This work was supported in part by funding from the National Science Foundation (#1827521, #1827521, #1450370)&#160;and the National Institutes of Health (U01HL127518). Photolithography services were provided by the University of Louisville Micro/Nano Technology Center.

Whole blood donations were collected from healthy donors following protocols approved by the institutional review board at the University of Louisville.
1.&#160;Fabrication&#160;of&#160;acoustofluidic&#160;device
<ol>
	<li>Obtain a photomask with a concentric spiral design containing channels with a diameter of 500 &#181;m. 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 writer.</li>
	<li>Prepare a mold of the concentric spiral design on a photoresist-coated silicon wafer using standard photolithography techniques.
	<ol>
		<li>Add approximately 2 Tbsp (~30 mL) of SU-8 2100 to a 100 mm silicon wafer.</li>
		<li>Spin-coat the wafer on a spinner at a speed of 150 rpm for 30 s to spread out the photoresist, then increase the speed to 1,200 rpm for 60 s to yield a thickness of 200 &#181;m.</li>
		<li>Cure the photoresist-coated wafer in a polyimide vacuum oven with a 30 min ramp up and 30 min dwell at 115 &#176;C, then ramp down for 30 min.</li>
		<li>Expose the photoresist-coated wafer for 130 s using a mask aligner with the photomask from step 1.1.</li>
		<li>Bake the wafer after exposure following the same process described in step 1.2.3.</li>
		<li>Develop the photoresist in SU-8 developer solution for approximately 8 min. 
		CAUTION: Only use developer solution in a well-ventilated chemical fume hood.</li>
	</ol>
	</li>
	<li>Silanize the mold to make the surface more hydrophobic. Place the photoresist-coated wafer into a desiccator and add a 20 &#181;L drop of chlorosilane (C8H4Cl3F13Si). Apply vacuum to the chamber for 30 s, then seal the chamber and leave overnight. 
	CAUTION: Chlorosilane is very hazardous and flammable. Exposure causes severe burns and eye damage.</li>
	<li>Combine 54 g of polydimethsiloxane (PDMS) base and 6 g of curing agent in a cup and mix vigorously and thoroughly with a spatula for at least 1 min.</li>
	<li>Place the cup containing the PDMS solution into a desiccator for approximately 30 min or until remnant air bubbles are removed from the solution.</li>
	<li>Place photoresist-coated wafer with the patterns facing upward in a 150-mm Petri dish.</li>
	<li>Pour the PDMS solution over the mold inside the 150-mm Petri dish.</li>
	<li>If needed, place the 150-mm Petri dish inside a desiccator and apply vacuum until remnant air bubbles disappear.</li>
	<li>Transfer the 150-mm Petri dish into a lab oven and bake for 2 h at 60 &#176;C to cure the PDMS.</li>
	<li>After curing, carefully remove the PDMS from the Petri dish by cutting around the edges of the wafer using a razor blade.</li>
	<li>Cut out each individual device using a knife or razor blade.</li>
	<li>Punch holes through the inlet and outlet ports of each device using a 2.5-mm biopsy punch.</li>
	<li>Place each PDMS device in a plasma asher with channels exposed (facing upward). Apply oxygen plasma treatment (100 W for 45 s, 500 mbar O2) then immediately place each PDMS device onto a clean soda lime glass microscope slide (75 mm x 25 mm x 1 mm) with channels facing the glass surface.</li>
	<li>Let devices bond overnight at room temperature.</li>
	<li>Gently apply silicone to the surface of the 1-cm diameter piezo transducer at a thickness of ~1-2mm, then carefully align the transducer with the concentric spiral and gently press it onto the bottom of the glass microscope slide (opposite side from the PDMS device).</li>
</ol>
2.&#160;Assembly&#160;and&#160;operation&#160;of&#160;acoustofluidic&#160;system
<ol>
	<li>Connect a microcontroller to a computer using a USB A to B cable. A green power LED indicator (labeled PWR) should illuminate.</li>
	<li>Use the associated program on the computer to upload a program which generates an 8 MHz signal. An example program is provided in the&#160;Supplemental&#160;Files. After uploading the program, it will be stored into microprocessor memory and will not need to be uploaded again.</li>
	<li>Solder a 1&#34; 22G wire to the end of each wire on the PZT transducer.</li>
	<li>Connect the negative (black) terminal wire of PZT transducer to a GND pin via the soldered wire.</li>
