Name: establishing single-cell based co-cultures in a deterministic manner with a microfluidic chip
Uploaded: 2019-09-27T14:30:00
Description: Watch this Scientific Journal Video about Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip at JoVE.com

这项工作得到了科技部（105-2628-E-400-001-MY2）和组织工程和再生医学博士项目、国立中兴大学和国家卫生研究院的资助。

1. 用软光刻制造晶圆模具
注：掩码模式数据可在我们以前的出版物14中提供。
<ol><li>在 120°C 烤箱中脱水 4 英寸硅晶圆 15 分钟。</li>
	<li>在 1，000 rpm 转速下将 4 g SU-8 2 负光刻胶旋转到 4 英寸硅片上，30 s 可形成 5 μm 厚的层（层#1）。</li>
	<li>软烘烤层#1在65°C的热板上1分钟，然后将层#1转移到95°C的热板3分钟。</li>
	<li>冷却层#1室温，将其放在半自动蒙版校准?...

<ol>
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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=Tumour+cell-derived+WNT5B+modulates+in+vitro+lymphangiogenesis+via+induction+of+partial+endothelial-mesenchymal+transition+of+lymphatic+endothelial+cells.">Tumour cell-derived WNT5B modulates in vitro lymphangiogenesis via induction of partial endothelial-mesenchymal transition of lymphatic endothelial cells.</a> Oncogene. 36, 1503 (2016).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+Blue+Exclusion+Test+of+Cell+Viability.">Trypan Blue Exclusion Test of Cell Viability.</a> Current Protocols in Immunology. 111 (1), A3.B.1-A3.B.3 (2015).</li><li>Kim, J. B., Stein, R., O'hare, M. J. <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+in+vitro+tissue+culture+models+of+breast+cancer-a+review.">Three-dimensional in vitro tissue culture models of breast cancer-a review.</a> Breast Cancer Research and Treatment. 85 (3), 281-291 (2004).</li><li>Shin, Y., 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=Microfluidic+assay+for+simultaneous+culture+of+multiple+cell+types+on+surfaces+or+within+hydrogels.">Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels.</a> Nature Protocols. 7 (7), 1247-1259 (2012).</li><li>Wu, M. H., Huang, S. B., 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>Hong, J. W., Song, S., Shin, J. H. <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+microfluidic+co-culture+system+for+investigation+of+bacterial+cancer+targeting.">A novel microfluidic co-culture system for investigation of bacterial cancer targeting.</a> Lab on a Chip. 13 (15), 3033-3040 (2013).</li></ol>

肿瘤微环境中各种细胞间的相互作用对肿瘤17的进展起着重要作用。为了理解细胞-细胞相互作用的机制，共培养系统作为一种常见的分析方法。然而，多种细胞类型和细胞本身的异质性导致了实验的复杂性和分析困难。
流体动力穿梭芯片允许通过确定性方法在培养室中进行多个单元加载，而不受稀释方法和微孔平台中的泊森分布限制的限制。通过提供高三?...

