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

<ol>
	<li>Claesson-Welsh, L., Dejana, E., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+the+Endothelial+Barrier:+Identifying+and+Reconciling+Controversies.">Permeability of the Endothelial Barrier: Identifying and Reconciling Controversies.</a> Trends in Molecular Medicine. 27 (4), 314-331 (2021).</li><li>Vera, D., 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=Engineering+tissue+barrier+models+on+hydrogel+microfluidic+platforms.">Engineering tissue barrier models on hydrogel microfluidic platforms.</a> ACS Applied Materials &amp; Interfaces. 13 (12), 13920-13933 (2021).</li><li>Wang, Y. I., Abaci, H. E., Shuler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+blood-brain+barrier+model+provides+in+vivo-like+barrier+properties+for+drug+permeability+screening.">Microfluidic blood-brain barrier model provides in vivo-like barrier properties for drug permeability screening.</a> Biotechnology and Bioengineering. 114 (1), 184-194 (2017).</li><li>Sakolish, C. M., Esch, M. B., Hickman, J. J., Shuler, M. L., Mahler, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+barrier+tissues+in+vitro:+methods,+achievements,+and+challenges.">Modeling barrier tissues in vitro: methods, achievements, and challenges.</a> eBioMedicine. 5, 30-39 (2016).</li><li>Kaisar, M. 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=New+experimental+models+of+the+blood-brain+barrier+for+CNS+drug+discovery.">New experimental models of the blood-brain barrier for CNS drug discovery.</a> Expert Opinion on Drug Discovery. 12 (1), 89-103 (2017).</li><li>Tan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+microfluidic+microphysiological+system+(PREDICT-96)+to+recapitulate+hepatocyte+function+in+dynamic,+re-circulating+flow+conditions.">A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions.</a> Lab on a Chip. 19 (9), 1556-1566 (2019).</li><li>Ayuso, J. M., Virumbrales-Mu&#241;oz, M., Lang, J. M., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+microfluidic+systems+in+precision+medicine.">A role for microfluidic systems in precision medicine.</a> Nature Communications. 13 (1), 3086 (2022).</li><li>Katt, M. E., Shusta, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier:+building+in+physiological+complexity.">In vitro models of the blood-brain barrier: building in physiological complexity.</a> Current Opinion in Chemical Engineering. 30, 42-52 (2020).</li><li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organs-on-chips+for+disease+modelling,+drug+development+and+personalized+medicine.">Human organs-on-chips for disease modelling, drug development and personalized medicine.</a> Nature Reviews Genetics. 23 (8), 467-491 (2022).</li><li>Duncombe, T. A., Tentori, A. M., Herr, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+reframing+biological+enquiry.">Microfluidics: reframing biological enquiry.</a> Nature reviews. Molecular cell biology. 16 (9), 554-567 (2015).</li><li>Williams, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost,+rapidly+integrated+debubbler+(rid)+module+for+microfluidic+cell+culture+applications.">A low-cost, rapidly integrated debubbler (rid) module for microfluidic cell culture applications.</a> Micromachines. 10 (6), 360 (2019).</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=Engineering+fiber+anisotropy+within+natural+collagen+hydrogels.">Engineering fiber anisotropy within natural collagen hydrogels.</a> American Journal of Physiology-Cell Physiology. 320 (6), C1112-C1124 (2021).</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=Microengineered+3D+collagen+gels+with+independently+tunable+fiber+anisotropy+and+directionality.">Microengineered 3D collagen gels with independently tunable fiber anisotropy and directionality.</a> Advanced Materials Technologies. 6 (4), 2001186 (2021).</li><li>&#321;ach, A., Wnuk, A., W&#243;jtowicz, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+to+study+the+functions+of+the+blood-brain+barrier.">Experimental models to study the functions of the blood-brain barrier.</a> Bioengineering. 10 (5), 519 (2023).</li><li>McCloskey, M. C., 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=The+Modular+&#181;SiM:+A+mass+produced,+rapidly+assembled,+and+reconfigurable+platform+for+the+study+of+barrier+tissue+models+in+vitro.">The Modular &#181;SiM: A mass produced, rapidly assembled, and reconfigurable platform for the study of barrier tissue models in vitro.</a> Advanced Healthcare Materials. 11 (18), 2200804 (2022).