，请将 PDMS 缓慢倒入每个空腔中。如果浇注后存在气泡，请将模具放入真空室中并再次脱气。</li>
	<li>在70°C的电炉上固化PDMS1小时，然后使其冷却至室温。</li>
	<li>使用平头镊子从模具型腔中提取模板。</li>
</ol>2. 流量模块的制造
注：流量模块与核心模块的三叶草形孔具有相似的占地面积，并包括一个模制微通道（宽度 = 1.5 mm，高度 = 0.2 mm，长度 = 5 mm）。三叶草形状有助于在多孔培养区域上对齐通道（图5）。
<ol><li>使用标准软光刻技术18 创建硅模具，其特征由 补充编码文件3 （图4D）中提供的设计定义。</li>
	<li>将 130 μm PSA 薄膜连接到 3.2 毫米厚的亚克力板上。</li>
	<li>根据 补充编码文件 4 中给出的设计激光切割亚克力板，以创建三叶草形空腔阵列（图 4E）。 注意： 型腔的高度决定了流量模块的高度，流量模块的高度必须略高于核心模块井的高度（0-0.1 毫米），以确保正确密封。</li>
	<li>从 PSA 薄膜上取下保护层，然后通过将硅模具上的三角形对准标记与激光切割片上的对齐标记，将激光切割片连接到硅模具上。 注意：与步骤1.4类似，确保激光切割的亚克力板与硅模具对齐，以便模具特征在每个型腔内居中（图4F）。此步骤确定微通道相对于封装的位置。</li>
	<li>将脱气后的PDMS缓慢倒入模具型腔上，并用平整的边缘将其平整。 注意： 如果浇注后存在气泡，请对模具进行脱气，直到去除气泡。</li>
	<li>将模具放在电炉上，在70°C下烘烤PDMS1小时，然后让模具冷却至室温。</li>
	<li>使用平头镊子小心地从模具型腔中取出流量模块。</li>
	<li>将流量模块定向，使微通道特征朝上，并使用活检打孔将核心入口和出口放置在通道末端（参见 材料表）。</li>
</ol>3. 下部和上部亚克力外壳的制造
注意： 核心模块安装在下壳体中。外壳中嵌入式磁铁之间的吸引力将流量模块压缩并密封到核心模块（图 6）。
<ol><li>使用 补充编码文件 5 中提供的设计对 2 mm 亚克力板进行激光切割，以创建带有用于磁铁插入孔的上壳体。</li>
	<li>将 130 μm PSA 薄膜连接到 3.5 mm 亚克力板上。</li>
	<li>根据 补充编码文件 6 中给出的设计对亚克力板进行激光切割，以创建带有用于磁铁插入的孔的下壳体。 注意： 下壳体的厚度是一个关键参数，必须与核心模块的整体厚度相匹配，以防止在流动过程中泄漏。</li>
	<li>取下 PSA 保护层并将下部外壳连接到玻璃盖玻片上。</li>
	<li>将 4.75 mm 镀镍钕磁铁（见 材料表）压入外壳的激光切割孔中。 注意： 确保下部和上部外壳中的磁铁以相反的极性放置，以确保磁吸力（图 6）。</li>
</ol>4. 流路的制造
注：闭环流路包含两个样品收集瓶作为储液器（图7）。入口储液器具有聚偏二氟乙烯 （PVDF） 过滤器，使细胞培养基与培养箱中的 CO2 浓度平衡。
<ol><li>使用 1 mm 活检打孔器在样本收集瓶的盖子上打两个孔。</li>
	<li>使用活检打孔器在单独的样本收集瓶的盖子上开两个孔（1 mm 和 4 mm），并在该小瓶的下部开一个 1 mm 的孔。</li>
	<li>将 20胶头插入每个 1 mm 孔中，并用热胶密封。</li>
	<li>将 0.22 μm PVDF 过滤器（参见 材料表）放入 4 mm 孔中并用热胶密封。</li>
	<li>使用 补充编码文件 7 中提供的设计激光切割 1.5 mm 亚克力板，以创建样品保存台。</li>
	<li>将储液器放置在亚克力平台上。</li>
	<li>使用微流量管连接点胶吸头（参见 材料表）。</li>
	<li>使用 21胶吸头将管子连接到流量模块的入口和出口。</li>
	<li>使用蠕动泵（见 材料表）进行流动循环。 注意： 如果需要，也可以使用注射器或气动泵来引入流量。流速应根据实验需要确定。 补充表 1 中提供了一个综合表格，该表格源自经过实验验证的 COMSOL 模型，以帮助用户选择必要的流速，以在单元单层16 处实现所需的剪切应力。</li>
</ol>5. 细胞接种
注意：与传统的膜插入物类似，可以在纳米膜上培养不同的细胞类型。二级细胞类型也可以在膜的另一侧的底部通道15中共培养。
<ol><li>在室温下用5μg/ cm2 纤连蛋白（参见 材料表）涂覆膜1小时以改善细胞附着。 注意：所选的电池类型可能会影响所需的涂层和电池密度。此处描述的程序适用于人脐静脉内皮细胞（HUVEC，参见 材料表）。</li>
	<li>使用P200移液器吸出涂层溶液，并在核心模块孔中放置图案模板。</li>
	<li>将细胞介质添加到设备的孔和底部通道。</li>
	<li>将密度为 40,000 个细胞/cm2 的 HUVEC 接种到孔中。 注意：该密度的HUVEC通常在孵育24小时后形成汇合单层16,19。</li>
	<li>将设备放入培养皿中。将装有去离子水的 15 mL 锥形管盖加入培养皿中，以保持局部湿度。</li>
	<li>将培养皿转移到培养箱中。 注意：设备顶部孔和底部通道中的细胞培养基应每 24 小时更换一次。要更换底部通道中的介质，请轻轻地将新介质注入其中一个端口，以从另一个端口替换旧介质。</li>
</ol>6. 重新配置为微流控模式
<ol><li>在组装前，将上部外壳、下部外壳、流量模块、储液器、管子和分配吸头放入紫外线灭菌室中 30 分钟，对它们进行消毒。循环70%乙醇，然后在流路中循环无菌PBS。</li>
	<li>用内皮细胞生长培养基填充储液器和管（参见 材料表）。</li>
	<li>将下壳体放在样品台上。将开井岩心模块插入下部外壳。</li>
	<li>从模块的孔中抽吸细胞培养基。将流量模块放入孔中。</li>
	<li>放置上壳体，将流量模块密封在核心模块井中。将入口和出口点胶吸头连接到流量模块端口。</li>
	<li>启动蠕动泵，将流体流引入系统。</li>
</ol>7. 引入水流后以开井形式进行下游分析
注意：这里的培养时间取决于实验目标。用户可以根据自己的喜好以开孔或微流控形式进行下游分析（例如，免疫细胞化学、RNA 提取）。例如，如果首选开孔形式，则应重新配置系统以根据标准方案16,19进行分析。
<ol><li>当达到所需的剪切流暴露时间时，停止泵。拆下入口和出口点胶吸头。</li>
	<li>拆下上壳体。用镊子轻轻地从孔中取出流量模块。</li>
	<li>向孔中加入 100 μL 细胞培养基。根据标准方案进行所需的检测。</li>
</ol>

