这项工作是由美国国家卫生学院授予＃1R01CA120825-01A1，生物医药合作研究发展计划（CBR）在马萨诸塞州阿姆赫斯特大学，和普山J. Toley的的伊森伯格奖学金为支持。我们非常感谢詹姆斯·谢弗，摄影师，叙述者，该视频的编辑作出的宝贵贡献。

1。设备制造在弹性体材料中的微流体的功能的基础上复制达菲等人所描述的方法。 <ol><li>的混合弹性体（聚二甲基硅氧烷PDMS）和固化剂的有机硅弹性体套件（ 道康宁密歇根州米德兰市）9:1的重量比，倒在主形成一个4毫米厚的一层。脱气以除去气泡，并在60℃下固化该混合物5小时。剥离固化的PDMS从模具上的弹性体中的流动特性，得到一张邮票。 </li><li>使用1.5毫米活检冲头（MILTEX，纽约，宾夕法尼亚州）的入口和出口的打孔安装在一台钻床。拉出任何碎片。 </li><li>主题特性方面的邮票和干净的载玻片上的氧等离子体中的氧等离子体刻蚀机8分钟。立即接触带来的表面经过处理的，在它们之间形成一个键。幻灯片上的回暖至少5个小时，保持在60°C的组装s到加强债券。 </li><li> 0.032“ID PTFE管（ 科尔帕默 ，弗农山，IL）使用的接口阳鲁尔锁连接器连接到刺阴鲁尔锁连接器（Qosina，旅游Edgewood，NY）的入口和出口。 </li><li>设置使用关断阀和一个Y形连接件（ 厄普丘奇科学 ，Oak Harbor的，WA，图1）的流动组件。安装设备的显微镜。将注射器管使用20G 1.5“针（BD生物科学中心，Rockville，MD）。 </li></ol> 2。形成统一的细胞球体椭球粒子的形成的悬滴法2,3。 <ol><li> Trypsinize细胞在无菌的细胞培养罩。 </li><li>胰蛋白酶处理的细胞以2000rpm离心5分钟，并再悬浮在6毫升新鲜培养基。原液稀释到所需的最终浓度（表1）。 </li><li>取下盖子的48孔板，将其倒置在科技教育rilized罩。对应于每口井的圆形区域是显而易见的封面上。填充的板的各孔用1ml无菌水以保持湿度。将20微升滴的每一个圆形区域，用微量稀释的细胞液中。小心地翻转盖，并把它放在一个孔板，确保滴不触摸各孔的边缘。 </li><li>在37°C孵育孔板为指定的天数（表1）。球体将在每个悬滴形成。 </li></ol> 3。的细胞球体的介绍到设备请参阅图1和表2本节。 <ol><li>消毒装置，由通过流入口引入用70％乙醇冲洗。随后，冲洗用PBS，然后由HEPES缓冲的细胞培养基。发表中期注射器S F连接管。 </li><li>绘制2-3球体注射器S P，点击注射器，以消除气泡，附加到该装置的包装入口。 </li><li>打开进水阀V 引脚和密切的V 翅 。打开出口阀V POUT和密切的V FOUT。握住包装的注射器针朝下垂直;观看作为球体定居到路厄氏锁定连接器的针的注射器底部。在包装上的注射器和手表球体推动柱塞进入到器件中的油管和流量。因为只有包装出口阀是打开的，椭球体将输入的装置的腔室，并保留在背面（图2）内的职位。 </li><li>关闭进水阀V 引脚和出口阀V 功率输出 。安装的注射器注射泵，S F。打开阀V FIN和：阀V FOUT。 3μl/ min的流介质进入的移动设备。 </li></ol> 4。介绍细胞凋亡检测剂（CaspGLOW） 在与球体的包装室，让平衡为up到24小时，以建立营养梯度和微环境之前，引入细胞凋亡检测或治疗剂。由于腔室中的组织接触坚不可摧从顶部和底部壁，有没有营养梯度沿厚度（0.15毫米）的组织。温育24小时后，所有的电池层沿着组织的厚度因此，等效的，在远离流路的方向的唯一的非均质性。 <ol><li>关闭V 翅 ，停止注射泵，取出注射器S F，取而代之的是从泵的注射器中含0.25微升/毫升的CaspGLOW红活性caspase-3的标记（红色-DEVD-FMK， 描述BioVision，山景， CA）。 </li><li> 0.7毫升上述溶液通过手动冲洗设备以确保位移较旧的介质。安装在泵上的注射器，重新启动流程，开V 翅 。经过一段5-8小时，细胞凋亡检测剂扩散到T他组织和荧光亮度在细胞凋亡的地区。细胞凋亡检测剂在此浓度维持在随后的所有流程解决方案到设备。 </li></ol>注： <ol class="lroman"><li>组织异质性可以确认获得线性荧光强度分布在第6。配置文件应该显示在细胞凋亡中的空间梯度，确认该组织是异质性细胞活力。 </li><li> CaspGLOW格林主动Caspase-3的标记物（荧光素-DEVD-FMK;产品描述BioVision）也可以被用于检测细胞凋亡。此标记包含了绿色荧光的荧光，而不是红色荧光的罗丹明。 </li></ol> 5。治疗剂简介<ol><li>按照程序步骤4.1-4.2介绍10μM盐酸阿霉素（阿霉素，Sigma-Aldrich公司， 圣路易斯 ，MO）细胞凋亡检测剂。 </li><li>继续流动的therapeutic剂溶液一段固定的时间。切断阀V 翅关闭注射泵。更换新鲜培养基含有细胞凋亡检测剂的注射器治疗注射器。连续监测在显微镜下的组织的同时，允许流动的新鲜培养基中为24 -36小时。 </li></ol> 6。