这项工作一直由德国联邦教育与研究，GO-生物批准号:0315569。

注:与知情同意和伦理委员会批准（伦理委员会的Charité大学医学院，德国柏林）获得人类幼年包皮，符合有关法律，从常规包皮环切后，小儿外科。 1.生产组织等效的培育，在MOC <ol><li>聚集HepaRG和HHSteC在悬滴板以产生肝球体。 <ol><li>为了达到在细胞培养瓶中生长的HepaRG细胞的胰蛋白酶（75 平方厘米），除去从汇合培养基，分化单层培养，洗净用PBS两次，并加入3毫升0.05％胰蛋白酶/ EDTA中。孵育3至5分钟，在37℃，使反应停止通过加入6 ml胰蛋白酶抑制剂。 </li><li>离心机HepaRG细胞在150×g离心5分钟，除去上清，重悬细胞沉淀在1ml HepaRG细胞培养基，并计数细胞。细胞生存力应&gt; 90％。 </li><li>在为了实现HHSteC细胞的胰蛋白酶处理，除去从单层培养的培养基，用PBS洗两次，并加入3毫升0.05％胰蛋白酶/ EDTA中。孵育5分钟，在37℃，使反应停止通过加入6 ml胰蛋白酶抑制剂。 </li><li>离心机的HHSteC细胞在150×g离心5分钟，除去上清，重悬细胞沉淀在1ml HepaRG细胞培养基，和计数细胞。 </li><li>在HepaRG细胞培养基1:联合HepaRG和HHSteC以24比。这样做，通过在HepaRG细胞培养基稀释细胞悬浮液调整细胞数，并添加1×10 5个细胞/ ml HHSteC至4.8×10 6细胞/ ml的HepaRG细胞。仔细混合。 </li><li>加入2毫升的PBS以悬滴和接收机板制备悬滴板。 </li><li>吸管20微升细胞悬浮液放入悬滴板的各孔中。总是准备约10％的悬滴比你需要检索聚集，一些聚集丢失杜里纳克的过程。小心地将板在37℃的孵化器。等待48小时的球体形成。 </li><li>为了获取球体，一定要使用枪头宽尖结局或削减1毫升枪头用无菌刀技巧，以扩大开放为约2〜3毫米。使用这些枪头处理球体，而不破坏它们。 </li><li>通过反复加入1毫升介质悬滴板的使用移液管的孔的顶端从悬滴板仔细洗掉球体。洗板，直到所有的球体都名落孙山。球状​​体是稍有盘形为300至400微米的中径为200至300微米，在这一点上的高度。 </li><li>收集的球状体，在接收器板，并将其与最多20球状体每使用制备的移液器吸头以及转移到24孔超低附着板上。调整每个媒体卷井0.5毫升。使用20到聚集INOculate 1 MOC电路，以获得1 / 100,000关于体内细胞数的小型化速率。 </li><li>孵育球状体在37℃和5％的CO 2，直至在信息操作中心进一步使用。不要使用前超过三天存储的球状体，以保证可比性。培育的聚集体为至少一天在超低附着板来检索均匀球状体。 </li></ol></li><li>追求两种方法来产生皮肤组织等同物:使用钻取活组织检查（1.2.1）或利用现成体外组织模型（1.3.1）的。 <ol><li>切转孔与托架下面白炽灯刀以制备在无菌条件直至进一步使用下96孔细胞培养插入和存储。 </li><li>消毒在80％的乙醇包皮样品30秒和切断环打开。样品应当具有2mm的平均高度。 </li><li>用活检穿孔削减4.5毫米直径活检获得平等miniaturiza灰比为肝脏和皮肤。加载活检入制备96孔Transwell小钳插入。照顾与表皮朝上放置活检。 </li><li>地方的细胞培养插入用含有HepaRG细胞培养基并储存在37℃和5％的CO 2，直至在信息操作中心进一步使用活检中的接收机板。做得比2更长的时间3小时不存储样品。 </li></ol></li><li>整合现成的体外皮肤模型中，从各种供应商处购买，进入MOC，确保它们是在96孔Transwell小格式。使用由供应商，或者，如果与另一种组织的共存设想在进一步的步骤中，使用同时支持组织的基本培养基供给的细胞培养液中。测试在现有的静态试验的各基本培养基为它支持的组织的能力。 <ol><li>检索皮肤模型从保持架板和切断96-插入托架下面白炽灯刀。 </li&gt; <li>将插入件放回接收机板并储存在37℃和5％的CO 2，直至在信息操作中心进一步使用。不要储存样本超过一天时间。 </li></ol></li></ol> 2. MOC制造<ol><li>混合的PDMS和固化剂以10:1的比例（体积/体积）并在真空下放置该混合物15分钟以除去气泡。 </li><li>同时，把聚碳酸酯盖板与硅橡胶添加剂，在80℃进行20分钟。 </li><li>插入螺丝铁氟龙进入盖板的各个孔创建微型四个PDMS无细胞培养室和六个PDMS膜，500微米厚。 </li><li>插上准备盖板两个微血管电路的母模，注入脱气PDMS。小心不要气泡集成到系统中。如果气泡冒出，尝试通过倾斜装置将其删除。 </li><li>孵育体系在80℃下60分钟以固化硅橡胶层。 </li><li>除去母模，并从该设备的特氟隆螺钉和粘接的PDMS层与一个75×25 平方毫米的足迹使用低压等离子体氧化载玻片。 </li><li>螺丝特殊螺纹接头MOC所有四个细胞培养的盖板的隔间。 </li><li>连接含有培养基注射器阴鲁尔X¼-28的男性适配器和它们拧到盖板的MOC适配器。 </li><li>通过重复压下并拉动注射器柱塞注入介质到微流体电路。 </li><li>检查通道在显微镜下介质的适当填充。 </li></ol> 3.内皮医监<ol><li>之前endothelializing操作中心，冲洗每个MOC电路与内皮细胞生长介质和静态孵育它三天，在37℃和5％的CO 2。 <ol><li>消毒用的酒精擦拭MOCS并将其放置在层流工作台。我ñ此外，消毒两对镊子，为进一步利用2六角键。 </li><li>松开MOC的组织培养室的使用六角形按键帽使用镊子取下盖子。插入介质后，螺丝帽回到MOCS以相同的方式。 </li></ol></li><li>为了实现人皮肤微血管内皮细胞（HDMEC）的胰蛋白酶处理，取出来自单层培养物的培养基，用PBS洗两次，并加入3毫升0.05％胰蛋白酶/ EDTA中。孵育5分钟，在37℃，使反应停止通过加入6 ml胰蛋白酶抑制剂。 </li><li>离心机的HDMEC在220×g离心5分钟，除去上清，重悬细胞沉淀在1ml内皮细胞生长培养基中，计数细胞。细胞生存力应&gt; 90％。 </li><li>调整在细胞悬浮液中的细胞数为2×10 7个细胞/ ml的终浓度通过它与内皮细胞生长培养基稀释，并转移250微升它1ml注射器。应用细胞的该浓度来操作中心保持的1 / 100,000的所有器官的小型化速率。 </li><li>注射器连接到阴路厄X¼-28雄性适配器，排出空气从该嵌合，并且它拧到一个特殊的螺纹MOC适配器。该适配器连接到每个MOC电路的两个隔间之一。 </li><li>连接一个空的注射器相同的方式来操作中心电路的第二隔室。 </li><li>压下并拉动两个注射器柱塞几次以连续的方式注入的细胞均匀。控制细胞的显微镜下的输注。 </li><li>孵育MOC在37℃和5％CO 2的静态条件下进行3小时，以使细胞附着在通道壁上。 </li><li>从培养箱中取出芯片，并将其放置在层流工作台，并用特殊螺纹MOC细胞培养室更换注射器和MOC适配器。 </li><li>加入400微升新鲜培养基到一个COM每个MOC电路partment，让它通过渠道由静水压冲洗。此后，更换介质在这两个区室用300μl的新鲜培养基。 </li><li>使用瓶盖关闭隔间，如3.1.2所述。 </li><li>连接芯片到泵控制单元。调节泵送速度以0.475赫兹的频率和培育芯片在37℃和5％的CO 2。 </li><li>替换每个MOC隔室的每一到两天的介质和监测细胞形态​​学用光学显微镜。 </li></ol> 4.装载芯片<ol><li>将MOC的层流板凳下并打开它，按步骤3.1.2描述。 </li><li>从组织培养室中取出介质，并用300微升新鲜HepaRG细胞培养液进行更换。 </li><li>转让20预制球体一个组织培养用枪头具有广泛开放的每个MOC电路仓（请参阅步骤1.1.8 / 9）。关闭了C使用镊子和六角形按键AP。 </li><li>转让的96孔细胞培养插入物含有皮肤等同于使用镊子每个MOC电路的其余组织培养室中。请注意，以避免皮肤替代物的膜下面形成气泡。为了做到这一点，将转孔在略微倾斜的角度，推动轻轻放下。周围的透孔中取出多余的媒体被从下向上推用吸管。 </li><li>关闭使用镊子和六角形按键帽。 </li></ol> 5.连接芯片到泵控制单元<ol><li>在控制单元的值设置运行参数需要的。