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Bioengineering

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

Published: January 21st, 2021

DOI:

10.3791/62035

1Department of Bioengineering, University of Louisville, 2School of Medicine, University of Louisville, 3Department of Biology, University of Louisville

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

Viral and non-viral platforms have been utilized to enhance molecular delivery to cells. Viral delivery (transduction) is a common technique utilized in cell-based therapies requiring genomic modification. Limitations with viral delivery include potential insertional mutagenesis, limited transgenic capacity, and undesired multiplicity of infection1,2. Therefore, non-viral molecular delivery techniques are in development for a broad range of biomedical and research applications. Common techniques include mechanical, electrical, hydrodynamic, or the use of laser-based energy to enhance uptake of biomolecules int....

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Whole blood donations were collected from healthy donors following protocols approved by the institutional review board at the University of Louisville.

1. Fabrication of acoustofluidic device

  1. Obtain a photomask with a concentric spiral design containing channels with a diameter of 500 µm. A CAD file is provided in the supplemental files as an example. A custom photomask can be ordered from a commercial vendor or patterned using a mask writ.......

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An image of the acoustofluidic system assembled inside a 3D-printed case is shown in Figure 1. This protocol produces an acoustofluidic system that can be used to enhance intracellular molecular delivery in multiple cell lines using ultrasound contrast agents. 

Figure 2 demonstrates enhanced intracellular delivery of a fluorescent compound, fluorescein, to primary human T cells with acoustofluidic treat.......

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This protocol describes the assembly and operation of a low-cost acoustofluidic system which enhances intracellular delivery of biomolecules for research applications. There are several important factors to consider when assembling and operating this system. The acoustofluidic device is fabricated in PDMS, which is a biocompatible material that can easily be molded with consistent channel dimensions27.The device channels can be rinsed with 15 mL of 70% ethanol solution prior to acoustofluidic proc.......

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This work was supported in part by funding from the National Science Foundation (#1827521, #1827521, #1450370) and the National Institutes of Health (U01HL127518). Photolithography services were provided by the University of Louisville Micro/Nano Technology Center.

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Name Company Catalog Number Comments
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&region=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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