具有开孔和微流控模式的可重构屏障组织平台

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Reversibly+sealed,+easy+access,+modular+(seam)+microfluidic+architecture+to+establish+in+vitro+tissue+interfaces.">A Reversibly sealed, easy access, modular (seam) microfluidic architecture to establish in vitro tissue interfaces.</a> PLOS ONE. 11 (5), e0156341 (2016).</li><li>Ahmed, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+extensional+flows+promote+long-range+fiber+alignment+in+3D+collagen+hydrogels.">Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels.</a> Biofabrication. 14 (3), 035019 (2022).</li><li>Hasan, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+fabrication+of+flexible+nanotextured+PDMS+as+a+substrate+for+selective+cell+capture.">One-step fabrication of flexible nanotextured PDMS as a substrate for selective cell capture.</a> Biomedical Physics &amp; Engineering Express. 4 (2), 025015 (2018).</li><li>Ahmed, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microengineering+3D+collagen+hydrogels+with+long-range+fiber+alignment.">Microengineering 3D collagen hydrogels with long-range fiber alignment.</a> Journal of Visualized Experiments. 187, e64457 (2022).</li></ol>

J.L.M. 是 SiMPore， Inc. 的联合创始人，并持有该公司的股权。SiMPore正在将超薄硅基技术商业化，包括本研究中使用的膜。

该协议的目的是开发一种实用的方法，将流动能力整合到具有超薄纳米膜的开孔平台中。在这种设计中，采用了磁闭锁方法，允许在实验期间在开井和流体模式之间切换，并结合了两种方法的优点。与传统的永久粘合平台不同，磁性锁存允许在实验工作流程16,25,26,27的方便点拆卸平台。在这个平台中?...