修改的空气压力从0到8000毫巴，真空从0至-800毫巴，并泵送频率从0.24到2.4赫兹。顺时针或逆时针设置泵送方向。 </li><li>从层流长椅下除去含MOC组织等同物和将其连接到泵控制单元。 </li><li>继numerat离子的管，插入气压管上的MOC各自的配件。 </li><li>培育MOC在37℃和5％的CO 2在培养箱中，或在活组织成像的情况下，使用MOC支撑到芯片加热至37℃，培养培养箱外的芯片。使用温水支持标准的显微镜下培养的细胞在MOC。 </li></ol> 6.执行媒体交流，取样媒体和暴露于物质<ol><li>执行例行媒体交换每天或每隔一天，在考虑组织培养，细胞的代谢活性的类型。 <ol><li>检索从孵化器的MOC并观察它在显微镜控制媒体的流量和检查污染之下。 </li><li>拔下空气高压油管断开泵控制单元的MOC。使用酒精湿巾消毒MOC，并把它的层流板凳下。 </li><li>打开组织培养补偿artment含有肝球体，如在步骤3.1.2说明。 </li><li>去除高达200微升从使用移液管的隔间，而不破坏球状体和介质存放在深 - 孔平板的一个空的井。介质样品直接分析或关闭深孔板和存储介质的样品在-80℃下用于进一步分析。 </li><li>更换MOC的媒体多达250微升的新鲜细胞培养基，并关闭盖子。在除去培养基并换成占损失量由于少量关闭系统时泄漏出的芯片的差异。 </li><li>打开组织培养室保持皮肤当量在这一点上，以检查组织完整性的上限。小心不要引入气泡进入系统。关闭盖子。 </li><li>连接所述泵控制单元向信息操作中心的油管，根据步骤5.3，并放置MOC在培养箱。 </li></ol></li></ol> 7。分析日报传媒样品并进行在线分析<ol><li>上线使用活细胞成像或脱机，通过分析每天的媒体样本分析组织培养性能。执行后者通过标准常规酶测定（ 例如乳酸脱氢酶（LDH）活性）或酶联免疫吸附（ 例如白蛋白浓度）。在线分析是在下面说明。 </li><li>除去内皮MOC的介质，如在步骤6.1.1至6.1.4中描述，并且它在两个组织培养室用200μl的10微克替换/ ml的荧光团共轭乙酰低密度脂蛋白（LDL）的溶液（稀释在细胞培养基）。 </li><li>关闭帽。连接信息操作中心到泵控制单元，根据步骤5.3，和泵它为30分钟，在0.475 Hz到微流体回路内分布均匀溶液。 </li><li>停止泵送，并培育商务部静态3.5小时，在37℃和5％的CO 2。 </li><li>类似吨Ø步骤7.2，删除这两个车厢的乙酰化LDL的解决方案，并与400微升新鲜培养基在两个车厢的更换。 </li><li>等待3至5分钟，以允许通过所述微流体通道电路的静水压力驱动介质。 </li><li>更换已流经用300μl新鲜培养基，并填补上述第二组织培养室用300μl的新鲜培养基的培养基。 </li><li>关闭MOC，根据步骤3.1.2。把它在荧光显微镜下观察细胞的生长和生存能力。 </li><li>将染色MOC早在孵化器继续培养。除去污渍泄漏出的细胞与每个下列介质的更换。 </li></ol> 8.检索从MOC等效组织和执行终点分析<ol><li>检索组织当量从信息操作中心在端点分析实验结束。 <ol><li>为了检索肝脏和皮肤Ë从MOC quivalents，从每个组织培养舱取出介质，如步骤6.1.1到6.1.4描述。 </li><li>删除包含从使用镊子操作中心皮肤的96孔细胞培养插入。用钳子夹住它放在一边，拉下来仔细剥离从插入膜。小心不要失去皮肤相当于在这一点上。 </li><li>冷冻该膜保持皮肤等效于冷冻嵌入化合物，并将其存储在-80℃直至进一步分析。 </li></ol></li><li>通过使用切割枪头吸取他们的组织培养舱同样取下肝脏当量（请参阅步骤1.1.8 / 9）。 <ol><li>嵌入肝球体的冷冻嵌入化合物。小心不转让过多的液体，并删除任何多余的液体用吸管。放置在球体上的嵌入化合物并除去培养基后，添加更多的冷冻化合物到球状体的顶部，以完全封闭它们。 </li><li>冷冻肝脏当量，并将其存储在-80℃直至进一步分析。 </li></ol></li><li>通过切割所述组织当量在冷冻切片机至8微米的部分和染色用于组织特异性标志物，如在先前的协议10描述执行端点分析。 </li></ol>