高效捕获不同类型的单细胞，并提供足够的培养空间，为多种类型的单细胞的单个细胞共培养实验1。限制稀释是准备单细胞用于此类实验的最常见方法，因为所需的设备成本低。然而，由于泊森分布的限制，最大单细胞采集概率仅为37%，使得实验操作费力大，耗时2.相比之下，使用荧光活化细胞分拣（FACS）可以克服泊森分布限制，以高效制备单细胞3。然而，由于仪器仪表和维护费用高昂，一些实验室可能无法进入外地资产管制系统。微造设备最近被开发用于单细胞陷印4，单细胞配对5和单细胞培养应用。这些设备基于其准确操作单细胞6、执行高通量实验或减少样品和试剂消耗的能力而具有优势。然而，使用当前微流体装置对多种细胞类型进行单细胞共培养实验仍然具有挑战性，因为泊森分布1、7、8或不能设备捕获两种以上类型的单个细胞4，5，6，9，10。
例如，Yoon等人报告了用于细胞-细胞相互作用研究的微流体装置11。此设备使用概率方法在一个腔室中配对细胞。但是，由于设备结构的几何限制，它只能实现两种不同单元类型的配对。另一份来自Lee等人的报告展示了一种确定性的方法来捕获和配对单个细胞12。此设备通过确定性方法提高了配对效率，但受配对单元所需的长时间操作时间的限制。具体来说，第二个细胞捕获只能在24小时后将第一个捕获的细胞附着在表面后进行。 赵等人报告了一种基于液滴的微流体装置，以捕获两种类型的单个细胞13。可以发现，液滴基微流体装置仍局限于泊液分布，只能在非附着细胞上使用，在培养过程中无法改变培养液。
此前，我们已经开发出一种微流体"流体动力学穿梭芯片"，利用确定性流体动力将多种类型的单细胞捕获到培养室中，并随后进行细胞共培养实验以进行分析细胞-细胞相互作用下的单个细胞迁移行为14。流体动力穿梭芯片包括一组阵列单元，每个单元包含蛇形通路通道、捕获场和培养室。通过使用蛇形通路通道和培养室之间的流动电阻差，以及专门设计的操作程序，不同类型的单细胞可以反复捕获到培养室中。值得注意的是，培养室的充足空间不仅可以防止细胞在细胞捕获过程中被冲洗，而且为细胞扩散、增殖和迁移提供了足够的空间，从而可以观察活的单细胞相互作用。在本文中，我们将重点介绍此设备的生产和详细的协议步骤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3M Advanced Polyolefin Diagnostic Microfluidic Medical Tape</td>
			<td>3M Company</td>
			<td>9795R</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics</td>
			<td>Biowest</td>
			<td>L0014-100</td>
			<td>Glutamine-Penicillin-Streptomycin</td>
		</tr>
		<tr>
			<td>AutoCAD software</td>
			<td>Autodesk</td>
			<td>AutoCAD LT 2011</td>
			<td>Part No. 057C1-74A111-1001</td>
		</tr>
		<tr>
			<td>CellTracke Blue CMAC Dye</td>
			<td>Invitrogen</td>
			<td>C2110</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Green CMFDA Dye</td>
			<td>Invitrogen</td>
			<td>C7025</td>
			<td></td>
		</tr>
		<tr>
			<td>Conventional oven</td>
			<td>YEONG-SHIN company</td>
			<td>ovp45</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Bel-Art Products</td>
			<td>F42020-0000</td>
			<td>Space saver vacuum desiccator 190 mm white base</td>
		</tr>
		<tr>
			<td>DiIC12(3) cell membrane dye</td>
			<td>BD Biosciences</td>
			<td>354218</td>
			<td>Used as a cell tracker</td>
		</tr>
		<tr>
			<td>DMEM-F12 medium</td>
			<td>Gibco</td>
			<td>11320-082</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium MV 2</td>
			<td>PromoCell</td>
			<td>C-22022</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum Hyclone</td>
			<td>Thermo</td>
			<td>SH30071.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton 700 series Glass syringe ( 0.1 ml )</td>
			<td>Hamilton</td>
			<td>80630</td>
			<td>100 &#181;L, Model 710 RN SYR, Small Removable NDL, 22s ga, 2 in, point style 2</td>
		</tr>
		<tr>
			<td>Harris Uni-Core puncher</td>
			<td>Ted Pella Inc.</td>
			<td>15075</td>
			<td>with 1.5mm inner-diameter</td>
		</tr>
		<tr>
			<td>Harris Uni-Core puncher</td>
			<td>Ted Pella Inc.</td>
			<td>15071</td>
			<td>with 0.5mm inner-diameter</td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>YOTEC company</td>
			<td>YS-300S</td>
			<td></td>
		</tr>
		<tr>
			<td>Msak aligner</td>
			<td>Deya Optronic CO.</td>
			<td>A1K-5-MDA</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen plasma</td>
			<td>NORDSON MARCH</td>
			<td>AP-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Nordson</td>
			<td>AP-300</td>
			<td>Bench-Top Plasma Treatment System</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) kit</td>
			<td>Dow corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-tetrafluoroethene (PTFE)</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td>TFT-23T</td>
			<td>inner diameter, 0.51 mm; outer diameter, 0.82 mm</td>
		</tr>
		<tr>
			<td>Removable tape</td>
			<td>3M Company</td>
			<td>Scotch Removable Tape 811</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>Eltech corperation</td>
			<td>SPE0039</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Synrex Co., Ltd.</td>
			<td>SC-HMI 2&#34; ~ 6&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>Leica E24</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 10 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131259</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131240</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y111072</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>Grand Chemical Companies</td>
			<td>GP5002-000000-72GC</td>
			<td>Propylene glycol monomethyl ether acetate</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>703007</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichlorosilane</td>
			<td>Gelest, Inc</td>
			<td>SIT8174.0</td>
			<td>Tridecafluoro-1,1,2,2-tetrahydrooctyl. Hazardous. Corrosive to the respiratory tract, reacts violently with water.</td>
		</tr>
		<tr>
			<td>Trypsin Neutralizer Solution</td>
			<td>Gibco</td>
			<td>R-002-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing single-cell based co-cultures in a deterministic manner with a microfluidic chip

