We thank Sri Kosuri (Wyss Institute at Harvard University) for providing the GFP-labeled E. coli strain. This work was supported by the Defense Advanced Research Projects Agency under Cooperative Agreement Number W911NF-12-2-0036, Food and Drug Administration under contract #HHSF223201310079C, and the Wyss Institute for Biologically Inspired Engineering at Harvard University. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Office, Army Research Laboratory, Food and Drug Administration, or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation hereon.

1.肠道的单芯片器件的微细加工注意:肠道上的单芯片是由透明的，透气的聚硅氧烷聚合物（聚二甲基硅氧烷，PDMS）制成的微流体器件，包含由柔性分隔的两个平行微通道（1毫米宽×150微米的高度×1厘米长）多孔（10微米孔径，25微米间距的孔到孔）PDMS膜5,9。制造肠道上的单芯片（ 图1A，左）以下提供的步骤。 <ol><li>在肠道上的单芯片5,9的微细加工过程。 <ol><li>通过混合的PDMS预聚物和固化剂的制备未固化的，脱气的PDMS（15:1重量/重量），并且将其放置在真空干燥器30分钟。 </li><li>倾30克和3克未固化的，脱气的PDMS上具有上部的SU-8基于微图案和肠道上的单芯片的下微通道，分别各硅模具。然后在60℃的干燥烘箱中固化为L东四小时。 </li><li>通过使用外科手术刀周围边缘切割脱模从各硅模具的上部和下部的PDMS层。冲床用活检冲床（2.0毫米孔径）6孔（两个进口，两个出口和两个真空室）。 </li><li>通过含有交阵列与平面重叠圆形支柱（10微米的直径，在高度为25μm）硅烷化的硅晶片上浇注10克未固化的和脱气的PDMS制备多孔膜，硅烷化的PDMS支撑层（15:1，W / W; 1厘米厚）。放置在设定为3公斤的重量压并在60℃下固化该聚合物12小时或更长的时间。 </li><li>脱模通过仔细举起从晶片板坯的角部​​，和剥离，直至多孔PDMS膜被完全从晶片脱离粘附到从硅晶片的PDMS支承板坯的多孔PDMS膜的设置。 </li><li>露出的上部层的沟道侧（PDMS，15:1，W / W）和多孔膜侧到复由一手持电晕产生阿斯马贾为分别为1分钟，3秒。 </li><li>通过将并联每一块无任何气泡放置在一个共形接触的多孔PDMS膜和上微通道层的等离子体处理的表面。 </li><li>孵育80°CO / N整个安装两个PDMS层的粘接不可逆转。 </li><li>通过仔细地抬起支承层的角部，和剥离，直到支撑层完全从与膜的上层脱落剥离从与多孔膜的上层的组装与PDMS支撑层。 </li><li>撕下这张膜位于比用细尖镊子，使空心真空室真空室（位于主通道两侧的两个腔室）的部分。 </li><li>暴露附着在顶片和与刻在下部片以相同的方式在等离子体通道的一侧的膜的一侧所描述我n阶1.1.6 1分钟。 </li><li>通过将并联每一块未经立体镜之下的任何气泡对准的多孔膜的上微通道层和下部层用共形接触。 </li><li>固化在干燥炉中的整个设置在80℃CO / N以产生含有旁边的微流体单元沟道两个中空真空腔室的完整规模肠道上的单芯片微流体装置。 </li></ol></li><li>通过一个90°的弯曲不锈钢钝端针连接管将连接到上部或下部微通道在肠上的单芯片每个端口的入口和出口。 </li><li>连接管连接到连接于使用一90°的弯曲不锈钢钝端针头的真空​​室中的孔。 </li><li>由流用1毫升灭菌的一次性注射器70％（V / V）乙醇消毒微通道和管道。 </li><li>干出在60℃的烘箱O / N微通道和管道。 </li></ol> 2.微引擎的增长ERED肠绒毛在肠道上的单芯片器件<ol><li>种子肠上皮细胞（ 如 :的Caco-2BBE）上的肠上的单芯片微型5,9。 <ol><li>消毒装置，用70％（V / V）乙醇（参见步骤1.4），使用1毫升灭菌的一次性注射器，随后在60℃干燥烘箱õ干燥/ N（步骤1.5）之前的表面活化。 </li><li>暴露肠道上的单芯片微系统的完整的设置（ 即，一体的肠上的单芯片连接于管）于UV光（253.7纳米）和臭氧同时40分钟。 </li><li>冷静下来RT在该设备用于在生物安全柜（BSC）在UV光下15分钟。 </li><li>引入100微升的ECM涂覆的溶液 - 50微克/毫升的I型胶原并在无血清的Dulbecco改良的Eagle培养基（DMEM）稀释300微克/毫升的细胞外基质混合物的混合物 - 使用一次性成上下两个微通道无菌1ml注射器。 </li><li> Incuba忒整个设置在37℃，湿度5％， 二氧化碳培养箱1小时。 </li><li>德加50毫升的预热（37℃）完全培养基（DMEM，20％，体积/体积，胎牛血清（FBS），100单位/ ml青霉素和100微克/毫升链霉素;在一个3毫升的等分一次性注射器），使用在BSC 50毫升过滤系统（0.45微米孔径）为1分钟。 <ol><li>通过过滤系统通过预热介质和过滤介质上轻轻拍打1分钟以除去培养基中的任何气泡或溶解的气体。 </li></ol></li><li>放置脱气完全培养基的等分试样在3mL的一次性注射器和孵育在37℃潮湿的5％CO 2的培养箱中培养1小时。 </li><li>连接含有脱气23毫升一次性注射器，预热的完全培养基中的上和下微通道。 </li><li>使用注射泵在体积流动的脱气完整细胞培养基进入上部微通道在37℃流30微升/小时的速率（相当于剪切应力以0.02达因/ cm 2）的加湿5％CO 2培养箱O / N。 </li></ol></li><li>在肠道上的单芯片微工程的Caco-2绒毛的形成。 <ol><li>使用的Caco-2BBE细胞用50和65之间的通道数目。 </li><li>生长的Caco-2细胞在含有完全培养基在37℃的T75烧瓶加湿5％CO 2的培养箱中4-5天，以获得充分融合细胞。 </li><li>加入10 mL 的 Ca 2+和Mg 2+ -free PBS充分在T75烧瓶中生长汇合Caco-2细胞，洗涤细胞，然后吸出PBS中。重复此步骤两次。 </li><li>加入1ml 0.05％胰蛋白酶/ EDTA溶液孵育在37℃潮湿的5％CO 2培养箱5分钟。 </li><li>通过上下吹打三至五倍重悬离解的细胞用10ml预热完全细胞培养基（含FBS）。 </li><li>降速C语言对细胞悬液entrifugation在500 XG 5分钟，然后取出上清。 </li><li>悬浮再次用1ml预热完全培养基在器件〜1.5×10 5个细胞/ cm 2至调整细胞密度。使用血细胞计数器估算细胞密度。 </li><li>通过将附着于连接到上部微通道管的出口25的G5 / 8针1毫升一次性注射器注入100微升重新悬浮的Caco-2细胞进入上部微通道（内腔侧）。 </li><li>钳连接到用长尾夹在上下微通道的所有入口和管道的出口。 </li><li>孵育在37℃，湿润的5％CO 2培养箱整个设置1小时，以允许解离Caco-2细胞附着在多孔膜的表面上。 </li><li>除去夹具和使用注射泵，直至细胞形成24-36小时完整单层恢复培养基的流动仅在30微升/小时的上微通道。使用阶段反政府T或微分干涉对比（DIC）显微镜来确认在细胞单层细胞 - 细胞连接。 </li><li>当细胞形成单层，灌注在30微升/小时的相同的流速培养基成两个上（腔）和下（毛细管）微通道。 </li><li>机械变形模仿蠕动状运动5,9中的应用。 <ol><li>打开装有张力设备的真空泵。 </li><li>通过管道用不锈钢接头连接真空室与真空控制器。 </li><li>设置10％的平均细胞株的伸展运动在0.15赫兹与真空控制软件循环正弦函数模式的频率，然后单击"开始"。 </li></ol></li><li>维持培养基的两个上和下微通道下机械变形为〜100小时的恒定流量（30微升/小时）至汇合的Caco-2单层（10％，0.15赫兹）。 