Our research is focused on improving porous substrate electroporation. Research has shown major benefits from using micro and nano channels to localize electroporation, but there are still questions to be answered, especially regarding monitoring and characterizing effects during the intermediate stages of the process. Some of the most recent developments include our use of trans epithelial electrical impedance monitoring, as well as using nano channel based electroporation to create exosomes or measure tension in cell membranes.
PSCP experiments still rely heavily on before and after fluorescent imaging to determine delivery and cell viability outcomes. Additionally, many PSCP based studies under report pulse parameters and very little research has been done to verify optimal parameters for common cell types. The primary research gap we address is the lack of reporting on the effects of PSEP during the cell recovery stage.
However, our analysis of TEEI measurements correlated TEEI change with viability and delivery efficiency, which allowed us to identify optimal PSEP waveform parameters as well. Our primary focus moving forward will be to identify the mechanisms causing the TEEI increase. Ideally, those findings will help us fine tune the PSEP process for an even more efficient delivery.
To begin fibronectin coating, select 12 inserts and two 24 well plates. Place the inserts into one well plate, creating two rows of six. Prepare one microgram per milliliter fibronectin solution by mixing 52 microliters of fibronectin stock solution and 1.2 milliliters of PBS in a 1.5 milliliter tube.
Distribute 100 microliters of the fibronectin solution into each insert. Incubate the inserts in the well plate at 37 degrees Celsius for three hours. After incubation, remove the excess fibronectin solution from each insert.
Wash the inserts twice by adding 100 microliters of sterile distilled water to each insert. Remove the water in the same order as it was added to ensure consistent wash time between inserts. Now wash the inserts with 100 microliters of cell culture media, and then remove the media from the inserts.
Next seed, 200 microliters of A for 31 cell suspension into the inserts for negative control pipette 200 microliters of cell culture media into the inserts. Using a permanent marker, draw a line dividing the second well plate into two columns that are three wells wide. Separate each column into rows.
Label each region in the grid with relevant parameters. Add one milliliter of cell culture media to each well. Transfer the inserts from the preparation plate to their appropriate location in the labeled experiment plate and incubate at 37 degrees Celsius for 12 hours.
To begin pipette 1.5 milliliters of the 0.1 milligram per milliliter peridium iodide solution into each well in the electrode array. Place an insert into each well in the electrode array, fitting the feet of the insert into the alignment grooves, so the insert is flush with the upper surface of the well. Screw the top electrode printed circuit board to the top of the electrode array wells and connect the electrode array to the porous substrate electroporation or PSEP device.
Place the electrode array in the 37 degrees Celsius incubator for temperature equilibration. Click the dropdown next to membrane in the top left corner of the gooey and click on 400 nanometer GBO. On the right side of the gooey, type one into the post pulse time duration minute edit field.
Now click on the run button and enter appropriate names for wells one to three and four to six when prompted, then click on okay to start the experiment. After one hour, remove the electrode array from the incubator and transfer the inserts back into the original locations in the experiment plate. Mix two microliters of host 33342, and five microliters of calcium AM with 123 microliters of cell culture media in a 200 microliter tube.
Gently pipette 10 microliters of the stain solution into each post pulse insert and place the inserts back into the incubator for five minutes, transfer the well plate, to the plate holder of a fluorescent microscope with 5x magnification objective. Image using brightfield and the fluorescence of each stain. Pipette 1.5 milliliters of the cell culture media into each well in the electrode array.
Place cell sample inserts into wells one to three and control inserts into wells four to six fitting the feet of the insert into the alignment grooves. Screw the top electrode printed circuit board to the top of the electrode array wells and connect the electrode array to the PSEP device. Place the electrode array in the 37 degrees Celsius incubator for temperature equilibration.
Click on the dropdown next to membrane in the top left corner of the gooey and click on 400 nanometer GBO. Now click on the run button and enter appropriate names for wells one to three and four to six when prompted, then click on okay to start the experiment. After one hour, remove the electrode array from the incubator and transfer the inserts back into the original locations in the experiment well plate.
After preparing host 32342, calcium AM, and propidium iodide in cell culture media mix and gently pipette 10 microliters of the stain solution into each post pulse insert and place the inserts back into the incubator for five minutes. On a fluorescent microscope image the cell sample inserts using brightfield and the fluorescence of each stain, the optimized healthy curve showed no dip below the baseline and the largest increase in TEEI post PSEP imaging of the cell monolayer revealed negligible cell death and a healthy fully co fluent cell monolayer. The optimized unhealthy data resulted in a diminished TEEI response.
Post PSEP imaging revealed near zero cell death and reduced delivery efficiency due to fewer cells in the monolayer. Application of unoptimized waveforms resulted in significant decreases in TEEI response indicating substantial cell death and diminished delivery efficiency.
