Name: assembly and operation of an acoustofluidic device for enhanced delivery of molecular compounds to cells
Uploaded: 2021-01-21T13:30:00
Description: Watch this Scientific Journal Video about Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells at JoVE.com

这项工作得到了国家科学基金会（#1827521、#1827521、#1450370）和国家卫生研究院（U01HL127518）的资助。光刻服务由路易斯维尔大学微/纳米技术中心提供。

根据路易斯维尔大学机构审查委员会批准的协议，从健康献血者那里收集了全部献血。
1. 制造声毒流体装置
<ol><li>获得带有同心螺旋设计的光面，包含直径为 500 μm 的通道。以补充文件为例，提供 CAD 文件。定制光标可以从商业供应商订购或使用掩码编写器进行图案。</li>
	<li>使用标准光刻技术，在光耐药涂层硅晶片上准备同心螺旋设计的模具。<ol>
<li>在 100 mm ?...

<ol>
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N., 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=Acoustofluidic+sonoporation+for+gene+delivery+to+human+hematopoietic+stem+and+progenitor+cells.">Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells.</a> Proceedings of the National Academy of Sciences. 117 (20), 10976-10982 (2020).</li><li>Centner, C. 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=Ultrasound-induced+molecular+delivery+to+erythrocytes+using+a+microfluidic+system.">Ultrasound-induced molecular delivery to erythrocytes using a microfluidic system.</a> Biomicrofluidics. 14 (2), 024114 (2020).</li><li>Qi, J., Ding, C., Jiang, X., Gao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+developing+CAR+T-cell+therapy+for+HIV+cure.">Advances in developing CAR T-cell therapy for HIV cure.</a> Frontiers in Immunology. 11, 361 (2020).</li><li>Annesley, C. E., Summers, C., Ceppi, F., Gardner, R. 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本协议描述了低成本声学流体系统的组装和运行，该系统可增强用于研究应用的生物分子的细胞内输送。在组装和操作该系统时，需要考虑几个重要因素。共氟化装置是在PDMS中制造的，PDMS是一种生物相容材料，可以很容易地与一致的通道尺寸27成型。在进行声毒处理之前，设备通道可以用 15 mL 的 70% 乙醇溶液冲洗，以便在与培养细胞配合使用时增加不育性。乙醇清洗后，15 mL 的...