细胞共培养测定已广泛用于研究不同细胞类型之间的细胞-细胞相互作用，以更好地了解包括癌症在内的疾病的生物学。然而，使用传统的共生系统来澄清高异质细胞群中细胞间相互作用的复杂机制具有挑战性，因为细胞亚群的异质性被平均值所掩盖;传统的共培养系统只能用于描述总体信号，但不能跟踪单个细胞的行为。此外，传统的单细胞实验方法由于泊森分布而降低细胞操作效率。微制造器件是单细胞研究的新兴技术，因为它们能以高通量精确操作单个细胞，并能减少样品和试剂的消耗。在这里，我们描述了微流体芯片在多种单细胞共培养物中的概念和应用。该芯片可以有效地捕获培养室中的多种类型的单个细胞（+46%）并有足够的培养空间可用于研究细胞在单细胞级细胞-细胞相互作用下的行为（如迁移、增殖等）。淋巴内皮细胞和口腔鳞状细胞癌用于在微流体平台上进行单细胞共培养实验，进行活多细胞相互作用研究。

该器件具有三层结构，如切割 PDMS 器件的横截面照片所示（图1A）。第一层包含连接文化室和旁路通道的捕获位点（宽度为 6.0 μm，高度为 4.6 μm）。培养室和通路通道之间的流动电阻差异导致细胞流入捕获位置并填充小路径的入口。在捕获位置捕获单元后，小路径的流量阻力增加，导致下一个传入的单元朝向并通过旁路通道到达下一个?...

Watch this Scientific Journal Video about Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip at JoVE.com

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

本报告描述了一种基于微流体芯片的方法，用于建立单细胞培养实验，实现多个单细胞的高效配对和微观分析。

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 ...