<ol>
	<li>Kelm, J. M., Fussenegger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+tissue+engineering+using+gravity-enforced+cell+assembly.">Microscale tissue engineering using gravity-enforced cell assembly.</a> Trends in biotechnology. 22 (4), 195-202 (2004).</li><li>Marx, U., Walles, H., 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=Human-on-a-chip+Developments:+A+Translational+Cutting-edge+Alternative+to+Systemic+Safety+Assessment+and+Efficiency+Evaluation+of+Substances+in+Laboratory+Animals+and+Man.">Human-on-a-chip Developments: A Translational Cutting-edge Alternative to Systemic Safety Assessment and Efficiency Evaluation of Substances in Laboratory Animals and Man.</a> ATLA. 40 (5), 235-257 (2012).</li><li>Baudoin, R., Griscom, L., Prot, J. M., Legallais, C., Leclerc, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+HepG2/C3A+cell+cultures+in+a+microfluidic+bioreactor.">Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor.</a> Biochemical Engineering Journal. 53 (2), 172-181 (2011).</li><li>Dash, A., Inman, W., 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=Liver+tissue+engineering+in+the+evaluation+of+drug+safety.">Liver tissue engineering in the evaluation of drug safety.</a> Expert opinion on drug metabolism &amp; toxicology. 5 (10), 1159-1174 (2009).</li><li>Materne, E. -. M., Tonevitsky, A. G., Marx, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chip-based+liver+equivalents+for+toxicity+testing--organotypicalness+versus+cost-efficient+high+throughput.">Chip-based liver equivalents for toxicity testing--organotypicalness versus cost-efficient high throughput.</a> Lab on a chip. 13 (18), 3481-3495 (2013).</li><li>Huh, D., Torisawa, Y., Hamilton, G. a., Kim, H. J., 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=Microengineered+physiological+biomimicry:+organs-on-chips.">Microengineered physiological biomimicry: organs-on-chips.</a> Lab on a chip. 12 (12), 2156-2164 (2012).</li><li>Sin, A., Chin, K. C., Jamil, M. F., Kostov, Y., Rao, G., 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=The+design+and+fabrication+of+three-chamber+microscale+cell+culture+analog+devices+with+integrated+dissolved+oxygen+sensors.">The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors.</a> Biotechnology progress. 20 (1), 338-345 (2004).</li><li>Tatosian, D. a., 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=A+novel+system+for+evaluation+of+drug+mixtures+for+potential+efficacy+in+treating+multidrug+resistant+cancers.">A novel system for evaluation of drug mixtures for potential efficacy in treating multidrug resistant cancers.</a> Biotechnology and bioengineering. 103 (1), 187-198 (2009).</li><li>Schimek, K., Busek, 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=Integrating+biological+vasculature+into+a+multi-organ-chip+microsystem.">Integrating biological vasculature into a multi-organ-chip microsystem.</a> Lab on a chip. 13 (18), 3588-3598 (2013).</li><li>Wagner, I., Materne, E. -. 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=A+dynamic+multi-organ-chip+for+long-term+cultivation+and+substance+testing+proven+by+3D+human+liver+and+skin+tissue+co-culture.">A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver and skin tissue co-culture.</a> Lab on a chip. 13 (18), 3538-3547 (2013).</li><li>Vieira, U., 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=HepaRG+human+hepatic+cell+line+utility+as+a+surrogate+for+primary+human+hepatocytes+in+drug+metabolism+assessment+in+vitro.">HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro.</a> Journal of pharmacological and toxicological methods. 63 (1), 59-68 (2010).</li><li>Abu-Absi, S. F., Hansen, L. K., Hu, W. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+co-culture+of+hepatocytes+and+stellate+cells.">Three-dimensional co-culture of hepatocytes and stellate cells.</a> Cytotechnology. 45 (3), 125-140 (2004).</li><li>Leite, S. B., Wilk-Zasadna, I., 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=Three-dimensional+HepaRG+model+as+an+attractive+tool+for+toxicity+testing.">Three-dimensional HepaRG model as an attractive tool for toxicity testing.</a> Toxicological sciences. 130 (1), 106-116 (2012).</li><li>Chiquet-Ehrismann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenascins.">Tenascins.</a> The international journal of biochemistry &amp; cell biology. 36 (6), 986-990 (2004).</li><li>Sidgwick, G. P., Bayat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+molecules+implicated+in+hypertrophic+and+keloid+scarring.">Extracellular matrix molecules implicated in hypertrophic and keloid scarring.</a> Journal of the European Academy of Dermatology and Venereology JEADV. 26 (2), 141-152 (2012).</li><li>Ata&#231;, B., Wagner, I., 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=Skin+and+hair+on-a-chip:+in+vitro+skin+models+versus+ex+vivo+tissue+maintenance+with+dynamic+perfusion.">Skin and hair on-a-chip: in vitro skin models versus ex vivo tissue maintenance with dynamic perfusion.</a> Lab on a chip. 13 (18), 3555-3561 (2013).</li><li>Wu, M. -. H., Huang, S., Lee, G. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+cell+culture+systems+for+drug+research.">Microfluidic cell culture systems for drug research.</a> Lab on a chip. 10 (8), 939-956 (2010).</li><li>Schanz, J., Pusch, J., Hansmann, J., Walles, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularised+human+tissue+models+A+new+approach+for+the+refinement+of+biomedical+research.">Vascularised human tissue models A new approach for the refinement of biomedical research.</a> Journal of Biotechnology. 148 (1), 56-63 (2010).</li><li>Holnthoner, W., Hohenegger, 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=Adipose-derived+stem+cells+induce+vascular+tube+formation+of+outgrowth+endothelial+cells+in+a+fibrin+matrix.">Adipose-derived stem cells induce vascular tube formation of outgrowth endothelial cells in a fibrin matrix.</a> J Tissue Eng Regen Med. , (2012).</li><li>Dunn, J. C. Y., Yarmush, M. L., Koebe, H. G., Tompkins, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+function+and+extracellular+matrix+geometry:+long-term+culture+in+a+sandwich+configuration.">Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration.</a> FASEB Journal. 3, 174-177 (1989).</li><li>Leclerc, E., Sakai, Y., Fujii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+PDMS+(polydimethylsiloxane)+bioreactor+for+large-scale+culture+of+hepatocytes.">Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes.</a> Biotechnology progress. 20 (3), 750-755 (2004).</li><li>Kim, M. S., Yeon, J. H., Park, J. -. 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+microfluidic+platform+for+3-dimensional+cell+culture+and+cell-based+assays.">A microfluidic platform for 3-dimensional cell culture and cell-based assays.</a> Biomedical microdevices. 9 (1), 25-34 (2007).</li><li>Prot, J. -. M., Aninat, 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=Improvement+of+HepG2/C3A+Cell+Functions+in+a+Microfluidic+Biochip.">Improvement of HepG2/C3A Cell Functions in a Microfluidic Biochip.</a> Biotechnology and bioengineering. 108 (7), 1704-1715 (2011).</li><li>Riccalton-Banks, L., Liew, C., Bhandari, R., Fry, J., Shakesheff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+functional+liver+tissue:+three-dimensional+coculture+of+primary+hepatocytes+and+stellate+cells.">Long-term culture of functional liver tissue: three-dimensional coculture of primary hepatocytes and stellate cells.</a> Tissue engineering. 9 (3), 401-410 (2003).</li><li>Powers, M. J., Domansky, 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+Microfabricated+Array+Bioreactor+for+Perfused+3D+Liver+Culture.">A Microfabricated Array Bioreactor for Perfused 3D Liver Culture.</a> Biotechnology and Bioengineering. 78 (3), 257-269 (2002).</li></ol>