</li><li>Mansouri, M., 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=The+modular+&#181;sim+reconfigured:+integration+of+microfluidic+capabilities+to+study+in+vitro+barrier+tissue+models+under+flow.">The modular &#181;sim reconfigured: integration of microfluidic capabilities to study in vitro barrier tissue models under flow.</a> Advanced Healthcare Materials. 11 (21), 2200802 (2022).</li><li>Hudecz, D., 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=Modelling+a+human+blood-brain+barrier+co-culture+using+an+ultrathin+silicon+nitride+membrane-based+microfluidic+device.">Modelling a human blood-brain barrier co-culture using an ultrathin silicon nitride membrane-based microfluidic device.</a> International Journal of Molecular Sciences. 24 (6), 5624 (2023).</li><li>Joshi, I. M., 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+Matrices+with+Tumor-Mimetic+Gradients+in+Fiber+Alignment.">Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.</a> bioRxiv. , (2023).</li><li>Hsu, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miniaturized+3D+printed+pressure+regulator+(&#181;PR)+for+microfluidic+cell+culture+applications.">A miniaturized 3D printed pressure regulator (&#181;PR) for microfluidic cell culture applications.</a> Scientific Reports. 12 (1), 10769 (2022).</li><li>Rogers, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+microfluidic+bilayer+co-culture+platform+to+study+endothelial-pericyte+interactions.">A high-throughput microfluidic bilayer co-culture platform to study endothelial-pericyte interactions.</a> Scientific reports. 11 (1), 12225 (2021).</li><li>Wettschureck, N., Strilic, B., Offermanns, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passing+the+vascular+barrier:+endothelial+signaling+processes+controlling+extravasation.">Passing the vascular barrier: endothelial signaling processes controlling extravasation.</a> Physiological Reviews. 99 (3), 1467-1525 (2019).</li><li>Wang, Y. I., Shuler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniChip+enables+long-term+recirculating+unidirectional+perfusion+with+gravity-driven+flow+for+microphysiological+systems.">UniChip enables long-term recirculating unidirectional perfusion with gravity-driven flow for microphysiological systems.</a> Lab on a Chip. 18 (17), 2563-2574 (2018).</li><li>Nayak, L., Lin, Z., Jain, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Go+with+the+flow&amp;#34;:+how+Kr&#252;ppel-like+factor+2+regulates+the+vasoprotective+effects+of+shear+stress.">34;Go with the flow&amp;#34;: how Kr&#252;ppel-like factor 2 regulates the vasoprotective effects of shear stress.</a> Antioxidants &amp; Redox Signaling. 15 (5), 1449-1461 (2011).</li><li>Satoh, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatic+pressure-driven+multi-throughput+microfluidic+circulation+culture+system.">A pneumatic pressure-driven multi-throughput microfluidic circulation culture system.</a> Lab on a chip. 16 (12), 2339-2348 (2016).</li><li>Abhyankar, V. V., Wu, M., Koh, C. Y., Hatch, A. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 x 0.86 Micro Flow tubes</td>
			<td>Langer Instruments</td>
			<td>WX10-14 &#38; 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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#160;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&#34; x 12&#34; x 1/16&#34;</td>
		</tr>
		<tr>
			<td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td>
			<td>McMaster-Carr</td>
			<td>8589K31</td>
			<td>12&#34; x 12&#34; x 3/32&#34;</td>
		</tr>
		<tr>
			<td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td>
			<td>McMaster-Carr</td>
			<td>8560K191</td>
			<td>12&#34; x 12&#34; x 7.64&#34;</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&#38;A2</td>
			<td>SiMPore Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixture B1&#38;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&#38;J Magnetics</td>
			<td>D31</td>
			<td>3/16&#34; dia.&#160;x 1/16&#34; 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&#160;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&#160; 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

Research

JoVE Journal

Bioengineering

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

873 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

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

模型和方法来评价药物输送系统在整个细胞屏障的运输

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

阻隔组织破坏与有机电化学晶体管传感

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

基于琼脂糖组织模拟光学幽灵的漫反射光谱研究

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

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 ...

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 ...