	<li>Connect the positive (red) terminal wire of PZT transducer to the output pin (#9 in the provided example program) via the soldered wire.</li>
	<li>Optionally, mount the acoustofluidic device and the microcontroller in a 3D-printed case. CAD files are provided in the&#160;Supplemental&#160;Files&#160;as examples. Additional wires can be connected to other microcontroller pins to control an external LED indicator and on/off push button if desired.</li>
	<li>Cut 3-6&#34; sections of tygon PVC soft plastic tubing (1/16&#34; ID, 1/8&#34; OD) and push the tubing into the inlet and outlet ports. It may be necessary to rotate the tubing while applying pressure until it fits in the opening. Optionally, after inserting the tubing into each port, glue can be applied at the junction to bond the PDMS and tubing together.</li>
	<li>Assemble the microfluidic reservoir according to manufacturer&#39;s instructions.</li>
	<li>Cut a 3-6&#34; section of tygon PVC soft plastic tubing (1/16&#34; ID, 1/8&#34; OD) and push the tubing over the 1/32&#34; ID tubing from the microfluidic reservoir output tubing. Optionally, wrap the junction with paraffin film to prevent leakage.</li>
	<li>Fill a 60-mL syringe with ambient air (optionally, filter the air with a 0.2-&#181;m filter) and connect it to tygon PVC tubing (1/16&#34; ID, 1/8&#34; OD) on the side of the microfluidic reservoir.</li>
	<li>Set the syringe pump to a rate of 200 mL/h to push the cell/ultrasound contrast agent solutions through the acoustofluidic device at a volumetric flow rate of 50 mL/h and collect the samples from the output of the acoustofluidic device into a 50mL centrifuge tube. Optionally, rinse channel prior to acoustofluidic treatment with 15 mL of 70% ethanol solution to increase sterility of fluidic channels. Additionally, channels can be rinsed with 15 mL of deionized water to remove residual ethanol in the device prior to pumping cells through the system.</li>
</ol>
3.&#160;Preparation of ultrasound contrast agents
NOTE: Ultrasound contrast agents significantly enhance acoustofluidic delivery of molecular compounds by transiently increasing permeabilization of nearby cellular membranes19. Molecular delivery is very limited without ultrasound contrast agents in this system.
<ol>
	<li>Prepare a phospholipid solution in a 20mL scintillation vial containing the following mixture:
	<ol>
		<li>Add 25 mg of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).</li>
		<li>Add 11.6 mg of 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC).</li>
		<li>Add 0.26 mg of 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG).</li>
		<li>Add 0.88 mg of polyoxyethylene40 stearate.</li>
	</ol>
	</li>
	<li>Add chloroform until all phospholipids are dissolved (e.g., 3 mL of chloroform).</li>
	<li>Evaporate chloroform in a desiccator for 48 h to form a dry lipid film (evaporation under argon or with a rotary evaporator can be used to accelerate the drying process).</li>
	<li>Rehydrate the lipid film with 10 mL of sterile phosphate-buffered saline (PBS).</li>
	<li>Sonicate the lipid solution for 3 min at 40% amplitude to form a cationic micellar solution.</li>
	<li>After sonication store the phospholipid solution at 2-6 &#176;C for up to 1 month.</li>
	<li>To prepare ultrasound contrast agents, add 200 &#181;L of cationic micellar solution and 600 &#181;L of sterile PBS to a 2 mL glass septum vial.</li>
	<li>Seal the vial by crimping the cap.</li>
	<li>Use a 1.5&#34; 20G needle to fill the vial head space with decafluorobutane gas for 30 s.</li>
	<li>Amalgamate the vial for 45 s at 4,350 cpm to form perfluorobutane gas-filled ultrasound contrast agents.</li>
	<li>Add 25 &#181;L of ultrasound contrast agent solution per 1 mL of cell solution immediately before pumping the combined contrast agent/cell mixture through the acoustofluidic device. The cell solution can be modified as desired by the user, but in our studies the cell solution consisted of primary T cells in step 4.21, and A549 lung cancer cells in step 5.7, respectively.</li>
</ol>
4.&#160;Preparation of primary Tcells
<ol>
	<li>Isolate peripheral blood mononuclear cells (PBMCs) from whole blood solutions and store at -150 &#176;C. Density gradient separation containing a substrate is commonly utilized to separate PBMCs from whole blood24,25,26.</li>
	<li>Thaw frozen vial in 37 &#176;C water bath.</li>