血管屏障是将血室与周围组织分开的关键界面。它们通过吸引免疫细胞、控制分子通透性和屏蔽病原体侵入组织，在维持体内平衡方面发挥着关键作用 1,2。已经开发了体外培养模型来模拟体内微环境，从而能够系统地研究影响健康和疾病状态下屏障特性的因素和条件3,4。
对于这种培养模型，最广泛使用的方法是类似Transwell的“开孔”配置5，其中多孔的、迹状蚀刻的培养膜将充满培养基的隔室分开（图1A）。在这种形式下，细胞可以接种在膜的任一侧，并且已经开发了广泛的实验方案。然而，这些系统提供支持屏障成熟和模拟体内免疫细胞循环所必需的流体流动的能力有限 5,6。因此，它们不能用于需要引入药物剂量、机械刺激或流体诱导剪切应力的动态流动的研究 6,7,8。
为了克服开井系统的局限性，已经开发了将多孔培养膜与可单独寻址的流体通道相结合的微流体平台9。这些平台可精确控制流体路线、灌注和化合物的引入、受控剪切刺激和动态细胞添加功能7,10,11,12,13。尽管微流控平台提供了先进的功能，但由于复杂的微流控方案及其与既定的实验工作流程不兼容，它们尚未在生物科学实验室中得到广泛采用4,10,14。
为了弥合这些技术之间的差距，我们提出了一种协议，该协议采用磁性可重构的基于模块的系统。该系统可以根据实验的具体需要在开孔和微流控模式之间轻松切换。该平台具有一个开孔装置，称为m-μSiM（由硅膜实现的模块化微生理系统），具有100 nm厚的培养膜（纳米膜）。该纳米膜具有高孔隙率（15%）和玻璃状透明度，如图1B所示。它在物理上将顶部隔室与底部通道分开，允许跨生理长度尺度 15 的分子运输。与传统的轨迹蚀刻膜不同，传统的轨迹蚀刻膜在用明场成像对活细胞进行成像方面存在已知的挑战，纳米膜具有良好的光学和物理特性，可以清晰地看到膜表面两侧的细胞15,16,17。
本协议概述了专用播种和流动模块的制造，并解释了平台的磁性重构。它演示了如何在静态和动态条件下利用该平台建立内皮屏障。该演示表明，内皮细胞沿流动方向排列，在剪切刺激下剪切敏感基因靶标上调。

