Name: a microfluidic platform for high-throughput single-cell isolation and culture
Uploaded: 2016-06-16T15:00:00.000+00:00
Duration: 9 min 51 s
Description: Watch this Scientific Journal Video about A Microfluidic Platform for High-throughput Single-cell Isolation and Culture at JoVE.com

This work was supported by a grant from the National Health Research Institutes (03-A1 BNMP11-014).

注意:我们的微流体装置的制造的光掩模的设计通过使用计算机辅助设计（CAD）软件绘制。然后设计被用于制造使用的是商业服务镀铬光掩膜。 PDMS的设备用软光刻技术制成。11 1.制作母模的通过光刻<ol><li>光刻工艺12之前，使用4英寸硅晶片作为基板，并在120℃下脱水在常规烘箱晶片10分钟。 </li><li>通过在100瓦特使用氧等离子体处理中的等离子体清洁器30秒清洗脱水硅晶片。 </li><li>预热在65℃两个分别电炉和95℃，对下列烘烤过程。 </li><li>涂层5克，以通过旋涂机的清洗硅晶片负光致抗蚀剂（PR）的;旋在1,200rpm下（SU-8 50）30秒，以产生微通道层。 </li><li>放置PR在65℃涂覆的晶片上的预热加热板上12分钟，并在95℃下转移至另一预热加热板上33分钟（100微米厚的图案），以执行软烘烤过程。 </li><li>烘烤后，放置在一个半自动化掩模对准的支架的PR涂覆的硅晶片，并对准一个25400 dpi分辨率透明度光掩模。 </li><li>的剂量为500mJ / cm 2的中暴露的PR涂覆的硅晶片到UV光（365纳米）来创建在硅晶片上的PR图案。 </li><li>从对准器中取出晶片，并放置在烘烤后在95℃下12分钟的加热板上。 </li><li>浸泡在SU-8显影剂（丙二醇单甲醚乙酸酯，PGMEA）溶液晶片以洗掉未交联PR为12分钟，并用氮气轻轻擦干以暴露对准标记。 </li><li>再次，涂层5克，以通过旋涂在晶片的负光致抗蚀剂;旋在700rpm（SU-8 100）30秒和1,200rpm下（SU-8 10），用于为300 30秒＃181;米厚的格局和27微米厚的图案分别使微孔层。 </li><li>放置在电炉上的PR涂覆的晶片在65℃下进行4分钟，并在95℃下8分钟（27微米深捕集阱层）;并在65℃40分钟，并在95℃110分钟（300微米深培养阱层）。 </li><li>冷却后，放置在装有UV光掩模对准器的PR涂覆的硅晶片。 </li><li>剂量250毫焦耳/ 平方厘米（27微米厚的图案）和700兆焦耳/厘米2（300微米厚的图案）暴露PR涂硅片紫外光灯（365纳米）。 </li><li>烘烤在95℃下的晶片5分钟，（27微米厚图案）和30分钟（300微米厚图案），分别。 </li><li>由PGMEA为分别为6分钟（27微米厚图案）和25分钟（300微米厚图案），洗掉未交联PR，然后用氮气干燥。 </li><li>测量高度的图案展示了通过放置在晶片上扫描激光轮廓曲线的在xy阶段用扫描激光表面光度仪晶片。 <ol><li>调整焦平面清楚地显示了由一20X物镜下使用"相机视图"观察模式在晶片上的图案特征。 </li><li>从"摄像机视图"到"激光视图"切换观察模式，以及设置特征的上下位置。 </li><li>设定分别测量模式，面积，质量，和z音调高达透明（上），1线（1,024×1），高准确度和0.5微米，然后按下开始底部开始测量。 </li></ol></li></ol> 2.准备PDMS器械的单细胞分离<ol><li> PDMS铸造之前，silanize主模具与三氯硅烷以创建一个疏水性表面，这使得它更容易从主模具剥离PDMS复制品。 <ol><li>将主模具和含权舟三氯硅烷的200微升在干燥器，和15分钟，施加真空（-85千帕）。 </li><li>停止真空，然后离开主模具在干燥器中silanize在室温下将主模具至少1小时。 </li><li>从干燥器中取出主模具和各母模放置在一个10cm培养皿。 </li></ol></li><li>混合含有碱和以10比的固化剂17.6克PDMS聚合物试剂盒的总量:1，然后倒入PDMS上的培养皿的母模。 </li><li>放置培养皿在干燥器和1小时施加真空（-85千帕），以除去在PDMS中的气泡。 </li><li>从干燥器中取出培养皿并将其放置在常规烘箱中在65℃下3 - 6小时以固化的PDMS。 </li><li>从主模中取出固化的PDMS复制品，并通过与内径-0冲床冲两个孔作为入口和出口处的捕集阱阵列的PDMS复制品所述微通道的两端。