	<li>Dilute thawed PBMCs 1:10 with PBS in a 15mL centrifuge tube. Each 1mL vial contains approximately 10 million PBMCs.</li>
	<li>Centrifuge diluted PBMCs at 580 x&#160;g&#160;for 11 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and add 13 mL of MACs running buffer to resuspend the cells.</li>
	<li>Count the PBMCs with an automated cell counter or hemocytometer.</li>
	<li>Centrifuge the PBMCs again at 580 x&#160;g&#160;for 11 min at 4 &#176;C and aspirate the supernatant.</li>
	<li>Add 40 &#181;L of chilled running buffer per 10 million PBMCs.</li>
	<li>To isolate T cells, add 10 &#181;L of Pan T-Cell Biotin Antibody Cocktail per 10 million PBMCs.</li>
	<li>Gently agitate the PBMCs and store the solution at 4 &#176;C for 5 min per 10 million cells.</li>
	<li>Add 30 &#181;L of running buffer and 20 &#181;L of Pan T-Cell MicroBead Cocktail per 10 million PBMCs.</li>
	<li>Mix the PBMCs and beads thoroughly and incubate for an additional 15 min at 4 &#176;C.</li>
	<li>Add running buffer to reach a total volume of 500 &#181;L.</li>
	<li>Separate primary T cells with a commercially available benchtop magnetic sorting instrument using the &#34;depletes separation&#34; setting following manufacturer&#39;s protocol. This step should yield between 5-10 million T cells after cell sorting.</li>
	<li>Count T cells using an automated cell counter or hemocytometer.</li>
	<li>Dilute T cells in 10 mL of sterile PBS and centrifuge at 580 x g for 10 min at 4 &#176;C to pellet the cells.</li>
	<li>Aspirate the supernatant and resuspend T cells in 1 mL of PBS.</li>
	<li>Count T cells using an automated cell counter or hemocytometer and aliquot 1 million/mL for experiments.</li>
	<li>Prepare a 1 mg/mL fluorescein solution in PBS.</li>
	<li>Add 100 &#181;L of 1 mg/mL fluorescein solution per 1 mL of T cell solution (final fluorescein concentration = 100 &#181;g/mL) immediately prior to processing.</li>
	<li>Add 25 &#181;L of ultrasound contrast agent solution as previously described in step 3.11.</li>
	<li>Process 1mL aliquots of cells using the acoustofluidic system (see steps 2.10-2.11). This step enhances delivery of fluorescein into primary T cells.</li>
	<li>Immediately after treatment, wash cells three times via centrifugation at 580 x&#160;g&#160;for 10 min with 500 &#181;L of PBS to remove extracellular fluorescein. Cells should be washed within 10 min after adding fluorescein solution.</li>
	<li>After final washing step, resuspend cells in 250 &#181;L of PBS and measure fluorescence on flow cytometer.</li>
</ol>
5.&#160;Preparation of&#160;A549&#160;lung cancer cells
<ol>
	<li>Culture A549 (adenocarcinomic human alveolar basal epithelial) cells in complete DMEM media (10% fetal bovine serum, 1% penicillin/streptomycin) at 37 &#176;C and 5% CO2&#160;in a flat-bottom tissue culture flask.</li>
	<li>Harvest A549 cells when they reach 70-90% confluency. Aspirate media from the flask and wash the cells once with PBS to remove serum proteins.</li>
	<li>Add trypsin (0.25%) EDTA to the flask and incubate for 5 min at 37 &#176;C. Trypsin is a digestive enzyme which causes the cells to detach from the bottom surface of the tissue culture flask.</li>
	<li>Transfer trypsin solution to a 15mL centrifuge tube and neutralize it by adding complete DMEM media at a 1:3 ratio.</li>
	<li>Pellet the cells via centrifugation at 1,500 x&#160;g&#160;for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the pellet at a concentration of 100,000/mL in PBS solution containing 200 mM trehalose in 15-mL conical vial.</li>
	<li>Add 25 &#181;L of ultrasound contrast agent solution as previously described in step 3.11.</li>
	<li>Process 1mL aliquots of cells using the acoustofluidic system (see steps 2.10-2.11). This step enhances delivery of trehalose into A549 lung cancer cells.</li>
	<li>Immediately after treatment, wash cells three times via centrifugation with 500 &#181;L of PBS to remove extracellular trehalose. Cells should be washed within 10 min after adding trehalose solution.</li>
	<li>After final washing step, resuspend cells in 100 &#181;L of PBS.</li>
	<li>Add 11 &#181;L of 1% Triton X-100 solution to lyse cells and release intracellular trehalose.</li>
	<li>Vortex for 15 s, then incubate for 30 min at room temperature.</li>
	<li>Vortex again for 15 s, then measure trehalose concentration using commercially available trehalose assay following manufacturer&#39;s recommendation.</li>
</ol>