<table><tbody><tr><td>0.5 x 0.86 Micro Flow tubes</td><td>Langer Instruments</td><td>WX10-14 &amp;amp; DG Series</td><td></td></tr><tr><td>1 mm Disposable Biopsy Punches, Integra Miltex</td><td>VWR</td><td>95039-090</td><td></td></tr><tr><td>1x PBS 7.4 pH</td><td>ThermoFisher Scientific</td><td>10010023</td><td></td></tr><tr><td>20 GAUGE IT SERIES DISPENSING TIP</td><td>Jensen Global</td><td>JG20-1.5X</td><td></td></tr><tr><td>21 GAUGE NT PREMIUM SERIES ANGLED DISPENSING TIP</td><td>Jensen Global</td><td>JG21-1.0HPX-90</td><td></td></tr><tr><td>3M 467 MP Pressure senstitive adhesive (PSA)</td><td>DigiKey</td><td>3M9726-ND</td><td></td></tr><tr><td>3M 468 MP Pressure senstitive adhesive (PSA)</td><td>DigiKey</td><td>3M9720-ND</td><td></td></tr><tr><td>AlexaFluor 488 conjugated phalloidin</td><td>ThermoFisher Scientific</td><td>A12379&amp;nbsp;</td><td></td></tr><tr><td>Applied Biosystems&amp;nbsp;TaqMan Fast Advanced Master Mix</td><td>Thermo Fisher Scientific</td><td>4444556</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA), Fraction V, 98%, Reagent grade, Alfa Aesar, Size = 10 g</td><td>VWR</td><td>AAJ64100-09</td><td></td></tr><tr><td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td><td>McMaster-Carr</td><td>8560K171</td><td>12&quot; x 12&quot; x 1/16&quot;</td></tr><tr><td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td><td>McMaster-Carr</td><td>8589K31</td><td>12&quot; x 12&quot; x 3/32&quot;</td></tr><tr><td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td><td>McMaster-Carr</td><td>8560K191</td><td>12&quot; x 12&quot; x 7.64&quot;</td></tr><tr><td>Corning Fibronectin, Human, 1 mg</td><td>Corning</td><td>47743-728</td><td></td></tr><tr><td>Cover Glasses, Globe Scientific, L x W = 24 x 60 mm</td><td>VWR</td><td>10118-677</td><td></td></tr><tr><td>DOW SYLGARD 184 SILICONE ENCAPSULANT CLEAR 0.5 KG KIT</td><td>Ellsworth Adhesives</td><td>4019862</td><td></td></tr><tr><td>EGM-2 Endothelial Cell Growth Medium-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>Fixture A1&amp;amp;A2</td><td>SiMPore Inc.</td><td>NA</td><td></td></tr><tr><td>Fixture B1&amp;amp;B2</td><td>SiMPore Inc.</td><td>NA</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td><td>Thermo Fisher Scientific</td><td>4374966</td><td></td></tr><tr><td>Human umbilical vein endothelial cells (HUVEC)</td><td>ThermoFisher Scientific</td><td>C0035C</td><td></td></tr><tr><td>LIVE/DEAD Cell Imaging Kit (488/570)</td><td>Thermo Fisher Scientific</td><td>R37601</td><td></td></tr><tr><td>Molecular Probes Hoechst 33342, Trihydrochloride, Trihydrate</td><td>Thermo Fisher Scientific</td><td>H3570</td><td></td></tr><tr><td>Nickel-plated magnets (4.75 mm diameter, 0.34 kg pull force)</td><td>K&amp;amp;J Magnetics</td><td>D31</td><td>3/16&quot; dia.&amp;nbsp;x 1/16&quot; thick</td></tr><tr><td>Paraformaldehyde, 4% w/v aq. soln., methanol free, Alfa Aesar</td><td>Fisher Scientific</td><td>aa47392-9M</td><td></td></tr><tr><td>Peristaltic Pump</td><td>Langer Instruments</td><td>BQ50-1J-A</td><td></td></tr><tr><td>Photoresist SU-8 developer solution</td><td>Fisher Scientific</td><td>NC9901158</td><td></td></tr><tr><td>PVDF syringe filters</td><td>PerkinElmer</td><td>2542913</td><td></td></tr><tr><td>Silicon wafer</td><td>University wafer,USA</td><td>1196</td><td></td></tr><tr><td>SU-8 3050</td><td>Fisher Scientific</td><td>NC0702369</td><td></td></tr><tr><td>Target gene: eNOS (Hs01574659_m1)</td><td>ThermoFisher Scientific</td><td>4331182</td><td></td></tr><tr><td>Target gene: GAPDH (Hs02786624_g1)</td><td>ThermoFisher Scientific</td><td>4331182</td><td></td></tr><tr><td>Target gene: KLF2 (Hs00360439_g1)</td><td>ThermoFisher Scientific</td><td>4331182</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Pierce 20x PBS Tween 20</td><td>Thermo Fisher Scientific</td><td>28352</td><td></td></tr><tr><td>Transport Tube Sample White caps, 5 mL, Sterile</td><td>VWR</td><td>100500-422</td><td></td></tr><tr><td>TRI-reagent</td><td>ThermoFisher Scientific</td><td>AM9738</td><td></td></tr><tr><td>Ultrathin Nanoporous Membrane Chip</td><td>SiMPore Inc.</td><td>NPSN100-1L</td><td>The design is&amp;nbsp; compatible with all of SiMPore membranes</td></tr><tr><td>uSiM component 1</td><td>SiMPore Inc.</td><td>NA</td><td></td></tr><tr><td>uSiM component 2</td><td>SiMPore Inc.</td><td>NA</td><td></td></tr></tbody></table>

transforming static barrier tissue models into dynamic microphysiological systems