乌韦·马克思TissUse GmbH的产生和销售在文章中使用的多器官芯片平台CEO。 本出版物的经费是由授予康宁公司获奖

这里所描述的MOC平台代表了稳定而强大的工具，培育各种来源的组织，在动态中流动状况在长期的文化时期10,16。在本实施例中，平台被用于培养原代细胞（HDMEC），来自细胞系产生的组织等同物（肝聚集体），以及前述的共培养与组织活检。操作中心是能够维持三组织共培养长达28天的联合介质回路。以最好的作者的知识，这是第一次已经超过4周进行了多组织共同培养包括活检，原代细胞和细胞系。 一个微流体系统的主要缺点之一是小分子附着于流体回路的表面材料的亲合性。作为表面与体积之比是在微流体系统中特别高的，这样的效果变得更加明显17 </s了&gt;。稳定HDMEC覆盖的通道的，在这里引入，可能作为生物屏障防止分子的粘附到信息操作中心。此外，它可以作为全血循环一个血液相容容器中，防止血液凝固。然而，使用全血作为介质取代是不可行的器官当量的全血管尚未实现。现有的体外组织-生成的血管作品是有前途的，引导的方式继续深造18,19。 它是众所周知的，肝细胞倾向于在静态二维体外培养条件下20随时间失去肝特异性功能。代谢酶，如细胞色素P450家族，是特别重要，如果某药物的代谢是待研究。细胞色素P450 3A4，涉及到许多外源物的生物转化酶，和细胞色素P450 7A，瓦特HICH参与胆汁酸的合成，表达在肝凝集在MOC培养超过14天。这表明代谢活性的表型的保存，允许药物代谢研究。聚集体在比静态培养物中的MOC增加白蛋白生产速率是一个额外的指征适当的培养条件。在这项研究中观察到的白蛋白生产速度是相当的，甚至高于通过微流控芯片，包括HepG2细胞21获得先前报道的价值- 23，但是，值没有达到这些人原代肝细胞培养24。此外，MOC系统，在其临时布局，不允许胆汁的单独隔离。细胞在总量偏振光形成胆小管样结构，如由MRP-2染色。然而，这些小管没有连接到一个信道的技术收集胆汁。这种非生理混合荷兰国际集团与血液隔室中的胆汁在系统的未来重新设计加以解决。 的流量特性的调整是非常重要25，特别是关于组织敏感剪切应力，如肝脏。剪应力由组织所感知的量可以用两种方式进行修改:首先，用于向下推动泵的膜的空气压力可以被降低，在系统中减少峰值剪切应力值。其次，组织可以嵌入在细胞外基质分层或在Transwell小培养插入物进行培养。后者屏蔽从底层当前的组织中的多孔膜。这些调整必须在开始MOC实验前对每个器官相当于在个人基础上进行的。以2.4赫兹的脉动操作中，例如，其对应于144次高，但仍然生理，心脏活动/分钟在人类中，所测定的剪切应力微血管电路的渠道达到约25达因/厘米2。这对应于生理剪切应力在刻度中微血管的较高端的，因此，也适用于实验，包括所述信道的内皮化。然而，由于提出的MOC系统的当前微流体布局仅由一个连接两个器官室介质电路，一泵送速度和剪切应力速率已被选择为整个系统。因此，流动特性，以各单一器官的需要精确的调整并不总是可行的。 此外，保健已采取在调节细胞的共同平台。培养细胞在MOC在组合媒体电路，因此，可用于每种组织模型中没有个别的细胞培养基，如在体外细胞培养的标准。一个最小的组合培养基配方需要事先和T定义他的细胞需要调整逐步到这个新的介质。的80％/ 20％旧到新媒体两天的调整过程中，然后用50％/ 50％，其次是20％/ 80％，并充分交换总是导致培养物在我们手中的一个合理的细胞活力和功能。 信息操作中心系统的当前微流体布局允许多达三个组织的共培养。人体中的至少十个最重要的器官的共存是必要的，以达到动态平衡。因此，提出的系统是能够预测特定组织 - 组织相互作用，而不是一种物质的真实全身反应。商务部的进一步发展，包括更多的器官腔设想。此外，该系统的有效性，是使用一组参考化合物显示。优选地，在临床试验期间（如曲格列酮）已经失败的化合物是其在MOC性能进行测试。然而，这样的复杂系统的真实有效性仍是阻碍BŸ有关的生物标志物和端点的功能评价缺乏标准化，收集有关这一点，类似系统的毒性表现更多的数据将扩大其可靠性和应用领域。