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A., 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+targeted+microbubble+destruction-mediated+delivery+of+a+transcription+factor+decoy+inhibits+STAT3+signaling+and+tumor+growth.">Ultrasound targeted microbubble destruction-mediated delivery of a transcription factor decoy inhibits STAT3 signaling and tumor growth.</a> Theranostics. 5 (12), 1378-1387 (2015).</li><li>Bhutto, D. F., 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=Effect+of+molecular+weight+on+sonoporation-mediated+uptake+in+human+cells.">Effect of molecular weight on sonoporation-mediated uptake in human cells.</a> Ultrasound in Medical Biology. 44 (12), 2662-2672 (2018).</li><li>Forbes, M. M., Steinberg, R. L., O'Brien, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency-dependent+evaluation+of+the+role+of+definity+in+producing+sonoporation+of+Chinese+hamster+ovary+cells.">Frequency-dependent evaluation of the role of definity in producing sonoporation of Chinese hamster ovary cells.</a> Journal of Ultrasound in Medicine. 30 (1), 61-69 (2011).</li><li>Helfield, B., Chen, X., Watkins, S. C., Villanueva, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+insight+into+mechanisms+of+sonoporation.">Biophysical insight into mechanisms of sonoporation.</a> Proceedings of National Academy of Science U. S. A. 113 (36), 9983-9988 (2016).</li><li>Miller, D. L., Bao, S., Morris, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonoporation+of+cultured+cells+in+the+rotating+tube+exposure+system.">Sonoporation of cultured cells in the rotating tube exposure system.</a> Ultrasound in Medical Biology. 25 (1), 143-149 (1999).</li></ol>

