Name: generation of heterogeneous drug gradients across cancer populations on a microfluidic evolution accelerator for real-time observation
Uploaded: 2019-09-19T15:00:00
Description: Watch this Scientific Journal Video about Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation at JoVE.com

这项工作得到了NSF PHY-1659940的支持。

1. 微流体装置的制造
<ol><li>使用布局设计软件生成所需的微流体模式（参见补充材料）。</li>
	<li>制作照片蒙版。有关详细信息，请参阅材料表。<ol>
<li>利用激光书写器，在涂有 100 nm Cr 和 500 nm 光刻胶 AZ1518 的苏打石灰玻璃板上书写图案。</li>
		<li>与开发人员 AZ300MIF 共同开发光刻胶，使用 60 s。</li>
		<li>使用 Cr-7 铬蚀刻剂，无需保护光刻胶。</li>
		<li>在 70°C 下使用光胶?...

<ol>
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传统细胞培养技术是近一个世纪前发展起来的，尽管事实证明，它预测癌症临床结果的能力有限，但它仍然是生物医学研究中最常用的临床前模型。动物模型具有最高的生理相关性和合理的基因相似性，但长期以来，人们一直承认在预测人类结果方面存在重大局限性。在现有的所有临床前模型中，微流体癌片模型似乎是最有前途的候选者，以满足癌症研究未满?...