当前单层或悬浮细胞培养测定为药物开发没有能够模仿人类细胞微环境，因此，导致快速去分化和功能的原代人细胞培养物的损失。需要组织模型具有较高生理相关它们接纳于临床试验之前，预测化合物的功效和安全性。最近， 体外细胞培养技术的标准已经从二维单层培养进化朝向三维的多细胞模型，旨在模仿体内组织的微环境。这些系统已经显示出对化合物1,2的作用模式的更准确的预测重大改进。此外，适应在体外培养条件下细胞的高度专业化的需求是特别令人感兴趣的。 在标准的体外条件下，各种重要的尽头TURE参数，如营养和氧气供应，去除累积的产品，和机械力作用在细胞通常不能在大多数情况下，充分进行控制。许多器官具备的物质和溶解氧生理学相关浓度梯度。然而，这些高度调节和优化的条件，在体外条件下，明确反对周围组织中的不可控的扩散梯度，导致一个非常不稳定的环境中，以及限制细胞发展3。因此，更稳定，尤其是在体外条件更加量化的需要，以保持细胞的存活和分化的多长时间。灌注系统，其中培养基成分，定期删除和取代，往往是更好的表征和可控比关于直接周围组织的静态文化。在静态条件下，细胞的分泌物扩散梯度和培养液中的营养成分可能围绕培养细胞3。周围的组织引入良好的特点中型流速使细胞的分泌物混合通过灌注丰富的媒介。这使得能够确定蜂窝微环境的产生，从而确保了稳定的细胞表型，并在整个试验持续时间4代谢酶的表达。 在多器官芯片（MOC）的最新发展为基础的系统，结合了控制介质流动的周围组织工程与生物反应器微观的中小型和细胞质量的要求带来的好处，从而导致物质的测试过程中需要的量减少。几种微流体系统用于组织培养已经描述迄今5,6。这些系统在组织内对流体的比率起到模拟生理学相关细胞串扰一个特别重要的作用。但是，由于技术的限制，例如使用外部泵和介质储层，第相比于组织体积E综合循环介质在大多数系统体积过大。该集团舒勒等人都是第一次来开发，确保在细胞培养室和体内相关组织，以流体比7,8物质的适当停留时间的系统。这是通过按比例缩放所述外部容器下降到96孔板，也代表了"其他组织"隔室来实现的。为了最大限度地减少了操作中心平台中的循环介质体积，我们集成蠕动片上的微型泵，省去了外部介质电路。这个微型泵能够在介质的流速和剪切应力速率9的可选择数操作该系统。 500微米的宽度和100μm的高度的微流控通道系统互连两个标准化组织培养空间，每一个都具有一个96孔板的单个孔的大小。坚持行业标准孔板大小S允许在透孔格式制作已经存在的组织模型的集成。此外，transwell小细胞培养插入物的垂直位置是可调的，从而使组织模型它们不仅直接暴露于流体流的培养，但也可以被提起并从底层当前屏蔽。同样地，空气 - 液体界面的文化为使用本系统时是可行的。 信息操作中心平台由聚二甲基硅氧烷（PDMS）层2毫米高，并与一个足迹75×25平方毫米 ，这是由低压等离子体氧化永久性地粘接，以形成不透流体的微流体回路的玻璃显微镜载片制造。通过标准软光刻和副本模塑9制造含有的各通道和细胞培养室的PDMS层。在这项研究中所使用的MOC的微流体设计，包括了每个芯片两个独立的微电路，各持2单元Culture车厢由通道系统100微米高的互连。这允许使用一种多器官芯片两个单独的两组织共培养物的性能。泵送频率被调整，得到40μl/ min的介质的流速。 在生理流动条件下，结合媒体的电路，这两个组织MOC设计提供在共存肝球体和皮肤穿刺活检在不同的文化空间，能力虽然。分化HepaRG细胞用在24比人类肝星状细胞（HHSteC）聚合在一起:1，以形成均匀的球状体。此比率被发现是最佳的，因为在以前的实验中观察到10，即使，肝细胞的近两倍的数量被用来相对于在体内的情况。皮肤栽培在内侧的transwell小细胞培养插管的空气 - 液体界面，从而使局部物质暴露。这些组织模型共培养了28天在MOC AYS证明该系统的全面性。此外，该芯片的微流体通道电路被充分覆盖人皮肤微血管内皮细胞（HDMEC）更紧密地模拟血管系统。

<table><tbody><tr><td>HepaRG cells</td><td>Biopredic International</td><td></td><td>undifferentiated cells</td></tr><tr><td>HHSteC</td><td>ScienCell Research Laboratories</td><td></td><td>cells and all culture supplements</td></tr><tr><td>HepaRG Medium</td><td>Sigma-Aldrich</td><td></td><td>William&#039;s Medium E&lt;br /&gt;10% FCS&lt;br /&gt;100 U/ml penicillin&lt;br /&gt;100 &amp;micro;g/ml streptomycin&lt;br /&gt;5 &amp;micro;g/ml human insulin&lt;br /&gt;2 mM L-glutamine&lt;br /&gt;5 x 10&lt;sup&gt;-5&lt;/sup&gt; M hydrocortisone hemisuccinate</td></tr><tr><td>HDMEC Medium</td><td>PromoCell</td><td></td><td>Endothelial Cell Growth Medium MV2 with Supplement-Pack MV2 and 1% penicillin-streptomycin</td></tr><tr><td>Dimethyl sulfoxide</td><td>Carl Roth</td><td></td><td>add 2% to HepaRG media</td></tr><tr><td>Trypsin/EDTA</td><td>Biowest</td><td></td><td></td></tr><tr><td>Trypsininhibitor</td><td>Carl Roth</td><td></td><td></td></tr><tr><td>MAXYMum Recovery Tips</td><td>Corning</td><td></td><td>1,000 &amp;micro;l Pipet Tips Wide Bore</td></tr><tr><td>384-Well Hanging Drop Plate</td><td>3D Biomatrix</td><td></td><td>Perfecta 3D 384-Well Hanging Drop Plate</td></tr><tr><td>Tissue culture flasks</td><td>Corning</td><td></td><td>75 cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Ultra-low attachment plate</td><td>Corning</td><td></td><td>24-well</td></tr><tr><td>Transwell cell culture inserts</td><td>Corning</td><td></td><td>96-well unit, 0.4 &amp;micro;m pore size</td></tr><tr><td>Deep well plates</td><td>Corning</td><td></td><td>96-well, 1 ml</td></tr><tr><td>Biopsy punch</td><td>Stusche</td><td></td><td>4.5 mm</td></tr><tr><td>Glass microscope slide</td><td>Menzel</td><td></td><td>footprint of 75 x 25&amp;nbsp;mm</td></tr><tr><td>Polydimethylsiloxane</td><td>Dow Corning</td><td></td><td>Sylgard 184</td></tr><tr><td>Silicon rubber additive</td><td>Wacker Chemie</td><td></td><td>Wacker Primer G790</td></tr><tr><td>Tubes for air pressure</td><td>SMC Pneumatik GmbH</td><td></td><td>Polyurethan-Schlauch, metrisch</td></tr><tr><td>Alumin ELISA</td><td>Bethyl Laboratories</td><td></td><td>Human Albumin ELISA Quantitation Set</td></tr><tr><td>Lactate dehydrogenase assay</td><td>Stanbio Laboratory</td><td></td><td>LDH Liqui-UV kit</td></tr><tr><td>Alexa Fluor 594 acetylated LDL</td><td>Invitrogen</td><td></td><td>1 mg/ml</td></tr></tbody></table>

the multi-organ chip - a microfluidic platform for long-term multi-tissue coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

