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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 infection^1,2. 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 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 compounds^4,5,6,7,8,9. 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 pores^4,7,8.

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 ("sonoporation") allowing rapid intracellular uptake of biomolecules via passive or active transport mechanisms^10,11,12. 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 channels^14,15,16,17. 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 membranes^22,23. 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.

Protokół

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

1. Fabrication of acoustofluidic device

1. Obtain a photomask with a concentric spiral design containing channels with a diameter of 500 µm. A CAD file is provided in the supplemental files as an example. A custom photomask can be ordered from a commercial vendor or patterned using a mask writer.
2. Prepare a mold of the concentric spiral design on a photoresist-coated silicon wafer using standard photolithography techniques.
   1. Add approximately 2 Tbsp (~30 mL) of SU-8 2100 to a 100 mm silicon wafer.
   2. 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 µm.
   3. Cure the photoresist-coated wafer in a polyimide vacuum oven with a 30 min ramp up and 30 min dwell at 115 °C, then ramp down for 30 min.
   4. Expose the photoresist-coated wafer for 130 s using a mask aligner with the photomask from step 1.1.
   5. Bake the wafer after exposure following the same process described in step 1.2.3.
   6. Develop the photoresist in SU-8 developer solution for approximately 8 min.
      CAUTION: Only use developer solution in a well-ventilated chemical fume hood.
3. Silanize the mold to make the surface more hydrophobic. Place the photoresist-coated wafer into a desiccator and add a 20 µL 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.
4. 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.
5. Place the cup containing the PDMS solution into a desiccator for approximately 30 min or until remnant air bubbles are removed from the solution.
6. Place photoresist-coated wafer with the patterns facing upward in a 150-mm Petri dish.
7. Pour the PDMS solution over the mold inside the 150-mm Petri dish.
8. If needed, place the 150-mm Petri dish inside a desiccator and apply vacuum until remnant air bubbles disappear.
9. Transfer the 150-mm Petri dish into a lab oven and bake for 2 h at 60 °C to cure the PDMS.
10. After curing, carefully remove the PDMS from the Petri dish by cutting around the edges of the wafer using a razor blade.
11. Cut out each individual device using a knife or razor blade.
12. Punch holes through the inlet and outlet ports of each device using a 2.5-mm biopsy punch.
13. 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.
14. Let devices bond overnight at room temperature.
15. 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).

2. Assembly and operation of acoustofluidic system

1. Connect a microcontroller to a computer using a USB A to B cable. A green power LED indicator (labeled PWR) should illuminate.
2. Use the associated program on the computer to upload a program which generates an 8 MHz signal. An example program is provided in the Supplemental Files. After uploading the program, it will be stored into microprocessor memory and will not need to be uploaded again.
3. Solder a 1" 22G wire to the end of each wire on the PZT transducer.
4. Connect the negative (black) terminal wire of PZT transducer to a GND pin via the soldered wire.
5. Connect the positive (red) terminal wire of PZT transducer to the output pin (#9 in the provided example program) via the soldered wire.
6. Optionally, mount the acoustofluidic device and the microcontroller in a 3D-printed case. CAD files are provided in the Supplemental Files as examples. Additional wires can be connected to other microcontroller pins to control an external LED indicator and on/off push button if desired.
7. Cut 3-6" sections of tygon PVC soft plastic tubing (1/16" ID, 1/8" 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.
8. Assemble the microfluidic reservoir according to manufacturer's instructions.
9. Cut a 3-6" section of tygon PVC soft plastic tubing (1/16" ID, 1/8" OD) and push the tubing over the 1/32" ID tubing from the microfluidic reservoir output tubing. Optionally, wrap the junction with paraffin film to prevent leakage.
10. Fill a 60-mL syringe with ambient air (optionally, filter the air with a 0.2-µm filter) and connect it to tygon PVC tubing (1/16" ID, 1/8" OD) on the side of the microfluidic reservoir.
11. 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.

3. 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.