癌症已日益被公认为是一个复杂的生态系统，它不仅取决于突变细胞群的持续调节，还取决于癌细胞与宿主微环境之间的重要相互作用。从这个意义上说，癌症在一个适应性的景观上进化，表现为多种因素，包括异质肿瘤微环境和与各种宿主细胞的串扰，所有这些都为进一步的遗传或表观遗传变化1，2，3。在实体肿瘤的情况下，化疗和其他资源梯度分布不均匀，有助于其分子异质性，并可能在耐药性的发展中发挥作用，增加特定肿瘤的血管生成亚种群，甚至转移4，5，6。传统的体外二维细胞培养研究，虽然拥有大规模、方便的实验能力，但提供均场、均匀和固定条件，往往缺乏真正所需的精确的空间和时间环境控制模拟体内肿瘤动力学。因此，需要更具代表性的外体模型在药物开发管道中的动物模型之前重现肿瘤微环境，以便更好地预测癌症进展以及动态压力内对药物的反应景观。微流体学已被提出来弥合二维细胞培养研究与更复杂的体内动物研究之间的差距，这些研究可能无法支持可控的定量研究7、8、9。
一个理想的体外系统来描述癌细胞动力学应该具有产生异构微环境的能力，以模拟在肿瘤中可能发生的适应性细胞反应，并允许在单细胞分辨率。在本文中，我们描述了一个微流体细胞培养平台，一种基于PDMS的设备，称为"进化加速器"（EA），它允许在细胞分辨率下对癌细胞动力学进行平行体外研究，并实时采集周，同时稳定地保持整个文化景观的压力梯度。这个平台的设计是基于我们以前的工作，其中生物体的进化动力学在元种群可以加速10，11。具体来说，在一组空间分离的种群中，当暴露于异质应力景观时，与大型统一种群相比，最适合的物种可以更快地在本地种群中占据主导地位。有利的物种然后迁移到邻近的微生境，以寻找资源和空间，并最终主宰整个种群。如图1所示，微流体EA芯片的图案由（i）一对蛇形通道组成，这些通道提供新鲜的介质循环，为化学扩散构造固定边界条件;（ii）六边细胞培养区它由109个相互连接的六角形和24个半六角形室组成，中间类似蜂窝结构。芯片深度为100μm。介质通道和细胞培养区域与小切线（约15μm宽）相连，防止直接介质流动和产生的剪切应力穿过细胞培养区，但仍允许化学物质通过小切线扩散并交换营养成分，代谢废物等。化学梯度的产生如图1B所示，其中一个介质通道含有0.1 mM的荧光，而另一个通道不含荧光。细胞在气体渗透膜上培养，通过膜上对芯片的正背压由微观结构封装。图2说明了器件支架的部件，实验设置如图3所示，在37°C的倒置显微镜上保持培养，相对湿度超过85%，并在诺莫夏气体成分。
该系统通过明场和荧光通道提供局部细胞相互作用的详细观察，并允许在空间上解决下游测定，如免疫荧光、西显孔或质谱。我们之前已经证明，这种微流体癌片上模型在上皮和中位PC3前列腺癌细胞12的长期共培养，以及耐药性多倍体巨的出现，作为原理的证明癌细胞使用上皮PC3细胞系13。尽管我们介绍了该平台的应用，以了解上皮PC3和间质性前列腺癌细胞在多西他塞的应力梯度下的时空动力学，但微流体系统可以很容易地应用于细胞系的任意组合和资源（即药物、营养、氧气）梯度。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL BD Luer-Lok tip syringes</td>
			<td>BD</td>
			<td>14-823-16E</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>1x anti-anti</td>
		</tr>
		<tr>
			<td>AZ 300 MIF</td>
			<td>Merck KGaA</td>
			<td>18441123163</td>
			<td>Photoresist developer</td>
		</tr>
		<tr>
			<td>AZ1518</td>
			<td>Merck KGaA</td>
			<td>AZ1518</td>
			<td>Photoresist</td>
		</tr>
		<tr>
			<td>AZ4330</td>
			<td>Merck KGaA</td>
			<td>AZ4330</td>
			<td>Photoresist</td>
		</tr>
		<tr>
			<td>Cr Chromium Etchant</td>
			<td>Sigma-Aldrich</td>
			<td>651826</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Life Technologies Corporation</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Heidelberg DWL 66+ laserwriter</td>
			<td>Heidelberg Instruments</td>
			<td>DWL66+</td>
			<td>Writing photomask</td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Sigma-Aldrich</td>
			<td>379212</td>
			<td>For photoresist adhesion enhancement</td>
		</tr>
		<tr>
			<td>Hollow steel pins</td>
			<td>New England Small Tube</td>
			<td>NE-1300-01</td>
			<td>&#160;.025 OD .017 ID x .500 long / type 304 WD fullhard</td>
		</tr>
		<tr>
			<td>ibidi Heating System, Multi-Well Plates, K-Frame</td>
			<td>ibidi</td>
			<td>10929</td>
			<td>On-stage incubator&#160;</td>
		</tr>
		<tr>
			<td>Luer-Lok 23 G dispensing needle</td>
			<td>McMaster-Carr</td>
			<td>75165A684</td>
			<td>To connect syringes and tubings</td>
		</tr>
		<tr>
			<td>Lumox dish 35</td>
			<td>Sarstedt</td>
			<td>94.6077.331</td>
			<td>Gas-permeable cell culture dish</td>
		</tr>
		<tr>
			<td>Microposit Remover 1165</td>
			<td>Dow Electronic Materials</td>
			<td>Microposit Remover 1165</td>
			<td>Photoresist stripper</td>
		</tr>
		<tr>
			<td>Microseal B Adhesive Sealer</td>
			<td>Bio-Rad Laboratories</td>
			<td>MSB1001</td>
			<td>Adhesive sealer</td>
		</tr>
		<tr>
			<td>O-Ring (for Lumox plate sealing)</td>
			<td>McMaster-Carr</td>
			<td>9452K114</td>
			<td>Dash No. 27; 1-5/16&#34; ID x 1-7/16&#34; OD; Duro 70</td>
		</tr>
		<tr>
			<td>O-Ring (for bottom glass window sealing)</td>
			<td>McMaster-Carr</td>
			<td>9452K74</td>
			<td>Dash No. 20; 7/8&#34; ID x 1&#34; OD; Duro 70</td>
		</tr>
		<tr>
			<td>Plasma-Preen Plasma Cleaning/Etching System</td>
			<td>Plasmatic Systems, Inc</td>
			<td>Plasma-Preen</td>
			<td>Oxygen plasma system</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies Corporation</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Samco RIE800iPB DRIE</td>
			<td>Samco</td>
			<td>RIE800iPB</td>
			<td>Deep reactive-ion etching system</td>
		</tr>
		<tr>
			<td>Suss MA6 mask aligner</td>
			<td>SUSS MicroTec</td>
			<td>MA6</td>
			<td>Mask aligner&#160;</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer</td>
			<td>Fisher Scientific</td>
			<td>NC9285739</td>
			<td>PDMS elastomer</td>
		</tr>
		<tr>
			<td>TePla M4L plasma etcher</td>
			<td>PVA TePla</td>
			<td>M4L</td>
			<td>Plasma etcher</td>
		</tr>
		<tr>
			<td>Trichloro-1H,1H,2H,2H-perfluorooctyl-silane (PFOTS)</td>
			<td>Sigma-Aldrich</td>
			<td>448931</td>
			<td>For silicon wafer silanization</td>
		</tr>
		<tr>
			<td>Tygon microbore tube (0.020&#34; x 0.060&#34;OD)</td>
			<td>Cole-Parmer</td>
			<td>EW-06419-01</td>
			<td>Tubings for media delivery</td>
		</tr>
	</tbody>
</table>

