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 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

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

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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

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

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

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

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

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

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

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

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

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

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

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

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

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

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

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

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

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

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

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

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

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

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

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