标准的体外组织培养物是在静态条件下进行的，这限制了氧气和营养供应到组织的扩散。流体系统，表现出改善电源特性，常常被其庞大的中等要求的阻碍，有非生理高，中到组织的比率。因此，代谢物和稀释的细胞不能够调节他们的周围环境。在本研究中提出的信息操作中心连接两个独立的组织培养室中，一个标准的96孔板的单个孔的每个的大小，通过微流体通道系统。在小规模的系统，并在芯片上的泵的整合允许系统在只有200至800微升介质卷操作。这对应于一个总的全身介质8的组织比:1至31:1，分别为肝脏和皮肤组织共培养物（具有约26微升的总的组织体积）。在一个人体重7总细胞外液容量3千克是14.6升，虚耗损intercapillary流体体积是5.1升，导致生理细胞外液为1组织的比例:4。因此，媒体在整个循环系统中MOC的量仍然较大相比的生理状况;然而，它代表了最小的媒体组织比迄今为止所报道的多器官系统5。作为工业标准的组织培养格式被保留，研究人员能够在一个共同的流体流动结合现有的和已验证的静态组织模型。 图1示出的实验装置的可能的MOC单一组织或多层组织共培养物的示意图。主组织切片和体外组织-生成当量从细胞系或原代细胞可以培养或者使用96孔细胞培养插入或通过直接将它们放置到组织培养室中。作为信道系统互连的细胞培养室仅为100μ米高，组织现金等价物超过这些尺寸将保持文化车厢内。商务部电路主要HDMECs的内皮使迈向更符合生理培养条件进一步措施提供了一个生物血管结构。 <img alt="图1" src="/files/ftp_upload/52526/52526fig1highres.jpg" width="700" /> 图1:MOC培养物的示意图组织等同物标准的体外条件下制备，接种到信息操作中心，并培养作为动态条件下的单培养物或共培养物。每日媒体样品和端点分析执行。气压驱动泵是通过从上面连接到MOC的三个蓝色管子应用。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52526/52526fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </a&gt; 继内皮协议，是在四天内动态培养所获得的微流体通道电路的汇合HDMEC覆盖， 如图2。细胞容易粘附到操作中心通道的壁中，创建一个汇合单层，并沿剪切伸长应力（ 图2B）。此外，细胞覆盖的信道的全周，如先前报道9。四天后，培养，观察直到培养结束内皮形态没有进一步的变化。 <img alt="图2" src="/files/ftp_upload/52526/52526fig2.jpg" /> 图2:内皮MOC通道人皮肤微血管内皮细胞（HDMEC）形成在微流体回路的汇合单层。细胞后MOC文化23天沾上乙酰化LDL。 （A）整米icrovascular电路嵌着沿剪切应力伸长细胞和（B）的细胞。标尺:（A）1000μm和（B）100微米。 在进一步的实验中，一致的盘形肝细胞的球状体是由HepaRG和HHSteC期间悬滴培养两天形成，因为这个模型系统先前报道的适用于药物代谢研究11 - 13。出于演示的目的，每个MOC电路的一个组织培养室中，接种20球状体在96孔细胞培养插入和组织中使用非内皮MOCS栽培动态条件下超过14天。任何数量的聚集体或原材料的量可以被集成或者直接进入隔室或使用细胞培养插入。从MOC检索后球体的免疫荧光染色显示出强大的，同质的表达李VER-典型细胞角蛋白8/18和I期代谢酶细胞色素P450 3A4和7A1（ 图3A和3B）。泪小管转运多药耐药蛋白2的染色（MRP-2）揭示了偏振光的表型和基本的胆小管状网络的存在（ 图3C）。 <img alt="图3" src="/files/ftp_upload/52526/52526fig3.jpg" /> 图3:。培养人人工肝微组织在MOC肝聚集在MOC培养14天被染色（A）细胞角蛋白8/18（红色）及（B）细胞色素P450 3A4（红色）和7A1（绿色）。的小管转运MRP-2（绿色）（C）的表达，蓝色的核染色。比例尺:100微米。 由于生产白蛋白是肝组织文化的一个基本前提，它有蜂N的监视，在MOC肝典型活动。白蛋白生产分析每日媒体样本示出了在生产速率在​​MOC培养相比静态培养物（ 图4）和以文献报道11值显著增加。在白蛋白合成率的增加可能是由于增加的氧气和营养供应在MOC培养物。因此，信息操作中心是能够维持肝聚集体超过14天的培养期间在代谢活跃的状态，增强肝典型行为，如白蛋白的生产。 <img alt="图4" src="/files/ftp_upload/52526/52526fig4.jpg" /> 图4:在MOC十四日肝脏球体性能白蛋白产量的肝脏单一组织培养在MOC和静态文化。数据是平均值±SEM（N = 4）。 澈Subsystemic重复给药毒性试验micals和化妆品中的动物，需要接触21至28日，由OECD方针定义没有。 410"重复剂量皮肤毒性:二十八分之二十一天的研究。"长期皮肝共培养这里列举的28天，以应对监管要求。通过培养皮肤活检在96孔细胞培养插入提供购买真皮物质暴露的空气 - 液体界面。在共培养实验中示例性地执行在内皮化MOCS证明三组织共培养以组合媒体电路是否可以保持可行的和代谢活性超过28天。 在首八天培养的，其中在约80单位/升之后（ 图5）保持不变在媒体上清液中LDH活性的分析表明，一个稳定下降的水平。这表示一个人工的但稳定组织转化系统中的在稍后的时间点。比较这三个组织共培养肝单-tissue和肝脏内皮细胞共培养实验中，显著降低LDH水平可以发现，特别是在培养物中不包括皮肤的第一天。高LDH活性的这个第一周期内的细胞死亡主要发生于皮肤培养室中，如皮肤组织的单MOC培养显示（数据未示出）。这可能是由于周围活检作为皮肤的冲压的结果而受伤区域。 <img alt="图5" src="/files/ftp_upload/52526/52526fig5.jpg" /> 图5:在肝脏单一组织培养（MOC李），肝文化传媒上清液中内皮MOCS（MOC李-VA）和肝，皮肤在共同培养内皮MOC 建设部 LDH活性十五日组织绩效 （MOC李-va-SK）。数据是平均值±SEM（N = 4）。 在28天的培养期间，肝脏球状体附着在MOC一个底部第二细胞生长出来，形成相邻的球状体之间的多层连接。这并没有妨碍组织功能。通过免疫荧光端点分析表明，肝球状体仍然具有代谢活性后MOC共培养28天，如由细胞色素P450 3A4染色（ 图6A）。 HHSteC被分布在整个肝等效整体，如图波形蛋白染色（ 图6B）。在波形蛋白染色强度的增加可能在细胞已经生长出球体的区域进行观察。染色为血管性血友病因子（vWF）表明，内皮细胞没有深入到组织，但均与外肝细胞（ 图6C）的直接细胞-细胞接触。 皮肤活检的免疫组化染色可见腱C和在基底膜（ 图6D），Ⅳ型胶原的表达，而静态控制染色显示elevat编辑水平腱C（ 图6E）的。腱生蛋白C已被证实的伤口愈合，炎性过程和间质纤维化过程中被上调，提示诱导静电纤维化过程中，但不是在动态培养14,15。 稳定的细胞生存力和在28天共培养在MOC后组织的功能性证明，该系统能够保持在一个共同的媒体电路最多三个组织的结合。主细胞，以及组织模型和活检，可以在信息操作中心系统同时栽培。 <img alt="图6" src="/files/ftp_upload/52526/52526fig6highres.jpg" width="700" /> 图6:多组织培养的性能在28天肝现金等价物和皮肤活检培养于内皮化MOC和细胞功能所表现出的（A）相免疫酶我的细胞色素P4503A4（红色），（B）的波形蛋白（红色），（C）的角蛋白8/18（红色）和vWF（绿色）肝组织中。共培育在静态条件下操作中心或（E）26天（D）的皮肤活组织检查中染色腱生蛋白C（红色）和IV型胶原（绿），蓝核染色。 （F）H＆E后，MOC培养28天皮肤染色。比例尺:100微米。