75毫米;流体通道（ 图2A）。 </li><li>使用磁带清洁PDMS复制品的表面上，然后将PDMS复制品在短暂氧等离子体处理（100瓦14秒）的等离子体清洁器。 </li><li>除去由氧等离子体机器与PDMS复制品。 </li><li>对齐的顶（包含捕获微孔）和一个底部PDMS（含有培养微孔）复制品用手在立体显微镜下，并使其接触。 </li><li>放置在烘箱对准PDMS复制品在65℃下放置24小时以实现PDMS复制品之间的永久接合，以形成最终器件。 </li><li>浸泡PDMS设备在去离子（DI） - 水填充容器，并将该容器在干燥器真空（-85千帕）下15分钟，以从PDMS设备的微通道除去空气。 </li><li>放置的DI水填充PDMS设备在组织培养罩，并使用UV光（光的波长:254nm），以30英里消毒装置的ñ。 </li><li>在PDMS设备与在1×PBS溶液，5％牛血清白蛋白（BSA）替换DI水，并在37℃下孵育30分钟，以防止细胞粘到PDMS表面。 注意:BSA涂层是至关重要的，以提高从分离的细胞的转移效率捕获孔到培养孔。 </li><li>替换在PDMS设备用无菌1×PBS溶液中的5％BSA溶液。 </li></ol> 3.单细胞悬浮液的制备<ol><li>培养的神经干细胞KT98和人癌A549细胞和MDA-MB-435，我们在常规细胞培养孵育箱（37℃，5％CO 2和95％湿度）的培养皿来制备细胞用于PDMS设备细胞实验。 </li><li>卸下并丢弃废培养基从生长至70细胞培​​养皿（含10％胎牛血清和1％抗生素的供给DMEM基础培养基） - 80％汇合。 </li><li>轻轻洗涤细胞用无菌PBS清洗3次。&lt;/ LI&gt; <li>卸下并丢弃PBS，加入2毫升蛋白，胶原溶解和DNA酶的活动重组酶混合物（请​​参阅材料清单的详细信息）。 </li><li>在室温下孵育培养皿5分钟，然后点击培养皿，以促进细胞脱落。 </li><li>添加4毫升灭菌PBS以分散细胞，然后将细胞悬浮液转移到15毫升锥形管中。 </li><li>离心管中，在300×g离心3分钟，去除上清。 </li><li>轻轻重悬在1ml的灭菌PBS细胞沉淀和计数使用标准台盼蓝排除法13的活细胞数目。 注意:此悬浮步骤是制备以及解离的单细胞悬浮液，以提高单细胞分离效率是至关重要的。 </li></ol> 4.单细胞的分离和克隆培养<ol><li>负载50微升细胞悬浮液中的2.2的浓度 - 2.5×10 <suP&gt; 6个细胞/ ml成经由与手持移液器设备的出口孔与PDMS装置的微通道。 注意:将细胞悬浮液装载吸管需要停止，并在第一停止位置保持，以避免引入气泡进入微通道。 </li><li>通过进口孔加载其他50微升细胞悬液，以均匀地填充了细胞的整个微通道。 </li><li>用密封塞（3毫米长，直径1毫米的尼龙切鱼线），以避免流由从入口和出口孔滴静水压力感应出水孔。 注意:插头是紫外线使用前组织培养罩内灭菌。 </li><li>填补了用培养基1毫升无菌注射器，从中弹出气泡，并将其设置在注射器泵。 </li><li>经由23政钝针和聚四氟乙烯（PTFE）管道连接介质装载注射器与PDMS装置的入口孔。 </li><li>从出线孔，所有拔下插头流2分钟的时间间隔，使细胞沉淀成重力单细胞捕获井。 </li><li>洗去未捕获的细胞用300μl培养基以600微升/分钟通过注射器泵驱动的流速。 </li><li>等待2分钟，以稳定装置，和密封带插头的入口和出口孔，以形成一个封闭的培养系统。 </li><li>手工翻转设备将捕获的单细胞转移到培养微孔。 </li><li>放置PDMS设备在100毫米组织培养皿，并在该装置周围添加灭菌的PBS 10毫升，以避免从所述微通道培养基蒸发。 </li><li>移动至培养皿的常规细胞培养孵育箱（37℃，5％CO 2和95％湿度）为单细胞的克隆培养。 </li></ol> 5.培养基补货<ol><li>培养1天后，具有提高细胞PR新鲜培养基替换PDMS设备培养基oliferation。 </li><li>放置在邻近所述入口孔和出口孔的区域的PDMS设备的顶部培养基的两个液滴以避免引入气泡进入所述微通道在执行下一个步骤。 </li><li>从微通道作为入口的两端附近的PDMS装置的顶部和用于所述微通道的出口冲两个孔。 注意:不要通过PDMS设备的整个两层冲;相反，仅仅通过与PDMS的顶层打孔。 </li><li>通过连接一个23政钝针和PTFE管含有对进口新鲜培养基1毫升的塑料注射器。 </li><li>流120微升新鲜培养基到设备5分钟，更换旧培养基。 </li><li>插入两个塞子以密封所述入口和出口孔，并且该设备返回到细胞培养孵化器。 </li><li>通过除去该密封塞，然后通过重复步骤5.4至5.5刷新培养基中培养的期间每2天。 </li></ol>