注意:Caco-2细胞SPONtaneously经历与波纹三维凸起延伸朝向上皮微通道5,8-10的内腔绒毛形态。 </li></ol></li><li>与在肠上的单芯片10流明毛细管组织界面模拟人体活肠的器官级的功能，按照步骤如上所述。 <ol><li>重复步骤2.1至2.2微工程增长的Caco-2绒毛。 </li><li>流的预热共培养培养基（完整的Caco-2细胞培养基和完整HMVECs培养基的混合物，1:1体积/体积）在30微升/小时的上下微通道。 </li><li>引入100微升解离的正常人毛细血管微血管内皮细胞（HMVECs;最终细胞密度，〜5.0×10 5个细胞/ cm 2）到由在步骤从2.2.3至2.2.9中描述的相同方法下微通道。 </li><li>放置固化矩形的PDMS片（0.5厘米×1厘米×1厘米;宽×长度×高度; 15:1，W/ w的弹性体:固化剂）在肠道上的单芯片器件的顶部，然后翻转整个设备设置倒挂（ 即上微面临的下行，下微面临上档）。 </li><li>孵育在37℃，湿润的5％CO 2培养箱设置为1小时，以允许解离HMVECs附着在下部微多孔膜的表面上。 </li><li>取出从二氧化碳培养箱整体设置，并翻转安装一遍。 </li><li>流预热共培养培养基（完整的Caco-2细胞培养基和完整HMVECs培养基的混合物，1:1体积/体积）为在30微升/小时以机械拉伸运动上下两个微通道（ 10％，0.15 Hz），并且至少三天以形成内皮细胞单层的细胞 - 细胞连接。 </li></ol></li></ol> 3.主机肠道微生物共培养在肠道上的单芯片微型<ol><li>执行预培养ØF中的细菌细胞如下的微工程小肠绒毛共同培养共生肠道微生物。 <ol><li>重悬在10ml的混合物的冷冻干燥益生菌粉末混合物:高压灭菌乳酸菌MRS肉汤和钢筋梭菌培养基（1:1，V / V）。 </li><li>通过在10ml的一次性无菌管中添加介质的适量，然后等分3毫升微生物细胞悬浮液调整最终细胞密度约为0.2光密度单位（在600nm）。 </li><li>含有悬浮的微生物细胞在厌氧容器地方管。添加厌氧气体生成小袋的两个包在容器中。紧紧关闭容器盖和孵化容器设置而没在37℃下振荡加湿5％ 二氧化碳培养箱O / N。 </li></ol></li><li>微生物组和共培养肠上皮细胞5,10接种。 <ol><li>准备包含脱气antibioti3毫升一次性注射器的c-无细胞培养基（ 即，DMEM，用20％FBS）中。对于培养液的脱气过程见2.1.6。 </li><li>取出从二氧化碳培养箱含微工程绒毛肠道的单芯片器件的整体设置，然后进入到生物安全柜。 </li><li>移除连接到管道连接到上部和下部微注射器。连接3.2.1准备设备注射器，带回的二氧化碳培养箱，那么这种流动不含抗生素的培养基中培养12小时微生物播种之前。 </li><li>降速预培养的益生菌细菌混合物的细胞（见3.1的步骤）以10,000 xg离心5分钟。吸出使用真空上清，然后重悬在无抗生素的DMEM培养基中（终细胞密度，〜1.0×10 7 CFU / ml）中。 </li><li>注入细胞悬液到使用连接到25 1毫升一次性注射器含有"无菌"肠绒毛的微通道的内腔侧G5 / 8针。允许微生物细胞的肠绒毛的顶端表面的粘附为〜而不流1.5小时通过夹紧到所有管端。 </li><li>灌注预热无抗生素培养基成上下两个微通道，在40微升/小时与环状节奏变形（10％，0.15赫兹）。 </li><li>绿色荧光蛋白的共培养（GFP）标记的大肠杆菌大肠杆菌 （GFP 大肠杆菌 ;非致病性DH5-α- 大肠杆菌宿主）-10,11-细胞用微工程绒毛，预培养的GFP E.大肠杆菌细胞中，在37℃下12小时振荡条件（200rpm）下蒸压LB培养基。重复该过程从3.2.5至3.2.6进行GFP 大肠杆菌的共培养大肠杆菌细胞与微工程绒毛。 </li></ol></li><li>细胞图像5,8-10 <ol><li>记录微生物和上皮形态5,8,10执行DIC，萤光，或激光扫描共聚焦显微镜。 <ol><li>对于DIC成像，取出肠的单芯片微系统的设置从二氧化碳培养箱，将设备安装在显微镜的阶段，然后记录细胞形态。 </li><li>对于绒毛上皮荧光成像，流PBS中洗涤，在100微升/小时的细胞10分钟。 <ol><li>固定绒毛用4％（重量/体积）低聚甲醛15分钟。然后流PBS中洗涤，在100微升/小时的细胞10分钟。 </li><li>用0.3％的透化绒毛（体积/体积）的Triton X-100稀释在含有PBS中2％（重量/体积）牛血清白蛋白（BSA）10分钟。然后流PBS中洗涤，在100微升/小时的细胞10分钟。 </li><li>块的细胞，用2％（重量/体积）1小时的PBS BSA溶液。然后流PBS中洗涤，在100微升/小时的细胞10分钟。 </li><li>添加300nM的4&#39;，6-二脒基-2-苯基二盐酸盐（DAPI）溶液在PBS中稀释为下光保护的核染色。 </li><li>添加25单位/毫升荧光鬼笔的（鬼笔环肽-CF647共轭）溶解在PBS的F-肌动蛋白染色光下的保护。 </li><li>使用激光扫描共聚焦显微镜荧光染色的细胞图像记录。 注:25X的目标是在激光共聚焦显微镜在适当的光学变焦应用。在图3B中，使用约525X放大倍数。 </li></ol></li></ol></li></ol></li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+characteristics+of+digesta+and+their+influence+on+flow+and+mixing+in+the+mammalian+intestine:+a+review.">Physical characteristics of digesta and their influence on flow and mixing in the mammalian intestine: a review.</a> J Comp Physiol B. 178, 673-690 (2008).</li><li>Granato, 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=Cell+surface-associated+lipoteichoic+acid+acts+as+an+adhesion+factor+for+attachment+of+Lactobacillus+johnsonii+La1+to+human+enterocyte-like+Caco-2+cells.">Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells.</a> Appl Environ Microbiol. 65, 1071-1077 (1999).</li><li>Dewhirst, F. E., 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+human+oral+microbiome.">The human oral microbiome.</a> J Bacteriol. 192, 5002-5017 (2010).</li><li>Grice, E. A., Segre, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+skin+microbiome.">The skin microbiome.</a> Nat Rev Microbiol. 9, 244-253 (2011).</li><li>Hay, P. E., 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=Abnormal+bacterial+colonisation+of+the+genital+tract+and+subsequent+preterm+delivery+and+late+miscarriage.">Abnormal bacterial colonisation of the genital tract and subsequent preterm delivery and late miscarriage.</a> Br Med J. 308, 295-298 (1994).</li></ol>