病毒和非病毒平台已被利用，以提高分子输送到细胞。病毒传递（转导）是一种常见的技术，用于细胞为基础的治疗，需要基因组修饰。病毒传播的限制包括潜在的插入性突变，有限的转基因能力，和不受欢迎的感染1，2的多重性。因此，非病毒分子传递技术正在开发广泛的生物医学和研究应用。常见的技术包括机械、电气、流体动力学，或利用激光能量来增强生物分子进入细胞3。电聚变是一种常用的非病毒分子传递平台，能够诱导等离子体膜中瞬态穿孔，用于分子化合物4、5、6、7、8、9的细胞内输送。然而，等离子体膜的瞬态穿孔是一个随机的过程，通过电聚变的分子吸收通常依赖于在瞬态膜孔隙4、7、8的被动扩散。
另一种方法是利用超声波通过超声波对比剂（即充满气体的微泡）的空腔增强细胞内分子传递。微泡空腔在周围介质中诱导微流效应，可导致附近等离子体膜（"声像"）的瞬时穿孔，从而通过被动或主动传输机制10、11、12快速吸收生物分子。声波是快速分子输送到细胞的有效技术，但这种方法往往需要昂贵的设备和批量处理方法，这些方法受到超声波暴露条件下产量较低和变异性较高的限制。为了解决这些限制，声透流体装置目前正在开发中，这种装置能够对悬浮细胞进行一致的声扑。
气流是一个扩展的领域，集成超声波和微流体技术，用于各种应用。这种方法以前曾用于粒子分离，通过应用连续超声波能量诱导流体通道内的站立声波14、15、16、17。粒子根据粒子大小、密度和相对于中等16的可压缩性等多种特性对设备的不同部分进行排序。声透流技术也在开发中，使各种细胞类型能够快速分子传递，用于研究应用和制造细胞疗法18。最近，我们使用基于PDMS的声学流体装置19演示了增强分子传递到红细胞。在声学平台中，可以操纵细胞和微泡动力学来诱导物理相互作用，增强生物分子的传递能力。通过优化细胞和微泡之间的距离，可以潜在地提高细胞内分子传递的效率和一致性。
声毒介质声扑的一个重要应用是将生物分子输送到原发性人类T细胞中。基于收养T细胞转移的免疫疗法，如奇美利安抗原受体T细胞（CAR T）疗法，正在迅速出现，用于治疗各种疾病，包括癌症和艾滋病毒20等病毒。CAR T疗法在小儿急性淋巴细胞白血病（ALL）患者中效果特别显著，完全缓解率为70-90%21。然而，这些疗法的T细胞制造通常取决于病毒转导，这是受潜在的插入突变，漫长的加工时间，以及提供非遗传生物分子的挑战，如蛋白质或小分子1的限制。以气管为媒介的分子输送方法有可能克服这些限制，改善T细胞疗法的制造。
声毒介质声质的另一个重要应用涉及防腐剂化合物的细胞内输送，如三卤素，在冷冻和干燥期间保护细胞。三卤素是由一些生物在自然界中产生的，通过保护它们的细胞膜22，23，帮助他们耐受冷冻和干燥。然而，三卤素不是由哺乳动物细胞产生的，并且对哺乳动物细胞膜不透水。因此，为了达到保护内部细胞膜所需的足够细胞内三卤素水平，有必要采用有效的分子传递技术，如声波。目前正在开发这种方法，用于各种细胞类型的干保存。
本协议详细描述了由微控制器驱动的相对低成本的声学流体系统的组装和运行情况。超声对比剂用于在流体通道内诱导声波，并使各种细胞类型（包括T细胞和癌细胞）能够快速分子传递。这种声毒流体系统可用于各种研究应用，也可作为原型系统来评估声扑方法，以改进细胞治疗制造工艺。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fabrication of Acoustofluidic Device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DOW SYLGARD 184 SILICONE ENCAPSULANT CLEAR 0.5 KG KIT</td>
			<td>Ellsworth Adhesives</td>
			<td>4019862 (SKU)</td>
			<td>https://www.ellsworth.com/products/by-market/consumer-products/encapsulants/silicone/dow-sylgard-184-silicone-encapsulant-clear-0.5-kg-kit/</td>
		</tr>
		<tr>
			<td>Harris Uni-Core (2.5 mm)</td>
			<td>Electron Microscopy Sciences</td>
			<td>69039-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic Reservoir for 15 mL Falcon Tube - S (2/4 port)</td>
			<td>Darwin Microfluidics</td>
			<td>LVF-KPT-S-2 (SKU)</td>
			<td>https://darwin-microfluidics.com/products/15-ml-falcon-tube-microfluidic-reservoir-s-2-4-port</td>
		</tr>
		<tr>
			<td>Microscope Slide</td>
			<td>VWR</td>
			<td>16004-430</td>
			<td>https://us.vwr.com/store/product/4646174/vwr-vistavisiontm-microscope-slides-plain-and-frosted-premium</td>
		</tr>
		<tr>
			<td>trichlorosilane</td>
			<td>Gelest</td>
			<td>105732-02-3 (Cas. No.)</td>
			<td>Chlorosilane is very hazaradous and flammable. Exposure causes severe burns and eye damage.&#160;</td>
		</tr>
		<tr>
			<td>Tygon PVC soft plastic tubing (1/16&#34; ID, 1/8&#34; OD)</td>
			<td>McMaster-Carr</td>
			<td>5233K51 (Part #)</td>
			<td>https://www.mcmaster.com/pvc-tubing/soft-tubing-for-air-and-water/</td>
		</tr>
		<tr>
			<td>Assembly of Acoustofluidic System</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino Uno</td>
			<td>Arduino</td>
			<td>7630049200050 (Barcode)</td>
			<td>https://store.arduino.cc/usa/arduino-uno-rev3</td>
		</tr>
		<tr>
			<td>Preparation of Ultrasound Contrast Agents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC)</td>
			<td>Avanti Lipids</td>
			<td>890703P-25mg (SKU)</td>
			<td>https://avantilipids.com/product/890703</td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)</td>
			<td>Avanti Lipids</td>
			<td>850365P-25mg (SKU)</td>
			<td>https://avantilipids.com/product/850365</td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG)</td>
			<td>Avanti Lipids</td>
			<td>840465P-25mg (SKU)</td>
			<td>https://avantilipids.com/product/840465</td>
		</tr>
		<tr>
			<td>APF-140HP (decafluorobutate gas)</td>
			<td>FlouroMed</td>
			<td>355-25-9 (Cas No.)</td>
			<td>http://www.fluoromed.com/products/perfluorodecalin/</td>
		</tr>
		<tr>
			<td>DB-338 Amalgamators&#160;</td>
			<td>COXO</td>
			<td></td>
			<td>https://www.coxotec.com/coxo/db-338-amalgamators/</td>
		</tr>
		<tr>
			<td>polyoxyethylene 40 stearate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P3440-250G (SKU)</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/p3440?lang=en&#38;region=US&#38;gclid= Cj0KCQjwy8f6BRC7ARIsAPIXOjjj Jh_151mYVEUyLZRavt4re9YQMLS vID64X-1KbO3LUKGjVUwb PDAaAqvOEALw_wcB</td>
		</tr>
		<tr>
			<td>Q125 Sonicator</td>
			<td>Qsonica</td>
			<td>Q125-110 (Ref.)</td>
			<td>https://www.sonicator.com/products/q125-sonicator?_pos=1&#38;_sid=406df3776&#38;_ss=r</td>
		</tr>
		<tr>
			<td>Preparation of Primarty T Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>autoMACs running buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221 (Order No.)</td>
			<td>https://www.miltenyibiotec.com/US-en/products/automacs-running-buffer-macs-separation-buffer.html#gref</td>
		</tr>
		<tr>
			<td>Pan T Cell Isolation Kit, human (Pan T-Cell Biotin Antibody Cocktail &#38; Pan T-Cell MicroBead Cocktail)&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-535 (Order No.)</td>
			<td>https://www.miltenyibiotec.com/US-en/products/pan-t-cell-isolation-kit-human.html#130-096-535</td>
		</tr>
		<tr>
			<td>magnetic cell sorter (autoMACS Pro Separator)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-545 (Order No.)</td>
			<td>https://www.miltenyibiotec.com/US-en/products/automacs-pro-separator-starter-kit.html#130-092-545</td>
		</tr>
		<tr>
			<td>Preparation of A549 Lung Cancer Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose Assay Kit&#160;</td>
			<td>Megazyme</td>
			<td>K-TREH (Cat. No.)</td>
			<td>https://www.megazyme.com/trehalose-assay-kit</td>
		</tr>
		<tr>
			<td>Trypan blue (0.4% in aqueous solution Ready-to-Use, sterile)</td>
			<td>VWR</td>
			<td>97063-702 (Cat. No.)</td>
			<td>https://us.vwr.com/store/product/7437427/trypan-blue-0-4-in-aqueous-solution-ready-to-use-sterile</td>
		</tr>
	</tbody>
</table>