generation of heterogeneous drug gradients across cancer populations on a microfluidic evolution accelerator for real-time observation

传统的细胞培养仍然是最常用的临床前模型，尽管它被证明是有限的能力，预测癌症的临床结果。提出了微流体癌片模型，以弥合过于简化的传统二维培养法与更复杂的动物模型之间的差距，后者产生可靠和可重复的定量结果的能力有限。在这里，我们提出了一个微流体癌片模型，该模型以全面的方式再现复杂肿瘤微环境的关键成分，但简单到足以提供可靠的癌症动力学定量描述。这种微流体癌片模型"进化加速器"将大量癌细胞分解成一系列相互关联的肿瘤微环境，同时生成异构化疗应激环境。癌症在药物梯度反应下的进展和进化动态可以实时监测数周，并且可以进行许多下游实验，以补充实验过程中拍摄的延时图像。

芯片上最佳细胞生长的验证 实验平台的主要目标是在复杂的肿瘤微环境中全面再现关键成分和相互作用，但简单到足以提供定量、可靠和可重复的数据。只有完全控制物理和生化环境因素，才能实现这一目标。我们必须排除不需要的因素，或者找出一种方法，将无法控制的因素纳入定量模型，以正确解释人口行为。
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Watch this Scientific Journal Video about Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation at JoVE.com

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

我们提出了一个微流体癌片模型，即"进化加速器"技术，它为在单细胞明确定义的环境条件下癌症动力学的长期实时定量研究提供了一个可控的平台水平。这项技术有望成为基础研究或临床前药物开发的体外模型。

在微流体进化加速器上生成跨癌症人群的异构药物梯度，用于实时观察

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

这种微流体癌片上模型，我们称之为进化加速器，为在单细胞水平上在定义良好的环境条件下对癌症动力学进行长期、实时定量研究提供了一个可控的平台。该技术使我们能够在肿瘤样应力地貌下探索癌症的进化动力学。在细胞水平上提供包括形态、种群、运动和迁移的信息。凭借在良好控制压力梯度下控制和监测混合肿瘤群体行为的能力，我们的技术可能为临床前药物开发提供一个更生理相关的模型。提出的压力密封包装方法也可以用于其他需要复杂调整环境气体成分和下游实验能力的微流体系统。成功进行长期实验的关键是确保物理和生化稳定的环境。应时刻监控温度、湿度和气体成分等因素。首先，制造一个板架，足以在 3D 打印机上同时容纳气体渗透培养皿。用螺丝刀拧下定制的三个井样品板的部件。通过紫外线照射对部件进行消毒至少一小时，并在无菌环境中离开。开始细胞系播种，在10厘米培养皿中，将5毫升的尝试素添加到每个PC3-Epi或PC3-EMT细胞培养中，并在37摄氏度的培养箱中运行5分钟的尝试素化。将培养皿中的细胞转移到15毫升离心管中，然后以150倍g离心3分钟。丢弃上一杯。使用血细胞计重新悬浮15毫升离心管中的每个细胞颗粒，并计算每个PC3-MP或PC3-EMT的细胞。将每个单元格类型的四个单元格类型总共隔离 2.5 倍 10。混合两种隔离细胞类型，并加入两毫升培养培养。将细胞培养的两毫升种子放入三种气体渗透培养皿中。将气体在97摄氏度的培养箱中保持渗透，使细胞附着。通过紫外线照射对 PDMS 设备进行消毒至少一小时，并在无菌环境中离开。现在建立一个气体供应系统，包括二氧化碳和氧气控制单元、气体泵、气体混合室、加湿器或气泡器，以及三套独立的气体阀门和压力表。或者使用提供无氧条件的供气系统。确保混合室的相对湿度增加到 85% 以上，在 37 摄氏度下设置一个在 37 摄氏度的台式孵化热控制单元，为盖子和底板提供单独的加热子单元。第二天，从孵化器中取出气体渗透培养皿，确定详细的组件井内有一个气体渗透培养皿架、一个PDMS芯片架、一个玻璃窗架和一对35毫升宽的玻璃窗。使用安装的螺钉组装这些部件，并夹紧气体渗透培养皿。然后将预热培养介质转移到真空室中，以脱气 20 分钟。在氧气等离子体系统中处理 PDMS 芯片 30 秒，以保持亲水性。接下来用10毫升的脱气生长培养剂缓慢加载两个注射器，另外两个注射器用10毫升的脱气所需试剂。例如，媒体和媒体与药物。通过 23 表点胶针将每个单独的注射器连接到 50 厘米的管子中，并插入一个空心钢销。将空心钢销插入油管的另一端。将介质推入管内以给油管上，并通过封盖层将钢销插入每个 PDMS 芯片中。填充储层层，用介质将 PDMS 型板层弄湿。储层层可用作片上气泡陷阱，以防止气泡进入微流体模式。然后用培养培养剂加载一毫升注射器。将注射器连接到一个 5 厘米的管子上，用 23 个仪表点胶针将注射器连接到一个空心钢销中。将空心钢销插入管的另一端，然后为油管进行上热。将空心钢销插入芯片的中心孔中，以在以后提取过多的介质。将每个芯片装载两个 10 毫升注射器，然后将另外两个注射器放在注射器系统的提取甲板上。为了避免在微流体图案中夹住微泡，将预加热和去气介质的一毫升分配到35毫升深气体渗透培养皿中。将芯片直接放在气体渗透培养膜上，芯片以 15 度倾斜角接近液体表面。使用 PDMS 芯片支架夹紧 PDMS 芯片，将 PDMS 器件向下推。将一块密封板放在 PDMS 设备的顶部，并夹住 PDMS 芯片支架，以防止芯片干燥。将阵列周围的媒体流速设置为每小时 20 微升。将整个板放在倒置显微镜的电动舞台上的台式培养箱中。通过气管将供气系统连接到气体通道，并保持表压力为 0.2 PSI。这可对已安装的 PDMS 芯片进行气体渗透培养膜加压，以确保器件密封。使用中心孔中的一毫升注射器缓慢提取芯片中的过多介质。在提取介质时观察显微镜下的芯片，然后在芯片密封时停止提取。将台式培养箱的温度感应单元连接到三个井板，并设置为 37 摄氏度。在这项研究中，PC3在进化加速器中培养，没有外部应力，在对齐时有生长曲线。表明细胞增殖轮廓作为整个芯片位置的函数的相同性。曲线的初始斜率显示了增殖率。设备中细胞的翻倍时间约为 24 小时。在进化加速器中，表达PC3-EMT和mCherry表达PC3-Epi细胞衬里的GPC是共同培养的。从40%的汇合开始，共培养在三天内增长40%。。而总汇合的增长曲线是物流增长模型的形式。PC3-EMT的种群继续扩大，PC3-Epi在无症状阶段受到抑制。PC3-Epi 和 PC3-EMT 电池密集地位于设备中心，允许自由迁移到空区域。两种表型速度的规范化直方图显示PC3-EMT速度明显高于PC3-Epi1.8倍。避免不惜一切代价制造微泡非常重要，而文化媒体应预先预热和去气化。PDMS 芯片应正确湿润并轻柔处理。由于压力密封方法的软再密封性质，许多下游实验都可以预制。如亚临床种群提取、局部代谢物分析、空间解决免疫荧光和测序等。我们的技术展示了一种在复杂的超微环境中全面重现关键组件和交互的方法。然而，它非常简单，能够提供定量、可靠和可重复的数据。

Research

JoVE Journal

Bioengineering

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

模型和方法来评价药物输送系统在整个细胞屏障的运输

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

科研

生物工程

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

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 ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

分层海藻酸钠构造：一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

A Protocol for Real-time 3D Single Particle Tracking

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...