Donald E. Ingber is a founder of Emulate Inc., holds equity in the company and chairs its Scientific Advisory Board. The other authors have no financial disclosures.

了解主机微生物的相互作用，是推进医药至关重要的;然而，在一个塑料盘或静态孔板进行传统的细胞培养模型不支持人肠细胞的稳定共培养与活肠道微生物超过1-2天，因为微生物细胞大多长满哺乳动物细胞在体外 。的过度生长的微生物数量迅速消耗氧气和营养物，随后产生代谢废物（ 例如，有机酸），其严重危及肠屏障功能和引起肠上皮细胞死亡的量过多。因此，防止了导致营养?...

人体肠道窝藏微生物物种的一个惊人的多样化（&lt;1000种）和微生物细胞的巨大数目（比人类宿主细胞的10倍）和基因（除人类基因组的100倍）1。这些人类微生物组起到代谢营养物和外源物，调节免疫应答，和维持肠稳态2关键作用。毫不奇怪，由于这些不同的功能，共生的肠道微生物广泛调节健康与疾病3。因此，了解肠道微生物与宿主-微生物相互作用的作用是非常重要的促胃肠（GI）的健康和探索新的治疗肠道疾病4。但是，现有的体外肠模型（ 例如，静态培养物）限制宿主微生物共培养到的短时间（&lt;1天），因为微生物细胞长满 ​​和妥协肠屏障功能5。替代动物模型（ 例如，无菌的6或遗传工程小鼠7）也不会通常用于研究宿主肠道微生物串扰，因为殖民化和人类肠道微生物稳定维持都很难。 为了克服这些挑战，我们最近开发出一种仿生人"的肠道上一个芯片"microphysiological系统（ 图1A，左）模拟发生在生活人体肠道5,8主机肠道微生物的相互作用。肠道上的单芯片微型包含由柔性，多孔，细胞外基质（ECM）涂覆的膜通过人类肠上皮Caco-2细胞内衬，模仿肠腔毛细管组织界面（ 图1A分开的两个平行的微流体通道，吧）9。真空驱动的周期性节奏的变形引起的生理机械变形模仿的变化通常INDUC通过蠕动（ 图1A，右图）主编。有趣的是，当Caco-2细胞在肠上的单芯片多100小时生长，它们自发地形成三维（3D）小肠绒毛与紧密连接，顶端刷状缘，不限于基底隐窝增殖性细胞，粘液产生，增加的药物代谢活性（ 例如，细胞色素P450 3A4，CYP3A4），和增强的葡萄糖再摄取8。在这种"无菌"微环境，这是可能的共同培养的益生菌鼠李糖乳杆菌 GG或长达两周5,10-治疗地层与宿主上皮细胞益生菌细菌的混合物。 在这项研究中，我们描述了详细的协议，以在肠上的单芯片设备执行主机肠道微生物共培养延长的时间。此外，我们讨论的关键问题和潜在的挑战，要考虑这个主机微生物共培养p的广阔的应用rotocol。

<table border="0" cellpadding="0" cellspacing="0" width="1454">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:509px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 25 mM glucose and 25 mM HEPES</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">10564-011</td>
			<td style="width:544px;">Warm it up at 37&#176;C in a water bath.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:509px;">Difco Lactobacilli MRS broth</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">288120</td>
			<td>Run autoclave at 121&#176;C for 15 min.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Poly(dimethylsiloxane)</td>
			<td style="width:227px;">Dow Corning</td>
			<td style="width:175px;">3097358-1004</td>
			<td>15:1 (w/w), PDMS : cureing agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:509px;">Caco-2BBE human colorectal carcinoma line</td>
			<td style="width:227px;">Harvard Digestive Disease Center</td>
			<td style="width:175px;"></td>
			<td>Human colorectal adenocarcinoma&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Heat-inactivated FBS</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">10082-147</td>
			<td>20% (v/v) in DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Trypsin/EDTA solution (0.05%)</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">25300-054</td>
			<td>Warm it up at 37&#8451; in a water bath.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Penicillin-streptomycin-glutamine</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">10378-016</td>
			<td>1/100 dilution in DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride</td>
			<td style="width:227px;">Molecular Probes</td>
			<td style="width:175px;">D1306</td>
			<td>Nuclei staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Phalloidin-CF647 conjugate (25 units/mL)</td>
			<td style="width:227px;">Biotium</td>
			<td style="width:175px;">00041</td>
			<td>F-actin staining</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:509px;">Flexcell FX-5000 tension system</td>
			<td style="width:227px;">Flexcell International Corporation</td>
			<td style="width:175px;">FX5K</td>
			<td>Peristalsis-like stretcing motion (10% cell strain, 0.15 Hz frequency)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Inverted epifluorescence microscope</td>
			<td style="width:227px;">Zeiss</td>
			<td style="width:175px;">Axio Observer Z1</td>
			<td>Imaging, DIC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Scanning confocal microscope</td>
			<td style="width:227px;">Leica</td>
			<td style="width:175px;">DMI6000</td>
			<td>Imaging, Fluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">UVO Cleaner</td>
			<td>Jelight Company Inc</td>
			<td style="width:175px;">342</td>
			<td>Surface activation of the gut-chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type I collagen&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:175px;">A10483-01</td>
			<td>Extracellular matrix component for cell culture into the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">354234</td>
			<td>Extracellular matrix component for cell culture into the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL disposable syringe</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">309628</td>
			<td>Cell and media injection stuff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25G5/8 needle</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">329651</td>
			<td>Cell and media injection stuff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump</td>
			<td>Braintree Scientific Inc.</td>
			<td style="width:175px;">BS-8000</td>
			<td>Injection equipment into the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VSL#3</td>
			<td>Sigma-Tau Pharmaceuticals</td>
			<td>7-45749-01782-6</td>
			<td>A formulation of 8 different commensal gut microbes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reinforced Clostridial Medium</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">218081</td>
			<td>Anaerobic bacteria culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GasPak EZ Anaerobe Container System with Indicator</td>
			<td style="width:227px;">BD</td>
			<td style="width:175px;">260001</td>
			<td>Anaerobic gas generating sachet&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td style="width:175px;">157-4-100</td>
			<td>Fixing the cells for staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:175px;">T8787</td>
			<td>Permeabilizing the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:175px;">A7030</td>
			<td>Blocking agent for staining of the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corona treater</td>
			<td>Electro-Technic Products</td>
			<td>BD-20AC</td>
			<td>Plasma generator for fabrication of the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Steriflip&#160;</td>
			<td style="width:227px;">Millipore</td>
			<td style="width:175px;">SE1M003M00</td>
			<td>Degasing the complete culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:509px;">Disposable hemocytometer</td>
			<td style="width:227px;">iNCYTO</td>
			<td style="width:175px;">DHC-N01</td>
			<td>For manual cell counting</td>
		</tr>
	</tbody>
</table>