时间推移显微镜和药物扩散系数的估计为了获得干净的荧光数据，获取所有影像聚焦显微镜组织的底层。因为有没有营养梯度在垂直方向上（参见第4节），底部层细胞是上述所有其它层的良好的代表。 <ol><li>使用自定义的脚本在IPLab（BD生物科学中心，Rockville，MD），整个收购过程实现了自动化。掌握在10倍放大倍率的填充的旋转椭球体，每30分钟（图3A）的透射光图像和荧光图像。 Acquire一个图像的背景荧光之前引入的Dox。为了适应大尺寸的腔室（1000微米×300微米），获得两个相邻的图像和瓷砖在一起。 </li><li>有关药物的扩散系数的数学估计，第一个步骤是，以产生平均的线性强度档案Dox诱导荧光使用ImageJ。选择一个矩形的感兴趣区域（ROI），涵盖了组织室。使用“剧情简介”命令来生成配置文件的平均强度为流道之间距离的函数。重复多达3个不同的时间点。 </li><li>获取的背景前，阿霉素来衡量组织的自体荧光的荧光图像。减去平均背景所获得的强度和荧光强度正常化的每个配置文件，以获得相应的最大强度<img src="/files/ftp_upload/2425/2425eq0.jpg" alt="Ç" /> （X，T）（图3的B）。 </li><li>接下来的步骤是，以评估肿瘤组织内的有效扩散系数 D的Dox。阿霉素的扩散可以由下面的式（5）表示 <img src="/files/ftp_upload/2425/2425eq1.jpg" alt="式（1）"/> 其中，erfc是余误差函数，x是从通道进入组织的距离，而t是时间后引入的Dox（参考图3）。使用下面的迭代计划，在每个时间点考虑： <ul><li>猜一个价值为 D </li><li>在每个位置 x，计算右侧的方程</li><li>计算双方之间的误差平方和（剩余） </li><li> 修改 D尽量减少剩余</li></ul></li><li>平均得到的D的最佳值，在每个时间点，在组织中估计的平均的有效扩散系数的Dox。 </li></ol> 7。代表性的成果： &lt;P类=“j​​ove_content”&gt;微流体装置提供1毫米所述0.3×0.15毫米光学访问文化室的三维肿瘤组织的生长。多细胞肿瘤球体流入这些商会，并保留过滤器由两个职位在后面。悬滴法允许从几个细胞系一致的大小和形状的球状体的快速形成。椭球粒子的设备上成功地生长至3天。在商会的增长是与内球体的微环境，可重复的修改。在细胞凋亡发生较少的附近的流道和更高的进入组织更深。该装置被用来估计在肿瘤组织中阿霉素的扩散系数。所获得的值与值9.1×10 -7 cm 2 s以下-1 8.75×10 -7 cm 2的-1同意在乳腺癌中的以前的报告6。 <table border="1"><tbody><tr><td> 细胞系 </td><td> 所需的细胞浓度 </td><td> 培养时间 </td></tr><tr><td> LS174T </td><td> 300个细胞/μL </td><td> 2-3天</td></tr><tr><td> T47D </td><td> 750个细胞/μL </td><td> 3-4天</td></tr><tr><td> MDA-MB-231 </td><td> 150个细胞/μL </td><td> 5-6天</td></tr></tbody></table> 悬滴细胞球体的参数见表1。 <table border="1"><tbody><tr><td> 部分 </td><td> 描述 </td></tr><tr><td> S F </td><td>流动注射</td></tr><tr><td> S P </td><td>包装注射器</td></tr><tr><td> V 翅 </td><td>流进气阀</td></tr><tr><td> V 引脚 </td><td>包装进气阀</td></tr><tr><td> V FOUT </td><td>流出口阀</td></tr><tr><td> V 功率输出， </td><td>包装出口阀</td></tr></tbody></table> 表2。注射器和阀门流量设置<img src="/files/ftp_upload/2425/2425fig1.jpg" alt="图1" /> 图1。的流量设置V 翅和V FOUT：流量入口和出口阀，V 引脚和V 功率输出 ：包装进，出口阀门，S F和S P：流量和包装注射器的示意图。入口和出口阀的填充注射器和包装时使用的旋转椭球体的腔室流入。的流量的注射器和流量的入口和出口阀被用于流动介质其后。 <img src="/files/ftp_upload/2425/2425fig2.jpg" alt="图2" /> 图2。被困在设备上的一个腔室的球状体的示意图。旋转椭球体流入到设备上的腔室和由两个过滤器位置被挡日ts在背面的腔室。 <img src="/files/ftp_upload/2425/2425fig3.jpg" alt="图3" /> 图3。设备上的肿瘤组织中阿霉素的扩散。 A.合并透射光及红色荧光图像的组织中，示出的位置和浓度的多柔比星（红色）。比例尺为250μm。B.归一化线性阿霉素荧光强度分布。