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The authors declare that they have no competing financial interests.

基于微孔的设备系统6,14已用于单细胞操纵和分析，例如大型单细胞俘获6和单造血干细胞增殖15。虽然井大小，数量和形状可以调节为特定的应用中，当孔的大小增加时，单细胞分离效率总是受到损害。9,15 为了克服此限制，Park等人报告了具有高的单细胞的捕集率（58.34％）的三角形的微孔的微流体芯片，而微孔尺寸放大以允许细胞扩散?...

目前将单个细胞单独在培养空间通常是通过使用有限稀释或荧光激活细胞分选（FACS）来实现的。对于许多实验室，有限稀释是一个方便的方法，因为它仅需要一个吸管和组织培养板中，这是容易获得的。在这种情况下，细胞悬浮液连续稀释至适当的细胞密度，然后通过使用手动移液管放置到培养孔。然后，这些隔间单细胞被用于细胞分析，如遗传异质性筛选1和集落形成2。然而，该方法是低通量和劳动密集的，不利用用于协助一机械臂，因为有限稀释法的泊松分布性质限制了单细胞事件的37％3的最大概率。 FACS机，集成机械臂可以通过准确PLAC克服泊松分布的限制ING在文化井一单细胞在同一时间4。然而，高机械剪切应力（因此，降低细胞活力）5和机购买和操作成本在许多实验室已经限制了它的使用。 为了克服上述限制，微型装置已经发展到高效的单细胞装入微孔6。然而，微孔不提供足够的空间用于装载的细胞增殖，由于需要使每个的尺寸微孔关闭到单个细胞的最大化单细胞加载的概率。作为培养测定在许多基于细胞的应用（ 例如，克隆形成实验7），较大微孔需要（从90 - 650微米的直径或边长）也被用于允许延长细胞培养物。然而，像有限稀释法，它们也具有低的单细胞负载效率，范围从10 - 30％的8，9 以前，我们已经开发出一种高通量微流体平台，单个细胞中分离出在个体微孔，并在分离的细胞的克隆形成实验证明其应用10的装置，用聚二甲基硅氧烷（PDMS）制成，并包括两组微孔阵列具有不同微孔尺寸，从而可以大大提高在微孔的大小加载单个细胞的效率是比电池显著大。值得注意的是，这种"双阱"的概念允许在不影响单细胞捕获效率，使得它直接地调整该装置的设计，以适应不同的细胞类型和应用程序灵活调整培养面积的大小。这种高效率的方法应该是对于长期的细胞培养实验对于细胞的异质性的研究和单克隆细胞系建立有用的。