有效结合捕获效率与动态活细胞图像观察，了解细胞-细胞相互作用对细胞行为的影响至关重要。该管可以高效捕获较大腔室中的多个单细胞，并提供足够的培养基，用于动态跟踪多个单细胞相互作用行为。首先准备 PDMS 设备。将晶圆模具放在称重盘中，在干燥器中加100微升三氯硅烷，并吸尘15分钟。在 37 摄氏度下停止真空，将干燥器中的晶圆模具盐化至少一小时。将 PDMS 基座和 PDMS 固化剂以 10 比 1 的比例混合，然后在 15 厘米的培养皿中将总共 20 克混合物倒入晶圆模具上。将 15 厘米的盘子放入干燥器中，并吸尘一分半钟。然后从干燥器中去除盘子，用氮气消除 PDMS 中的残余气泡。将盘子放在65摄氏度的烤箱中，加热PDMS两到四个小时。完成后，从晶圆模具中取出 PDMS 副本，并在 PDMS 上打孔 1.5 毫米入口和 0.5 毫米出口。使用可拆卸胶带清洁 PDMS 副本和滑动表面。用氧等离子体处理表面。然后，手动将 PDMS 副本与幻灯片对齐，并使其相互接触。将 PDMS 幻灯片放在 65 摄氏度的烤箱中一天。第二天，将 PDMS 设备浸入装满 PBS 的容器中。将其放入干燥器中，并吸尘 15 分钟。将设备移动到细胞培养罩，用紫外线消毒 30 分钟。将 PDMS 设备缓冲器更换为介质，并在 4 摄氏度下孵育一天。当细胞达到70至80%的汇合时，去除培养培养，用五毫升PBS轻轻洗涤三次。将含有一微摩尔荧光染料的一毫升DMEM/F-12介质加入WNT5B sh4和pLKO-GFP细胞，并在室温下孵育30分钟。孵育后，用五毫升无菌PBS轻轻清洗细胞三次，取出PBS，并加入两毫升0.25%的三辛-EDTA。在室温下孵育细胞四分钟，然后轻轻敲击培养皿，促进细胞分离。加入4毫升DMEM/F-12基基，分散WNT5B sh4和pLKO-GFP细胞，将细胞转移到15毫升管中，以300xg离心3分钟。取出上一提液，轻轻将细胞颗粒重新悬浮在一毫升DMEM/F-12培养基中。以每毫升三倍十至五细胞的浓度准备一毫升细胞悬浮液，并放在冰上以防止细胞聚集。在设备出口和注射器泵之间连接聚四氟乙烯管。取出介质，并将准备好的电池悬浮液的一微升添加到 PDMS 设备的入口中。接下来，使用注射器泵以每分钟 0.3 微升的流量将细胞悬浮液加载到设备中。将一微升 DMEM/F-12 介质添加到设备的入口中，然后使用注射器泵将介质加载到设备中。使用注射器泵将 0.3 微升 DMEM/F12 介质加载到设备中，流量为每分钟 10 微升。装入电池后，取出管子，用聚烯烃胶带密封入口和出口，以创建封闭的培养系统。将 PDMS 设备移动到 10 厘米培养皿中，并在设备周围添加 10 毫升无菌 PBS，以避免介质蒸发。将培养皿转移到三个单细胞培养的孵化器。PDMS 设备具有三层结构，具有连接到培养室和旁路通道的捕获站点。培养室和旁路通道之间的流量电阻差异允许单元流入捕获位置，这会导致以下单元通过旁通通道进入下一个捕获站点。回流步骤用于从捕获站点释放单元格并将其发送到培养室。在三种不同的细胞类型上评估了此协议的细胞捕获效率。人类淋巴内皮细胞，或LEC，人类口腔鳞状细胞癌T2.6细胞表达WNT5B特异性短发夹RNA，和pLKO-GFP病媒控制细胞。三个示范细胞的单细胞捕获效率为LEC的71%，WNT5B sh4细胞的74%，pLKO-GFP细胞的78%。三个单细胞捕获效率为48%该方法可用于淋巴内皮细胞和鳞状癌细胞的多个单细胞共培养，在显微镜下观察细胞的增殖和形态。确保准备好的细胞悬浮液具有最小的细胞聚合非常重要，因为细胞聚合不仅会降低单对对效率，而且会堵塞微通道。单个细胞之间的细胞相互作用在控制细胞行为方面起着重要作用。我们的方法可以提供一个有效的工具，帮助研究人员研究细胞-细胞在单个细胞水平，这可能会导致新的机制的发现，这是不容易获得基于人群的研究。

Research

JoVE Journal

Bioengineering

A Microfluidic-based Hydrodynamic Trap for Single Particles

一个单粒子为基础的微流控液力陷阱

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

科研

生物工程

On-Chip Endothelial Inflammatory Phenotyping

片上内皮炎症表型

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned 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 ...

A Microfluidic Chip for ICPMS Sample Introduction

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

A Microfluidic Platform for High-throughput Single-cell Isolation and 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 ...

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

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 ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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

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 ...