Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

The overall goal of this procedure is to deliver various types of biologically meaningful molecules in a sequential and dosage controllable manner with high efficiency using the Vortex assisted microfluidic porer, this is accomplished by first preparing four 50 milliliter centrifuge tubes individually containing DPBS solutions with cells and biomolecules, and attaching each tube to its respective vial holder connected to the pneumatic flow control system. The second step is to insert the inlet tubings from each respective vial into the microfluidic device with the embedded 15 pin electrodes. Next, the cells are flowed into the device where they're trapped in microscale vortices formed in the electroporation chambers.

The final step is to apply short electric pulses to the trapped cells promptly followed by injecting the solutions containing the biomolecules to be delivered into the cytosol. Ultimately, the cells obtained from this process can be released and collected for downstream analysis. The main advantage of this technique over existing methods is that the proposed technique is capable of sequentially delivering controlled amount of multiple molecules into a pre-selected identical cell population with high efficiency and viability.

Visual demonstration of this method is critical as the fluid exchange steps are difficult to learn because the timing of the cold flow step at each solution. Switching step determines stability of the cell trapping. A metastatic breast cancer cell line M-D-A-M-B 2 31 will be used in this experiment plate one times 10 to the fifth cells per milliliter in a volume of 10 milliliters per T 75 Tissue culture flask in Leibovitz's L 15 medium supplemented with 10%volume per volume, fetal bovine serum, and 1%penicillin.

Streptomycin incubate the cells in a humidified incubator at 37 degrees Celsius with a 0%carbon dioxide environment. Two days after seeding, harvest the cells for experiments. After washing the cells with bukos phosphate buffered saline or DPBS treat cells with 0.25%tripsin EDTA for two minutes.

Add eight milliliters of growth media to inactivate the enzymatic activity pellet cells by centrifusion for five minutes at 200 times G and resuspended media to a final concentration of five times 10 to the fifth cells per milliliter. The design and fabrication of the microfluidic electroporation device will not be shown in this video, but is described in the accompanying manuscript. The system consists of inlets for cells, molecules, and a flush solution.

Two straight channels where inertial focusing occurs. 10 electroporation chambers with electrodes and an outlet to set up for the flow experiments. Insert an outlet, poly ether, ether ketone, or peak tubing, and the 15 pin aluminum electrode for short pulse, high voltage application into the designated places via the holes in the microchannel.

The 15 pin electrode consists of 10 positive and five negative electrodes. Each positive electrode is spaced 1.5 millimeters apart from a negative electrode, and each electrode of the same polarity is spaced 1.35 millimeters apart. Connect the electrical equipment for generating high voltage short square wave pulses to the aluminum electrodes that are in contact with flowing solutions.

In the PDMS mold, the equipment should consist of a pulse generator and an in-house built high voltage amplifier. Prepare four 50 milliliter centrifuge tubes individually containing DPBS and solutions with cells and molecules. Attach each tube to its respective vial holder connected to the pneumatic flow control system.

Connect inlet peak tubing from the vial holders into the respective inlet holes in the microfluidic device. Next, set. The magnitude of square wave pulses volts to 100 volts.

In order to have the electric field strength across the electroporation chamber be equivalent to 0.7 kilovolts per centimeter. Set the pressure regulator to 40 PSIA single manually adjustable nitrogen source is used to uniformly pressurize all sample vials and utilizes a high speed manifold to timely activate individual solution ports. Using the custom built lab view software for valve control.

Open the lab view software labeled valve runner and click run from the dropdown menu entitled Operate. Click on the corresponding valve icon valve one to open the valve for the DPBS reservoir to prime the flow speed required for stable cell trapping vortex generation for 1.5 minutes. The valve icon should turn from gray to green when it is activated flow, both washing and cell solutions through the device simultaneously for 10 seconds prior to the cell trapping step.

To ensure undisrupted flow during the solution switching step, this brief co flow step should be repeated at each solution switching step. Switch the active solution port from the washing solution to the cell solution to trap cells in the electroporation chamber for 30 seconds. In this movie, the blue fluorescent signals represent viable hooks.

3, 3, 3, 4, 5 stage cells. Turn on the washing port and flush the device for 20 seconds. In order to remove nont trapped contaminating cells flow the solution containing the first molecule of interest.

Propidium iodide into the device for visualization purposes. In this demonstration, nucleic acid dyes are used instead of fluorescently tagged DExT strands because of the superior noise to signal ratio of the dyes apply five short pulses promptly after injection of the molecular solution, monitor the magnitude and number of the applied electrical pulses in real time using an oscilloscope, the fluorescent signals of the molecules can be visualized under the microscope, incubate the cells for 100 seconds in the molecular solution. Next flow, the solution containing the second molecule, the nucleic acid dye yo-yo one into the device.

In this demonstration, the second molecule is delivered without additional electrical pulse applications. This movie confirms that the cells now express all three fluorescent signals. Green for yo-yo, one, red for propidium, iodide, and blue for hooks.

3, 3, 3, 4, 5. Release the cells into a 96 well plate for downstream analysis by lowering the operating pressure below five PSI. Approximately 100 microliters of solution with 100 cells is collected from each release.

The electroporation procedure must be repeated at least three times to collect enough cells for flow cytometry centrifuge. The 96 well plate containing processed cells at 228 times G for five minutes. At room temperature, remove the supernatant that contains excess fluorescent molecules and resuspend the cells in DPBS.

If fluorescently tagged DExT strands were delivered, the cells are subsequently analyzed for molecular uptake. Efficiency by flow cytometry. Successful molecular delivery was qualitatively determined by monitoring changes in fluorescence intensity of electroporated orbiting cells in C two, which confirmed that 90%of treated cells uptake the 70, 000 Dalton an ionic DExT strand molecule.

The efficiency for each transferred dextran molecule defined as the ratio of the number of cells successfully taking up the molecule of interest to the total number of processed cells did not vary substantially depending on molecular weight or electrical charges. All tested dextran molecules were delivered into the cytosol with efficiency greater than 70%Shown here, our representative flow cytometry profiles for cells which were not treated with electroporation. The fluorescent threshold indicating successful molecular delivery is set from the data such that the signals from control samples are found below the threshold.

This representative flow cytometry data for sequentially electroporated cells displays the flow cytometry plot next to the fluorescent streak images, the green boxes represent signals from cells uptaking 3000, Dalton neutral dextran only, and the red boxes represent signals from cells. Uptaking 3000 Dalton, an ionic DExT strand, only fluorescent signals from cells uptaking, both DExT strand molecules shown in yellow indicate a dual molecule delivery efficiency of 56%After its development. This technique will be useful for researchers in the field of medicine, biotechnology, and pharmacology to explore combination of therapeutic reagents to achieve synergetic effects in treating complex disease.

Don't forget that working with high voltage electric pulses can be extremely hazardous, and precautions such as ensuring you're grounded should always be taken while performing this procedure.