This protocol describes the assembly and operation of an acoustofluidic system that can efficiently deliver biomolecules for a variety of research and cell-based therapeutic applications. The main advantage of this system is that it allows rapid delivery of biomolecules to cells while maintaining their viability. It is a platform technology that allows cells to be transformed for a variety of cellular therapeutic applications, such as cancer treatment.
Begin by combining 54 grams of PDMS base and six grams of curing agent in a cup. Mix vigorously and thoroughly with a spatula for at least one minute, then placed the cup with the PDMS solution into a desiccator for approximately 30 minutes or until remnant air bubbles are removed. Place a photoresist coated wafer with the patterns facing upward in a 150 millimeter Petri dish, then pour the PDMS solution over the mold.
If needed, place the Petri dish inside a desiccator and apply vacuum until remnant air bubbles disappear. Transfer the Petri dish into a lab oven and bake for two hours at 60 degrees Celsius to cure the PDMS. After curing, carefully remove the PDMS from the Petri dish by cutting around the edges of the wafer with a razor blade.
Cut out each individual device using a knife or razor blade, then punch holes through the inlet and outlet ports using a 2.5 millimeter biopsy punch. After treating the PDMS device with oxygen plasma, immediately place each device onto a clean soda lime glass microscope slide with channels facing the glass surface. Allow the devices to bond overnight at room temperature.
Gently apply silicone to the surface of the one centimeter diameter piezo transducer at a thickness of one to two millimeters, then carefully aligned the transducer with a concentric spiral and gently press it onto the bottom of the glass microscope slide. Connect a microcontroller to a computer using a USB A to B cable. A green power LED indicators should illuminate.
Use the associated software on the computer to upload a program that generates an eight megahertz signal. Solder a one inch 22 gauge wire to the end of each wire on the PZT transducer. Then use it to connect the negative terminal wire of the PZT transducer to a GND pin.
Connect the positive terminal wire of the PZT transducer to the output pin via the soldered wire. Cut three to six inch sections of tygon PVC soft plastic tubing 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, glue can be applied at the junction to bond the PDMS and tubing together. Optionally, mount the acoustofluidic device and the microcontroller in a 3D printed case. Assemble the microfluidic reservoir according to manufacturer's instructions.
Cut a three to six inch section of 1/16 inch inner diameter tygon PVC soft plastic tubing and push it over the 1/32 inch inner diameter tubing from the microfluidic reservoir output. Optionally, wrap the junction with paraffin film to prevent leakage. Fill a 60 milliliter syringe with ambient air on the side of the microfluidic reservoir.
Set the syringe pump to a rate of 200 milliliters per hour to push the contrast agent solutions through the acoustofluidic device at a volumetric flow rate of 50 milliliters per hour and collect the samples from the output of the device into a 50 milliliter centrifuge tube. Prepare a phopholipid solution in a 20 milliliters scintillation vial by adding 25 milligrams of DSPC, 11.6 milligrams of DSEPC, 0.26 milligrams of DSPG, and 0.88 milligrams of polyoxyethylene40 stearate. Add chloroform until all phospholipids are dissolved.
Evaporate chloroform in a desiccator for 48 hours to form a dry lipid film. Rehydrate the foam with 10 milliliters of sterile PBS, then sonicate the lipid solution for three minutes at 40%amplitude to form a cationic micellar solution. After sonication, the phopholipid solution can be stored at two to six degrees Celsius for up to one month.
To prepare ultrasound contrast agents, add 200 microliters of cationic micellar solution in 600 microliters of sterile PBS to a two milliliter glass septum vial. Seal the vial by crimping the cap. Use a 1.5 inch 20 gauge needle to fill the vial head space with decafluorobutane gas for 30 seconds.
Amalgamate the vial to form perfluorobutane gas-filled ultrasound contrast agents. Add 25 microliters of ultrasound contrast agent solution per one milliliter of cell solution. Then immediately pump the combined contrast agent and cell mixture through the acoustofluidic device.
This protocol produces an acoustofluidic system that can be used to enhance intracellular molecular delivery in multiple cell lines. Intracellular delivery of a fluorescent compound, fluorescein, to primary human T cells was improved with acoustofluidic treatment compared to an untreated control group. The fluorescence intensity of T cells increased by five fold after treatment, indicating enhanced delivery of fluorescein.
Cell viability decreased slightly after a acoustofluidic treatment, but remained above 80%Acoustofluidic treatment enhanced intracellular delivery of a preservative compound trehalose to human A549 lung carcinoma cells compared to flow alone and to cells in the untreated control group. Adding the cationic microbubbles to the cell solution is critical. Intracellular delivery of biomolecules will be limited without cationic microbubbles in solution.
This platform enables intracellular delivery of various biomolecules, which can alter cellular function for research or therapeutic purposes. After completing this procedure, additional cellular characterization can be performed to evaluate the impact of delivering various biomolecules.
