Published: January 21st, 2021
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....
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
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.......
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.......
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.....
|Fabrication of Acoustofluidic Device
|DOW SYLGARD 184 SILICONE ENCAPSULANT CLEAR 0.5 KG KIT
|Harris Uni-Core (2.5 mm)
|Electron Microscopy Sciences
|Microfluidic Reservoir for 15 mL Falcon Tube - S (2/4 port)
|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)
|5233K51 (Part #)
|Assembly of Acoustofluidic System
|Preparation of Ultrasound Contrast Agents
|APF-140HP (decafluorobutate gas)
|355-25-9 (Cas No.)
|polyoxyethylene 40 stearate
|Preparation of Primarty T Cells
|autoMACs running buffer
|130-091-221 (Order No.)
|Pan T Cell Isolation Kit, human (Pan T-Cell Biotin Antibody Cocktail & Pan T-Cell MicroBead Cocktail)
|130-096-535 (Order No.)
|magnetic cell sorter (autoMACS Pro Separator)
|130-092-545 (Order No.)
|Preparation of A549 Lung Cancer Cells
|Trehalose Assay Kit
|K-TREH (Cat. No.)
|Trypan blue (0.4% in aqueous solution Ready-to-Use, sterile)
|97063-702 (Cat. No.)
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