1. Prepare a phospholipid solution in a 20mL scintillation vial containing the following mixture:
   1. Add 25 mg of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
   2. Add 11.6 mg of 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC).
   3. Add 0.26 mg of 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG).
   4. Add 0.88 mg of polyoxyethylene40 stearate.
2. Add chloroform until all phospholipids are dissolved (e.g., 3 mL of chloroform).
3. 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).
4. Rehydrate the lipid film with 10 mL of sterile phosphate-buffered saline (PBS).
5. Sonicate the lipid solution for 3 min at 40% amplitude to form a cationic micellar solution.
6. After sonication store the phospholipid solution at 2-6 °C for up to 1 month.
7. To prepare ultrasound contrast agents, add 200 µL of cationic micellar solution and 600 µL of sterile PBS to a 2 mL glass septum vial.
8. Seal the vial by crimping the cap.
9. Use a 1.5" 20G needle to fill the vial head space with decafluorobutane gas for 30 s.
10. Amalgamate the vial for 45 s at 4,350 cpm to form perfluorobutane gas-filled ultrasound contrast agents.
11. Add 25 µL 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.

4. Preparation of primary Tcells

1. Isolate peripheral blood mononuclear cells (PBMCs) from whole blood solutions and store at -150 °C. Density gradient separation containing a substrate is commonly utilized to separate PBMCs from whole blood^24,25,26.
2. Thaw frozen vial in 37 °C water bath.
3. Dilute thawed PBMCs 1:10 with PBS in a 15mL centrifuge tube. Each 1mL vial contains approximately 10 million PBMCs.
4. Centrifuge diluted PBMCs at 580 x g for 11 min at 4 °C.
5. Aspirate the supernatant and add 13 mL of MACs running buffer to resuspend the cells.
6. Count the PBMCs with an automated cell counter or hemocytometer.
7. Centrifuge the PBMCs again at 580 x g for 11 min at 4 °C and aspirate the supernatant.
8. Add 40 µL of chilled running buffer per 10 million PBMCs.
9. To isolate T cells, add 10 µL of Pan T-Cell Biotin Antibody Cocktail per 10 million PBMCs.
10. Gently agitate the PBMCs and store the solution at 4 °C for 5 min per 10 million cells.
11. Add 30 µL of running buffer and 20 µL of Pan T-Cell MicroBead Cocktail per 10 million PBMCs.
12. Mix the PBMCs and beads thoroughly and incubate for an additional 15 min at 4 °C.
13. Add running buffer to reach a total volume of 500 µL.
14. Separate primary T cells with a commercially available benchtop magnetic sorting instrument using the "depletes separation" setting following manufacturer's protocol. This step should yield between 5-10 million T cells after cell sorting.
15. Count T cells using an automated cell counter or hemocytometer.
16. Dilute T cells in 10 mL of sterile PBS and centrifuge at 580 x g for 10 min at 4 °C to pellet the cells.
17. Aspirate the supernatant and resuspend T cells in 1 mL of PBS.
18. Count T cells using an automated cell counter or hemocytometer and aliquot 1 million/mL for experiments.
19. Prepare a 1 mg/mL fluorescein solution in PBS.
20. Add 100 µL of 1 mg/mL fluorescein solution per 1 mL of T cell solution (final fluorescein concentration = 100 µg/mL) immediately prior to processing.
21. Add 25 µL of ultrasound contrast agent solution as previously described in step 3.11.
22. 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.
23. Immediately after treatment, wash cells three times via centrifugation at 580 x g for 10 min with 500 µL of PBS to remove extracellular fluorescein. Cells should be washed within 10 min after adding fluorescein solution.
24. After final washing step, resuspend cells in 250 µL of PBS and measure fluorescence on flow cytometer.

5. Preparation of A549 lung cancer cells

1. Culture A549 (adenocarcinomic human alveolar basal epithelial) cells in complete DMEM media (10% fetal bovine serum, 1% penicillin/streptomycin) at 37 °C and 5% CO₂ in a flat-bottom tissue culture flask.
2. 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.
3. Add trypsin (0.25%) EDTA to the flask and incubate for 5 min at 37 °C. Trypsin is a digestive enzyme which causes the cells to detach from the bottom surface of the tissue culture flask.
4. Transfer trypsin solution to a 15mL centrifuge tube and neutralize it by adding complete DMEM media at a 1:3 ratio.
5. Pellet the cells via centrifugation at 1,500 x g for 5 min at 4 °C.
6. 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.
7. Add 25 µL of ultrasound contrast agent solution as previously described in step 3.11.
8. 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.
9. Immediately after treatment, wash cells three times via centrifugation with 500 µL of PBS to remove extracellular trehalose. Cells should be washed within 10 min after adding trehalose solution.
10. After final washing step, resuspend cells in 100 µL of PBS.
11. Add 11 µL of 1% Triton X-100 solution to lyse cells and release intracellular trehalose.
12. Vortex for 15 s, then incubate for 30 min at room temperature.
13. Vortex again for 15 s, then measure trehalose concentration using commercially available trehalose assay following manufacturer's recommendation.