Co-authors MAM and JAK hold ownership in DesiCorp which may financially benefit from products related to this research.

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...

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 into cells&#160;3. Electroporation is a commonly used non-viral molecular delivery platform which has the ability to induce transient perforation in the plasma membrane for intracellular delivery of molecular compounds4,5,6,7,8,9. However, the transient perforation of the plasma membrane is a stochastic process and molecular uptake via electroporation is generally dependent on passive diffusion across the transient membrane pores4,7,8.
An alternative method is the utilization of ultrasound for enhanced intracellular molecular delivery via cavitation of ultrasound contrast agents (i.e., gas-filled microbubbles). Microbubble cavitation induces microstreaming effects in the surrounding media which can cause transient perforation of nearby plasma membranes (&#34;sonoporation&#34;) allowing rapid intracellular uptake of biomolecules via passive or active transport mechanisms10,11,12. Sonoporation is an effective technique for the rapid molecular delivery to cells, but this approach often requires expensive equipment and bulk treatment methods which are limited by lower throughput and higher variability in ultrasound exposure conditions13. To address these limitations, acoustofluidic devices, which enable consistent sonoporation of cells in suspension, are currently in development.
Acoustofluidics is an expanding field that integrates ultrasound and microfluidic technologies for a wide variety of applications. This approach has previously been used for particle separation by applying continuous ultrasound energy to induce standing acoustic waves within the fluidic channels14,15,16,17. Particles are sorted toward different parts of the device based on a variety of properties such as particle size, density, and compressibility relative to the medium16. Acoustofluidic technologies are also in development to enable rapid molecular delivery to a variety of cell types for research applications and manufacturing of cell therapies18. Recently, we demonstrated enhanced molecular delivery to erythrocytes using a PDMS-based acoustofluidic device19. In the acoustofluidic platform, cell and microbubble dynamics can be manipulated to induce physical interactions that enable enhanced delivery of biomolecules. The efficiency and consistency of intracellular molecular delivery can potentially be increased by optimizing the distance between cells and microbubbles.
One important application for acoustofluidic-mediated sonoporation involves transport of biomolecules into primary human T cells. Immunotherapies based on adoptive T cell transfer, such as Chimeric Antigen Receptor T cell (CAR T) therapy, are rapidly emerging for treatment of various diseases, including cancer and viruses such as HIV20. CAR T therapy has been particularly effective in pediatric acute lymphoblastic leukemia (ALL) patients, with complete remission rates of 70-90%21. However, T cell manufacturing for these therapies generally depends on viral transduction which is limited by potential insertional mutagenesis, long processing times, and challenges of delivering non-genetic biomolecules such as proteins or small molecules1. Acoustofluidic-mediated molecular delivery methods can potentially overcome these limitations and improve manufacturing of T cell therapies.
Another important application for acoustofluidic-mediated sonoporation involves intracellular delivery of preservative compounds, such as trehalose, which protect cells during freezing and desiccation. Trehalose is produced by some organisms in nature and helps them tolerate freezing and desiccation by protecting their cellular membranes22,23. However, trehalose is not produced by mammalian cells and is impermeable to mammalian cell membranes. Therefore, effective molecular delivery techniques, such as sonoporation, are necessary in order to achieve sufficient intracellular trehalose levels required to protect internal cellular membranes. This approach is currently in development for dry preservation of various cell types.
This protocol provides a detailed description of the assembly and operation of a relatively low-cost acoustofluidic system driven by a microcontroller. Ultrasound contrast agents are utilized to induce sonoporation within the fluidic channels and enable rapid molecular delivery to various cell types, including T cells and cancer cells. This acoustofluidic system can be used for a variety of research applications and may also be useful as a prototype system to evaluate sonoporation methods for improved cell therapy manufacturing processes.