微生理系统是小型化的细胞培养平台，用于在实验室环境中模拟人体组织的结构和功能。然而，这些平台尚未在生物科学实验室中得到广泛采用，尽管缺乏流体流动能力，但基于膜的开放式方法仍是模拟组织屏障的黄金标准。这个问题主要归因于现有的微生理系统与为明井系统开发的标准协议和工具不兼容。
在这里，我们提出了一种协议，用于创建具有开井结构、流量增强能力以及与传统协议兼容的可重构膜平台。该系统采用磁性组装方法，可在开井和微流体模式之间进行可逆切换。通过这种方法，用户可以灵活地使用标准方案以开孔形式开始实验，并根据需要添加或删除流动功能。为了证明该系统的实际用途及其与标准技术的兼容性，以开孔形式建立了内皮细胞单层。该系统被重新配置以引入流体流动，然后切换到开孔形式以进行免疫染色和 RNA 提取。由于其与传统开井方案的兼容性和流量增强能力，这种可重构设计有望被工程和生物科学实验室采用。

如图6A所示，开井岩心模块最初位于由下部外壳和盖玻片形成的特定空腔内。随后，将包括微通道和接入端口的流量模块插入核心模块的孔中。如图6B所示，由于嵌入在下部和上部外壳中的磁体之间的磁吸引力，流量模块牢固地密封在膜的硅支撑层上。为了评估这种磁闭锁机制的有效性，进行了爆破压力测试，证明该系统可以承受高达 38.8 ± 2.4 kPa 的死?...

Watch this Scientific Journal Video about Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue at JoVE.com

作者聚焦：开发独特的模块化微生理系统来模拟人体屏障组织

该协议描述了一种可重新配置的基于膜的细胞培养平台，该平台将开孔形式与流体流动功能集成在一起。该平台与标准方案兼容，并允许在开孔和微流控培养模式之间进行可逆过渡，以满足工程和生物科学实验室的需求。

将静态屏障组织模型转化为动态微生理系统

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. ...

我们正在开发模拟人体屏障组织的模块化微生理系统。我们的系统是独一无二的，因为它可以从静态明井培养重新配置为流动增强系统。这种灵活性使该平台可用于生物科学和工程实验室。我们提出了一种协议，用于创建具有流动增强文化和生活方式成像的可重新配置的明井平台。它与传统的生物科学协议兼容。我们的可重构设计允许用户在实验期间在开孔和微流控模式之间切换，并允许用户使用他们熟悉的格式进行每个步骤。

transforming-static-barrier-tissue-models-into-dynamic

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

895 次观看.  Rochester Institute of Technology. 我们正在开发模拟人体屏障组织的模块化微生理系统。我们的系统是独一无二的，因为它可以从静态明井培养重新配置为流动增强系统。这种灵活性使该平台可用于生物科学和工程实验室。我们提出了一种协议，用于创建具有流动增强文化和生活方式成像的可重新配置的明井平台。它与传统的生物科学协议兼容。我们的可重构设计允许用户在实验期间在开孔和微流控模?...

视频: 作者聚焦：开发独特的模块化微生理系统来模拟人体屏障组织

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

细胞电微环境的有限元素建模

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

评估人体骨骼肌微组织骨骼肌健康的功能指标

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

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 ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

在开顶器官芯片上重建人上皮组织的细胞结构和功能

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

设计生物反应器以改善工程心脏组织的数据采集和模型通量

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

通过免疫荧光染色分析血脑屏障模型

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

作者聚焦：推进组织芯片技术在人体组织中的应用 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...

将静态屏障组织模型转化为动态微生理系统

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微机械动态细胞培养平台

细胞电微环境的有限元素建模

评估人体骨骼肌微组织骨骼肌健康的功能指标

在微流体平台内开发 3D 组织人类心脏组织

成纤维细胞衍生的人类工程结缔组织用于筛查应用

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

在开顶器官芯片上重建人上皮组织的细胞结构和功能

设计生物反应器以改善工程心脏组织的数据采集和模型通量

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

将静态屏障组织模型转化为动态微生理系统

副本

摘要

探索更多视频

此视频中的章节

微机械动态细胞培养平台

细胞电微环境的有限元素建模

评估人体骨骼肌微组织骨骼肌健康的功能指标

在微流体平台内开发 3D 组织人类心脏组织

成纤维细胞衍生的人类工程结缔组织用于筛查应用

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

在开顶器官芯片上重建人上皮组织的细胞结构和功能

设计生物反应器以改善工程心脏组织的数据采集和模型通量

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模