Wyniki

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

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

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Fabrication of Acoustofluidic Device
DOW SYLGARD 184 SILICONE ENCAPSULANT CLEAR 0.5 KG KIT	Ellsworth Adhesives	4019862 (SKU)	https://www.ellsworth.com/products/by-market/consumer-products/encapsulants/silicone/dow-sylgard-184-silicone-encapsulant-clear-0.5-kg-kit/
Harris Uni-Core (2.5 mm)	Electron Microscopy Sciences	69039-25
Microfluidic Reservoir for 15 mL Falcon Tube - S (2/4 port)	Darwin Microfluidics	LVF-KPT-S-2 (SKU)	https://darwin-microfluidics.com/products/15-ml-falcon-tube-microfluidic-reservoir-s-2-4-port
Microscope Slide	VWR	16004-430	https://us.vwr.com/store/product/4646174/vwr-vistavisiontm-microscope-slides-plain-and-frosted-premium
trichlorosilane	Gelest	105732-02-3 (Cas. No.)	Chlorosilane is very hazaradous and flammable. Exposure causes severe burns and eye damage.
Tygon PVC soft plastic tubing (1/16" ID, 1/8" OD)	McMaster-Carr	5233K51 (Part #)	https://www.mcmaster.com/pvc-tubing/soft-tubing-for-air-and-water/
Assembly of Acoustofluidic System
Arduino Uno	Arduino	7630049200050 (Barcode)	https://store.arduino.cc/usa/arduino-uno-rev3
Preparation of Ultrasound Contrast Agents
1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC)	Avanti Lipids	890703P-25mg (SKU)	https://avantilipids.com/product/890703
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)	Avanti Lipids	850365P-25mg (SKU)	https://avantilipids.com/product/850365
1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG)	Avanti Lipids	840465P-25mg (SKU)	https://avantilipids.com/product/840465
APF-140HP (decafluorobutate gas)	FlouroMed	355-25-9 (Cas No.)	http://www.fluoromed.com/products/perfluorodecalin/
DB-338 Amalgamators	COXO		https://www.coxotec.com/coxo/db-338-amalgamators/
polyoxyethylene 40 stearate	Sigma-Aldrich	P3440-250G (SKU)	https://www.sigmaaldrich.com/catalog/product/sigma/p3440?lang=en&region=US&gclid= Cj0KCQjwy8f6BRC7ARIsAPIXOjjj Jh_151mYVEUyLZRavt4re9YQMLS vID64X-1KbO3LUKGjVUwb PDAaAqvOEALw_wcB
Q125 Sonicator	Qsonica	Q125-110 (Ref.)	https://www.sonicator.com/products/q125-sonicator?_pos=1&_sid=406df3776&_ss=r
Preparation of Primarty T Cells
autoMACs running buffer	Miltenyi Biotec	130-091-221 (Order No.)	https://www.miltenyibiotec.com/US-en/products/automacs-running-buffer-macs-separation-buffer.html#gref
Pan T Cell Isolation Kit, human (Pan T-Cell Biotin Antibody Cocktail & Pan T-Cell MicroBead Cocktail)	Miltenyi Biotec	130-096-535 (Order No.)	https://www.miltenyibiotec.com/US-en/products/pan-t-cell-isolation-kit-human.html#130-096-535
magnetic cell sorter (autoMACS Pro Separator)	Miltenyi Biotec	130-092-545 (Order No.)	https://www.miltenyibiotec.com/US-en/products/automacs-pro-separator-starter-kit.html#130-092-545
Preparation of A549 Lung Cancer Cells
Trehalose Assay Kit	Megazyme	K-TREH (Cat. No.)	https://www.megazyme.com/trehalose-assay-kit
Trypan blue (0.4% in aqueous solution Ready-to-Use, sterile)	VWR	97063-702 (Cat. No.)	https://us.vwr.com/store/product/7437427/trypan-blue-0-4-in-aqueous-solution-ready-to-use-sterile