co-culture of living microbiome with microengineered human intestinal villi in a gut-on-a-chip microfluidic device

在这里，我们描述进行多品种的长期合作，培养人肠道微生物与人体肠道的单芯片器件microphysiological微工程小肠绒毛的协议。我们概括在一个微流体装置，其中，生理机械变形和流体剪切流动不断施加到模仿蠕动肠腔毛细管组织界面。在管腔微通道，人肠上皮细胞的Caco-2细胞进行培养，以形成一个"无菌"绒毛上皮和再生小肠绒毛。预培养的微生物细胞接种到腔侧建立主机的微生物生态系统。后的微生物细胞附着在绒毛的顶端表面，流体流动和机械变形被恢复，以产生一个稳定状态的微环境，其中新鲜培养基不断地供给和未结合的细菌（以及细菌废物）被连续除去。扩展共培养˚F后罗天至数周，找到多个小菌落被随机定位的绒毛之间，并且两者的微生物细胞和上皮细胞保持存活和功能的至少一个星期培养。我们的共培养协议可以适于提供用于可在各种人体器官中找到其他宿主微生物生态系统，其可在人的微生物组中编排健康和疾病中的作用的体外研究促进通用平台。

为了模拟在体外人体肠道主机微生物生态系统，它是 必要开发一种实验协议来重建的生理条件，例如蠕动状力学和流体流动下肠道细菌和人肠上皮细胞的长期稳定的共培养。这里，我们利用仿生肠道上的单芯片微型 （ 图1A）共同培养生活在直接接触的微生物细胞用活的人体绒毛为或体外周的周期以上。在肠上皮细胞中自发地形成?...

Watch this Scientific Journal Video about Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device at JoVE.com

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

我们描述的体外协议共同培养肠道微生物和小肠绒毛为使用人肠道上的单芯片microphysiological系统长时间。

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 ...

co-culture-living-microbiome-with-microengineered-human-intestinal

Research

JoVE Journal

Bioengineering

22.3K 次观看.  The University of Texas at Austin. 我们描述的体外协议共同培养肠道微生物和小肠绒毛为使用人肠道上的单芯片microphysiological系统长时间。 

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

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

由微型条纹分析技术在微流控设备混合不均匀

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

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

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微流控装置重新创建一个肿瘤微环境