assembly and operation of an acoustofluidic device for enhanced delivery of molecular compounds to cells

广泛的生物医学研究和基于细胞的治疗应用需要高效的细胞内生物分子输送。超声波介质是一种新兴的生物分子细胞内快速输送技术。当充满气体的微气泡在附近的细胞膜中形成瞬态孔隙时，就会发生声扑，从而能够从周围液体中快速吸收生物分子。目前悬浮中细胞的体外声波成像技术受到吞吐量缓慢、每个细胞超声波暴露条件下的变异性和高成本的限制。为了解决这些限制，开发出一种低成本的声学流体装置，它结合了基于PDMS的流体装置中的超声波传感器，在与超声波对比剂结合通过通道时诱导细胞的一致声波。该设备是使用标准光刻技术制造的，以生产基于PDMS的流体芯片。超声波皮佐盘传感器连接到设备上，由微控制器驱动。该组件可以集成在 3D 打印的箱子中以获得额外的保护。细胞和微泡使用注射器泵或连接到 PVC 管的渗透泵通过设备。通过这种声毒流体系统，可以证明生物分子能够增强地将生物分子输送到人类T细胞和肺癌细胞。与批量治疗方法相比，这种声毒流体系统可增加产量，降低变异性，从而改进生物医学研究应用和细胞治疗的制造细胞处理方法。

图1显示在3D打印外壳内组装的声透流体系统图像。此协议产生声透流体系统，可用于使用超声波对比剂在多个细胞系中增强细胞内分子传递。
图2  与未经治疗的对照组（p<0.05，n=3/组）相比，通过声毒素治疗，将荧光化合物荧光素增强到原发性人类T细胞的细胞内传递。T细胞在PBS中浓度为100万微米/mL，具有100微?...