<table><tbody><tr><td>&lt;strong&gt;Fabrication of Acoustofluidic Device&lt;/strong&gt;</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.&amp;nbsp;</td></tr><tr><td>Tygon PVC soft plastic tubing (1/16&quot; ID, 1/8&quot; 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>&lt;strong&gt;Assembly of Acoustofluidic System&lt;/strong&gt;</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>&lt;strong&gt;Preparation of Ultrasound Contrast Agents&lt;/strong&gt;</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&amp;nbsp;</td><td>COXO</td><td></td><td>https://www.coxotec.com/coxo/db-338-amalgamators/</td></tr><tr><td>polyoxyethylene 40 stearate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P3440-250G (SKU)</td><td>https://www.sigmaaldrich.com/catalog/product/sigma/p3440?lang=en&amp;amp;region=US&amp;amp;gclid=&lt;br/&gt;Cj0KCQjwy8f6BRC7ARIsAPIXOjjj&lt;br/&gt;Jh_151mYVEUyLZRavt4re9YQMLS&lt;br/&gt;vID64X-1KbO3LUKGjVUwb&lt;br/&gt;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&amp;amp;_sid=406df3776&amp;amp;_ss=r</td></tr><tr><td>&lt;strong&gt;Preparation of Primarty T Cells&lt;/strong&gt;</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 &amp;amp; Pan T-Cell MicroBead Cocktail)&amp;nbsp;</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>&lt;strong&gt;Preparation of A549 Lung Cancer Cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trehalose Assay Kit&amp;nbsp;</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

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.

An image of the acoustofluidic system assembled inside a 3D-printed case is shown in&#160;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.&#160;
Figure 2&#160;demonstrates enhanced intracellular delivery of a fluorescent compound, fluorescein, to primary human T cells with acoustofluidic treat...

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

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. ...

assembly-operation-an-acoustofluidic-device-for-enhanced-delivery

Research

JoVE Journal

Bioengineering

3.0K Views.  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.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.

In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping device.

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we ...

Engineering

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Biology

Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must be smaller than the incident wavelength of sound and the width of the fluidic channels are typically tens to hundreds of micrometers across, acoustofluidic devices typically use ultrasonic waves generated from a piezoelectric transducer pulsating at high frequencies (in the megahertz range). At characteristic frequencies that depend on the geometry of the device, it is possible to induce the formation of standing waves that can focus particles along desired fluidic streamlines within a bulk flow. Here, we describe a method for the fabrication of acoustophoretic devices from common materials and clean room equipment. We show representative results for the focusing of particles with positive or negative acoustic contrast factors, which move towards the pressure nodes or antinodes of the standing waves, respectively. These devices offer enormous practical utility for precisely positioning large numbers of microscopic entities (e.g., cells) in stationary or flowing fluids for applications ranging from cytometry to assembly.

Acoustofluidic devices use ultrasonic waves within microfluidic channels to manipulate, concentrate and isolate suspended micro and nanoscopic entities. This protocol describes the fabrication and operation of such a device supporting bulk acoustic standing waves to focus particles in a central streamline without the aid of sheath fluids.

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Genetics

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

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

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the mechanical properties of the cells is often challenging. Conventional measurement methods based on contact such as compression or stretching are prone to cause cell damage, affecting measurement accuracy and subsequent cell culture. Measurements in adherent state can also affect accuracy, especially after irradiation since ionizing radiation will flatten cells and enhance adhesion. Here, a cell mechanics measurement system based on acoustofluidic method has been developed. The cell compressibility can be obtained by recording the cell motion trajectory under the action of the acoustic force, which can realize fast and non-destructive measurement in suspended state. This paper reports in detail the protocols for chip design, sample preparation, trajectory recording, parameter extraction and analysis. The compressibility of different types of tumor cells was measured based on this method. Measurement of the compressibility of nucleus was also achieved by adjusting the resonance frequency of the piezoelectric ceramic and the width of the microchannel. Combined with the molecular level verification of immunofluorescence experiments, the cell compressibility before and after drug-induced epithelial to mesenchymal transition (EMT) were compared. Further, the change of cell compressibility after X-ray irradiation with different doses was revealed. The cell mechanics measurement method proposed in this paper is universal and flexible and has broad application prospects in scientific research and clinical practice.

Here a protocol is presented to build a fast and non-destructive system for measuring cell or nucleus compressibility based on acoustofluidic microdevice. Changes in mechanical properties of tumor cells after epithelial-mesenchymal transition or ionizing radiation were investigated, demonstrating the application prospect of this method in scientific research and clinical practice.

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the ...