Watch this Scientific Journal Video about The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture at JoVE.com

多器官芯片——用于长期多组织共培养的微流控平台|生物工程|视频

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

the-multi-organ-chip-microfluidic-platform-for-long-term-multi-tissue

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Biology

Bioengineering

Genetics

Biotechnology

SCIENTISTS IN ACTION

28.6K 次观看.  Technische Universität Berlin. Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

视频: The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

基于芯片的三维细胞培养，在灌注微生物反应器

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

科研

生物学

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 ...

生物工程

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

生成基于人类 iPSC 的血脑屏障芯片

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

发育生物学

<meta charset="utf8"></head><body>使用微生理系统进行肾癌和健康组织共培养

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating intricate morphological features in three-dimensional environments with dynamic interactions lacking in conventional models. We implemented a renal cell carcinoma (RCC) co-culture model to recreate the cross-talk between healthy and malignant renal tissue. 
This model is based on the referenced multi-organ platform and consists of co-culturing a reconstructed renal proximal tubule with RCC spheroids. Custom-designed 3D-printed chambers were used to culture human renal epithelial proximal tubule cells (RPTEC) and facilitate their self-assembly into a renal tubule contained in a collagen type I matrix. Caki-1 RCC cells were embedded in an agar collagen matrix, subsequently forming cancer spheroids. Both collagen and agar/collagen gels were optimized to maintain their integrity during cyclic perfusion and withstand shear stress during a minimum culture period of 7 days. 
The gels also enable adequate nutrient supply and cell secretions. Moreover, the agar/collagen gels limit the overproliferation of RCC cells, ensuring relatively homogeneous spheroid size. The MPS chip microfluidic circuits comprise two independent culture chambers with the size of a standard 96-microplate well. The renal tubule and RCC gels populate separate chambers and share the same culture media, which is recirculated approximately twice per minute. Under these conditions, we observed upregulation of immune factor expression and secretion in the renal tubules (interleukin-8 and tumor necrosis factor-alpha). The renal tubules also shift their metabolic activity towards glycolysis under the influence of RCC. This novel approach demonstrates that a co-culture-based MPS can amplify the complexity of RCC in vitro and be employed to study the impact of cancer on non-tumor cells.

<meta charset="utf8"></head><body>肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

Here, we present protocols that detail instructions for implementing 3D cell cultures using collagen and collagen-agarose matrixes in a microphysiological system. These protocols support renal proximal tubule and renal cell carcinoma spheroids co-culture, simulating in vivo conditions and enabling advanced investigation of kidney cancer cell interactions.

肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating ...

癌症研究

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

在微流体进化加速器上生成跨癌症人群的异构药物梯度，用于实时观察

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

多器官芯片 - 一种微流体平台的长期多组织共培养

摘要

探索更多视频

此视频中的章节

基于芯片的三维细胞培养，在灌注微生物反应器

高通量单细胞的分离和文化的微流体平台

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

生成基于人类 iPSC 的血脑屏障芯片

肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

可伸缩、双通道、微流控芯片制造

在微流体进化加速器上生成跨癌症人群的异构药物梯度，用于实时观察

使用微流体芯片以确定性方式建立基于单细胞的共培养物