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	<li>Duffy, D. C., McDonald, J. C., Schueller, O. J., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Prototyping+of+Microfluidic+Systems+in+Poly(dimethylsiloxane).">Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane).</a> Anal. Chem. 70, 4974-4984 (1998).</li><li>Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+generation+of+homogeneous+multicellular+tumor+spheroids+applicable+to+a+wide+variety+of+cell+types.">Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.</a> Biotechnol. Bioeng. 83, 173-180 (2003).</li><li>Timmins, N. E., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method.">Generation of multicellular tumor spheroids by the hanging-drop method.</a> Methods. Mol. Med. 140, 141-151 (2007).</li><li>Kasinskas, R. W., Forbes, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+specifically+chemotax+and+proliferate+in+heterogeneous+tumor+tissue+in+vitro.">Salmonella typhimurium specifically chemotax and proliferate in heterogeneous tumor tissue in vitro.</a> Biotechnol. Bioeng. 94, 710-721 (2006).</li><li>Walsh, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multipurpose+microfluidic+device+designed+to+mimic+microenvironment+gradients+and+develop+targeted+cancer+therapeutics.">A multipurpose microfluidic device designed to mimic microenvironment gradients and develop targeted cancer therapeutics.</a> Lab. Chip. 9, 545-554 (2009).</li><li>Lankelma, J., Fernandez Luque, R., Dekker, H., Schinkel, W., Pinedo, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mathematical+model+of+drug+transport+in+human+breast+cancer.">A mathematical model of drug transport in human breast cancer.</a> Microvasc. Res. 59, 149-161 (2000).</li><li>Less, J. R., Skalak, T. C., Sevick, E. M., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+architecture+in+a+mammary+carcinoma:+branching+patterns+and+vessel+dimensions.">Microvascular architecture in a mammary carcinoma: branching patterns and vessel dimensions.</a> Cancer. Res. 51, 265-273 (1991).</li><li>Brown, J. M., Giaccia, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unique+physiology+of+solid+tumors:+opportunities+(and+problems)+for+cancer+therapy.">The unique physiology of solid tumors: opportunities (and problems) for cancer therapy.</a> Cancer. Res. 58, 1408-1416 (1998).</li><li>Thomlinson, R. H., Gray, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histological+structure+of+some+human+lung+cancers+and+the+possible+implications+for+radiotherapy.">The histological structure of some human lung cancers and the possible implications for radiotherapy.</a> Br. J. Cancer. 9, 539-549 (1955).</li><li>Minchinton, A. I., Tannock, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+penetration+in+solid+tumours.">Drug penetration in solid tumours.</a> Nat. Rev. Cancer. 6, 583-592 (2006).</li></ol>

