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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 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。 在 100 mm ?…
Representative Results
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?…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Fabrication of Acoustofluidic Device | |||
| DOW SYLGARD 184 SILICONE ENCAPSULANT CLEAR 0.5 KG KIT | Ellsworth Adhesives | 4019862 (SKU) | https://www.ellsworth.com/products/by-market/consumer-products/encapsulants/silicone/dow-sylgard-184-silicone-encapsulant-clear-0.5-kg-kit/ |
| Harris Uni-Core (2.5 mm) | Electron Microscopy Sciences | 69039-25 | |
| Microfluidic Reservoir for 15 mL Falcon Tube – S (2/4 port) | Darwin Microfluidics | LVF-KPT-S-2 (SKU) | https://darwin-microfluidics.com/products/15-ml-falcon-tube-microfluidic-reservoir-s-2-4-port |
| Microscope Slide | VWR | 16004-430 | https://us.vwr.com/store/product/4646174/vwr-vistavisiontm-microscope-slides-plain-and-frosted-premium |
| trichlorosilane | Gelest | 105732-02-3 (Cas. No.) | Chlorosilane is very hazaradous and flammable. Exposure causes severe burns and eye damage. |
| Tygon PVC soft plastic tubing (1/16" ID, 1/8" OD) | McMaster-Carr | 5233K51 (Part #) | https://www.mcmaster.com/pvc-tubing/soft-tubing-for-air-and-water/ |
| Assembly of Acoustofluidic System | |||
| Arduino Uno | Arduino | 7630049200050 (Barcode) | https://store.arduino.cc/usa/arduino-uno-rev3 |
| Preparation of Ultrasound Contrast Agents | |||
| 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC) | Avanti Lipids | 890703P-25mg (SKU) | https://avantilipids.com/product/890703 |
| 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) | Avanti Lipids | 850365P-25mg (SKU) | https://avantilipids.com/product/850365 |
| 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) | Avanti Lipids | 840465P-25mg (SKU) | https://avantilipids.com/product/840465 |
| APF-140HP (decafluorobutate gas) | FlouroMed | 355-25-9 (Cas No.) | http://www.fluoromed.com/products/perfluorodecalin/ |
| DB-338 Amalgamators | COXO | https://www.coxotec.com/coxo/db-338-amalgamators/ | |
| polyoxyethylene 40 stearate | Sigma-Aldrich | P3440-250G (SKU) | https://www.sigmaaldrich.com/catalog/product/sigma/p3440?lang=en®ion=US&gclid= Cj0KCQjwy8f6BRC7ARIsAPIXOjjj Jh_151mYVEUyLZRavt4re9YQMLS vID64X-1KbO3LUKGjVUwb PDAaAqvOEALw_wcB |
| Q125 Sonicator | Qsonica | Q125-110 (Ref.) | https://www.sonicator.com/products/q125-sonicator?_pos=1&_sid=406df3776&_ss=r |
| Preparation of Primarty T Cells | |||
| autoMACs running buffer | Miltenyi Biotec | 130-091-221 (Order No.) | https://www.miltenyibiotec.com/US-en/products/automacs-running-buffer-macs-separation-buffer.html#gref |
| Pan T Cell Isolation Kit, human (Pan T-Cell Biotin Antibody Cocktail & Pan T-Cell MicroBead Cocktail) | Miltenyi Biotec | 130-096-535 (Order No.) | https://www.miltenyibiotec.com/US-en/products/pan-t-cell-isolation-kit-human.html#130-096-535 |
| magnetic cell sorter (autoMACS Pro Separator) | Miltenyi Biotec | 130-092-545 (Order No.) | https://www.miltenyibiotec.com/US-en/products/automacs-pro-separator-starter-kit.html#130-092-545 |
| Preparation of A549 Lung Cancer Cells | |||
| Trehalose Assay Kit | Megazyme | K-TREH (Cat. No.) | https://www.megazyme.com/trehalose-assay-kit |
| Trypan blue (0.4% in aqueous solution Ready-to-Use, sterile) | VWR | 97063-702 (Cat. No.) | https://us.vwr.com/store/product/7437427/trypan-blue-0-4-in-aqueous-solution-ready-to-use-sterile |
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Citazione di questo articolo
Centner, C. S., Murphy, E. M., Stamp, B. F., Priddy, M. C., Moore, J. T., Bates, P. J., Menze, M. A., Yaddanapudi, K., Kopechek, J. A. Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells. J. Vis. Exp. (167), e62035, doi:10.3791/62035 (2021).