Name: co-culture of living microbiome with microengineered human intestinal villi in a gut-on-a-chip microfluidic device
Uploaded: 2016-08-30T15:00:00
Description: 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

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?...

<ol>
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S., 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=Communicable+ulcerative+colitis+induced+by+T-bet+deficiency+in+the+innate+immune+system.">Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system.</a> Cell. 131, 33-45 (2007).</li><li>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=Gut-on-a-Chip+microenvironment+induces+human+intestinal+cells+to+undergo+villus+differentiation.">Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation.</a> Integr Biol. 5, 1130-1140 (2013).</li><li>Huh, D., Kim, H. 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=Microfabrication+of+human+organs-on-chips.">Microfabrication of human organs-on-chips.</a> Nat Protoc. 8, 2135-2157 (2013).</li><li>Kim, H. J., Li, H., Collin, J. 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=Contributions+of+microbiome+and+mechanical+deformation+to+intestinal+bacterial+overgrowth+and+inflammation+in+a+human+gut-on-a-chip.">Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip.</a> Proc. Natl. Acad. Sci. 113, E7-E15 (2016).</li><li>Miller, W. G., Lindow, S. 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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 ...

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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

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

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

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 ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

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

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 ...

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

使用高通量微流体设备生成动态环境条件

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

Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

生理尺寸微流控培养装置中上皮细胞单层的制备和结构评价

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as ...