没有利益冲突的声明。

在肿瘤的血管稀疏和虐待发达7,8。有些地区距离远（&gt; 100微米）的血管，是无法提供的营养物质和药物虽然血管9。异质微的效果有限，许多化疗药物10。这里开发的微流体装置，再现了异质性肿瘤微环境的特点是增殖，静态和凋亡或坏死的细胞，在体外。接近的通道中的单元是可行的，但扩散限制引起的营养缺陷凋亡区域远离通道。的流路表示血管和旋转椭球体表示相?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录号</th><th>评论</th></tr><tr><td>有机硅弹性体套件</td><td>安士澳粘合剂</td><td> 184银ELAST套件</td><td></td></tr><tr><td> MILTEX活检冲</td><td> MedexSupply </td><td> MTX-33-31AA </td><td> 1.5毫米</td></tr><tr><td> PTFE管</td><td>科尔帕默</td><td> EW-06417-31 </td><td> 0.032“ID </td></tr><tr><td>阳鲁尔锁连接器</td><td> Qosina </td><td> 65111 </td><td></td></tr><tr><td>刺阴鲁尔锁连接器</td><td> Qosina </td><td> 11556 </td><td></td></tr><tr><td>切断阀</td><td> IDEX健康与科学</td><td> P-721 </td><td></td></tr><tr><td> Y-连接器</td><td> IDEX健康与科学</td><td> P-513 </td><td></td></tr><tr><td> 20G 1.5“针</td><td> BD BiosciencË </td><td> 305176 </td><td></td></tr><tr><td>胰蛋白酶-EDTA </td><td> Invitrogen公司</td><td> 25300-054 </td><td></td></tr><tr><td> HEPES </td><td>西格玛</td><td> H-4034 </td><td></td></tr><tr><td> CaspGLOW荧光</td><td>产品描述BioVision </td><td> K183-25 </td><td></td></tr><tr><td> CaspGLOW红</td><td>产品描述BioVision </td><td> K193-25 </td><td></td></tr><tr><td>盐酸阿霉素</td><td>西格玛</td><td> 44583 </td><td></td></tr><tr><td> LS174T </td><td> ATCC </td><td> CCL-188 </td><td>人结肠癌细胞系</td></tr><tr><td> T47D </td><td> ATCC </td><td> HTB-133 </td><td>人类性导管癌的细胞株</td></tr><tr><td> MDA-MB-231 </td><td> ATCC </td><td> HTB-26 </td><td>人类乳腺腺癌细胞系</td></tr></tbody></table>

microfluidic device for recreating a tumor microenvironment in vitro

我们已经开发出一种微流体装置，模仿送货和药物异构三维肿瘤组织在体外的全身清除。交付血管的营养物质无法到达所有部位的肿瘤，可行的，静态的和坏死的细胞类型组成的异质性的微环境。许多治疗癌症的药物无法有效地渗透和治疗所有类型的细胞，因为这种异质性。单分子膜的癌细胞不模仿这种异质性，使其难以测试癌症药物与合适的体外模型。我们的编造出来的PDMS微流体装置使用软光刻技术。矩形室设备上的多细胞肿瘤球体，所形成的悬滴法，插入和约束，并保持连续介质灌注一侧。腔室在设备上创建的矩形形状的线性梯度在组织内。荧光染料被用来量化个Ë在组织内细胞凋亡的变化。在设备上的肿瘤治疗与荧光化疗药物阿霉素，时间推移显微镜被用来监测其扩散到组织中，和估计的有效​​扩散系数。悬滴法可以迅速形成均匀的球体从多种肿瘤细胞株。设备启用增长的球体3天。接近流动介质中最小的细胞凋亡细胞和，那些远离通道的凋亡，从而精确地模仿血管肿瘤邻近地区。与以前报道的价值在人类乳腺癌的阿霉素扩散系数的估计值一致。因为在实体肿瘤药物的渗透和保留会影响其功效，我们认为这是一个重要的工具，在了解药物的行为，并开发新的癌症疗法。

Watch this Scientific Journal Video about Microfluidic Device for Recreating a Tumor Microenvironment in Vitro at JoVE.com

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

我们的程序重新异质性肿瘤微环境的微流体装置，制造和操作<em在体外</em&gt;。肿瘤组织内的凋亡中的变异量化使用荧光染料和化疗药物到肿瘤组织中阿霉素的有效扩散系数进行了评价。

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 ...

microfluidic-device-for-recreating-a-tumor-microenvironment-in-vitro

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Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

11.5K 次观看.  University Of Massachusetts Amherst. 我们的程序重新异质性肿瘤微环境的微流体装置，制造和操作

视频: Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

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 ...

科研

生物工程

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation 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 ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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

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

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

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

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

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.

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 ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

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 ...

用于组合插头生产的微流体装置的设置

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

作者聚焦：在精准肿瘤学中整合计算和实验方法

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

用于组合塞生产的双层微流控装置

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...