Watch this Scientific Journal Video about Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells at JoVE.com

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

该协议描述了低成本声学流体装置的组装和运行，该装置通过超声波对比剂诱导的声波快速分子输送到细胞。

增强分子化合物输送到细胞的声透流体装置的组装和操作

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

本协议描述了声学流体系统的组装和运行，该系统可高效地为各种研究和基于细胞的治疗应用提供生物分子。该系统的主要优点是，它允许生物分子快速输送到细胞，同时保持其生存能力。它是一种平台技术，允许细胞被改造为各种细胞治疗应用，如癌症治疗。首先，将 54 克 PDMS 碱基和 6 克固化剂组合在杯子中。用铲子大力和彻底地混合至少一分钟，然后将带有PDMS溶液的杯子放入干燥器中约30分钟，或直到去除残余气泡。将带光耐光剂涂层的晶圆与朝上朝上的图案放在 150 毫米的 Petri 盘中，然后将 PDMS 溶液倒在模具上。如果需要，将培养皿放入干燥器中，并涂抹真空，直到残余气泡消失。将培养皿转移到实验室烤箱中，在60摄氏度下烘烤两小时，以治愈PDMS。固化后，用剃须刀刀片切开晶圆边缘，小心地从培养皿中取出PDMS。使用刀或剃刀刀片切除每个设备，然后使用 2.5 毫米活检冲孔打孔通过入口和出口端口。使用氧气等离子体处理PDMS设备后，立即将每个设备放在一个干净的苏打石灰玻璃显微镜滑动上，通道面向玻璃表面。允许设备在室温下隔夜粘合。轻轻地将硅胶涂抹在一厘米直径的 Piezo 传感器表面，厚度为一到两毫米，然后用同心螺旋小心地对齐传感器，轻轻按压到玻璃显微镜滑梯的底部。使用 USB A 到 B 电缆将微控制器连接到计算机。绿色动力 LED 指标应亮起。使用计算机上的相关软件上传生成八兆赫信号的程序。在 PZT 传感器上将一英寸 22 仪表线焊接到每根电线的末端。然后用它来连接 PZT 传感器的负端线到 GND 销。通过焊接线将 PZT 传感器的正端线连接到输出销。切开三到六英寸的 Tygon PVC 软塑料管，将管子推入进气口和出口端口。可能需要在施加压力时旋转管子，直到它适合打开。可选，可以在交界处涂上胶水，将PDMS和管子粘合在一起。可选地将声透流体设备和微控制器安装在 3D 打印外壳中。根据制造商的说明组装微流体储层。切割一个3至6英寸的1/16英寸内径TygonPVC软塑料管，并推动它从微流体储层输出的1/32英寸内径管。可选用石蜡薄膜包裹路口，以防止泄漏。在微流体储层的一侧填充一个60毫升的注射器，并充满环境空气。将注射器泵设置为每小时 200 毫升，以每小时 50 毫升的体积流量将对比剂解决方案通过声透流器，并将设备输出的样品收集到 50 毫升离心机管中。加入25毫克DSPC、11.6毫克DSPC、0.26毫克DSPG和0.88毫克聚氧乙烯40固醇，在20毫升闪烁瓶中加入磷脂溶液。加入氯仿，直到所有磷脂溶解。在干燥器中蒸发氯仿48小时，形成干脂膜。用10毫升无菌PBS补充泡沫，然后以40%的振幅将脂质溶液调谐三分钟，形成阴离子鼠溶液。声波化后，磷脂溶液可在摄氏2至6度下储存长达一个月。要制备超声波对比剂，在 600 微升无菌 PBS 中加入 200 微升的阴离子鼠片溶液，加入两毫升玻璃隔膜瓶中。通过压接盖住小瓶来密封小瓶。使用 1.5 英寸 20 量表针将小瓶头空间填充 30 秒的二氟丁烷气体。将小瓶合并，形成全氟丁烷气体填充超声波对比剂。每毫升细胞溶液添加25微升超声波对比剂溶液。然后立即泵送组合对比剂和细胞混合物通过声透流体设备。此协议产生声毒流体系统，可用于增强多个细胞系的细胞内分子传递。与未经治疗的对照组相比，通过声毒流体治疗，荧光化合物荧光素向原发性人类T细胞的细胞内输送得到了改善。T细胞的荧光强度在治疗后增加了五倍，表明荧光素的输送增强。细胞生存能力在声毒性治疗后略有下降，但与单独流动和未治疗对照组的细胞相比，仍然高于80%的血吸虫治疗，增强了防腐剂化合物三卤素对人体A549肺癌细胞的细胞的细胞内传递。将硅化微泡添加到细胞溶液中至关重要。如果没有溶液中的细胞微泡，生物分子的细胞内输送将受到限制。该平台可实现各种生物分子的细胞内传递，从而改变细胞功能，用于研究或治疗目的。完成此过程后，可以执行额外的细胞特征，以评估提供各种生物分子的影响。

Research

JoVE Journal

Bioengineering

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

科研

生物工程

Operation of a Benchtop Bioreactor

一个台式生物反应器的操作

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

电化学 DNA 生物传感器检测食源性致病菌的研究进展

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

细胞模拟支持和悬浮脂质双分子层模型的组装用于分子相互作用研究

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...