<table><tbody><tr><td>AutoCAD software</td><td>Autodesk</td><td>AutoCAD LT 2011</td><td>Part No. 057C1-74A111-1001</td></tr><tr><td>Silicon wafer&amp;nbsp;</td><td>Eltech corperation</td><td>SPE0039</td><td></td></tr><tr><td>Conventional oven</td><td>YEONG-SHIN company</td><td>ovp45</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>SU-8 50 negative photoresist</td><td>MicroChem</td><td>Y131269</td><td></td></tr><tr><td>SU-8 100 negative photoresist</td><td>MicroChem</td><td>Y131273</td><td></td></tr><tr><td>Spin coater</td><td>Synrex Co., Ltd.</td><td>SC-HMI 2&quot; ~ 6&quot;</td><td></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>SU-8 developer</td><td>Grand Chemical Companies</td><td>GP5002-000000-72GC</td><td>Propylene glycol monomethyl ether acetate</td></tr><tr><td>Scanning laser profilometer</td><td>KEYENCE</td><td>VK-X 100</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>Desiccator</td><td>Bel-Art Products</td><td>F42020-0000</td><td>Space saver vacuum desiccator 190&amp;nbsp;mm white base</td></tr><tr><td>Polydimethylsiloxane (PDMS) kit</td><td>Dow corning</td><td>Sylgard 184</td><td></td></tr><tr><td>Harris Uni-Core puncher</td><td>Ted Pella Inc.</td><td>15072</td><td>with 0.75 mm inner-diameter</td></tr><tr><td>Removable tape</td><td>3M Company</td><td>Scotch Removable Tape 811</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica Microsystems</td><td>Leica E24</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Bersing Technology</td><td>ALB001.500</td><td></td></tr><tr><td>DMEM basal medium</td><td>Gibco</td><td>12800-017</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Hyclone</td><td>SH30071.03HI</td><td></td></tr><tr><td>Antibiotics</td><td>Biowest</td><td>L0014-100</td><td>Glutamine-Penicillin-Streptomycin</td></tr><tr><td>Recombinant enzyme mixture</td><td>Innovative cell technology</td><td>AM-105</td><td>Accumax</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>Syringe pump</td><td>Harvard Apparatus</td><td>703007</td><td></td></tr><tr><td>Plastic syringe (1 ml)</td><td>BD Biosciences</td><td>309659</td><td></td></tr><tr><td>23 gauge blunt needles</td><td>Ever Sharp Technology, Inc.</td><td>TD21</td><td></td></tr><tr><td>Poly-tetrafluoroethene (PTFE) tubing</td><td>Ever Sharp Technology, Inc.</td><td>TFT-23T</td><td>inner diameter, 0.51&amp;nbsp;mm; outer diameter, 0.82&amp;nbsp;mm</td></tr></tbody></table>

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.

对于单细胞的分离和培养的微流体平台包括一个微通道（200微米的高度）与两套微孔阵列（ 图2A）。两套微孔阵列被称为捕捉阱（25微米直径27微米的深度），并培养良好（285微米直径300微米深度）分别为单细胞分离和培养，每从顶视图（ 图2B）观察时捕获阱被定位在培养孔的中心。对于设备操作（概略操作流程在图1中示出），所需?...

Watch this Scientific Journal Video about A Microfluidic Platform for High-throughput Single-cell Isolation and Culture at JoVE.com

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

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 ...

a-microfluidic-platform-for-high-throughput-single-cell-isolation

Research

JoVE Journal

Bioengineering

11.2K 次观看.  National Health Research Institutes, Taiwan. 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. 

视频: A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

科研

生物工程

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

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 ...

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.

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 ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

微流体平台精密小批量来样加工及它的使用尺寸分离生物颗粒的声微型

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

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 ...

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

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

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 ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

用于分离多功能循环肿瘤细胞的临床微流控芯片平台

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...

Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using The Single-cell Microliter-droplet Culture Omics System

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors are hindered by cumbersome procedures and low throughput, impeding the rapid progress of microbial culturomics research. To address these challenges, we had successfully developed the Single-cell Microliter-droplet Culture Omics System (MISS cell), an automated high-throughput platform that utilizes droplet microfluidic technology for microbial monoclonal isolation, cultivation, and screening. This system can generate a large number of single-cell droplets and cultivate, screen, and collect monoclonal colonies in a short time, facilitating an integrated process from microbial isolation to picking. In this protocol, we demonstrated its application using the isolation and cultivation of human gut microbiota as an example and compared the microbial isolation efficiency, monoclonal culture performance, and screening throughput using the solid-plate culture method. The experimental workflow was simple, and reagent consumption was very low. Compared to solid-plate culture methods, the MISS cell could cultivate a greater diversity of gut microbiota species, offering significant potential and value for microbial culturomics research.

This protocol describes how to use the Single-cell Microliter-droplet Culture Omics System (MISS cell) to perform microbial monoclonal isolation, cultivation, and picking. The MISS cell achieves an integrated workflow based on droplet microfluidic technology, which offers excellent droplet monodispersity, high parallel cultivation, and high-throughput biomass detection.

使用单细胞微升液滴培养组学系统的自动化和高通量微生物单克隆培养和挑取

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors ...