The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

This method allows researchers to determine optimal electroporation pulsing conditions for the desired cell type, while minimizing the need for the traditional empirical experimental approach. This method utilizes micro-electroporation techniques to manufacture a scalable microfluidic actuation device that is capable of electrically monitoring the degree of cell membrane permeabilization throughout the electroporation process. Demonstrating the procedure will be Kishankumar Busha, a master's student from my laboratory.

To begin, secure the silicon wafer to the chuck of the wafer spin coater using the spin coater's vacuum system. Then program the spinner. Next, dispense four milliliters of SU-8 2010 photo resist onto the center of the silicon wafer and run the program.

Once the system comes to a halt, turn off the vacuum. Then secure the photo mask with the 2D microfluidic channel designs onto the mask holder and insert the silicon wafer with the SU-8 coating facing upwards onto the wafer chuck. Set the exposure settings for 150 millijoules per square centimeter and run the machine.

After the UV exposure, submerge the silicon wafer in the SU-8 developer solution with gentle agitation for three to four minutes. Then remove the wafer from the solution and rinse the surface with isopropanol. Next, place the silicon wafer into a 150 degrees Celsius oven for 30 minutes for a hard bake.

Then allow it to cool down to room temperature before using stylus profilometry to measure the exact height and slope of the channel side walls. Next, dispense one milliliter of photo resist onto the surface of the glass slide and run the program once again. Once the system comes to a halt, turn off the vacuum and remove the glass slide.

After securing the photo mask with the 2D electrode designs onto the mask holder, insert and align the glass wafer with the S-1818 coating facing upwards onto the wafer chuck. Set the exposure settings for 215 millijoules per square centimeter and run the machine. After the exposure, submerged the glass slide in MF-319 developer solution for two minutes, applying gentle agitation.

Then remove the glass wafer and rinse its surface with deionized water. Finally, place the glass slide into the 150 degree Celsius oven for a hard bake, ensuring that the substrates surface of interest is facing up. After 30 minutes, remove the slide from the oven and protect it from light.

To etch the glass slide submerge it in a 10 to 1 buffered hydrofluoric acid solution for one minute in a polytetrafluoroethylene container. Then transfer and wash the glass slides in deionized water three times. Next, using a physical vapor deposition system sputter titanium for eight minutes at a rate of 100 angstroms per minute and platinum for 10 minutes at 200 angstroms per minute.

Then to lift off the photo resist, submerge the metal coated glass slides in an acetone bath for 10 minutes and sonicate the bath to introduce agitation. If necessary use an acetone soaked wipe to remove any residues. Next for polydimethylsiloxane replica molding makes the PDMS elastomer base with a hardener at a 10 to 1 weight ratio in a disposable container placed on top of an electronic balance.

Then, pour the PDMS solution over the silicon wafer and place the mixture under a vacuum to remove air bubbles. After curing the mixture at 65 degrees Celsius for a minimum of four hours, use the tip of a razor blade to cut out the molded PDMS and peel it from the silicon wafer. Then using a sharpened biopsy punch, remove the PDMS from the inlet and outlets of the device.

Next, program the plasma generator for PDMS bonding. Then place the PDMS and electrode glass slide into the system with the features facing up and run the program. After the program is complete, remove the devices and use a stereoscope to quickly align channel features to the electrodes.

Firmly apply pressure from the center of the PDMS towards the sides to remove any unwanted air bubbles at the bonding interface. Harvest the HEK293 cells, centrifuge them, and resuspend the pellet in electroporation buffer at approximately five million cells per milliliter. Next, add plasma DNA encoding for GFP to a final concentration of 20 micrograms per milliliter and mixed gently.

Then transfer the plasma-DNA cell suspension into a one cubic centimeter syringe for experimentation. Place the microdevice onto the stage of the microscope via a slide holder. Then turn on the charged coupled device, CCD camera, and bring the microfluidic channel into focus.

Next for single cell electroporation, set the syringe pump flow rate to 0.1 to 0.3 microliters per minute to ensure the flow of single cells through the electrode set. Then set the pulse parameters for the initial and lowest electrical energy electroporation pulse. Follow a predetermined number of cell detections per pulse application.

At the end of each tested condition, aspirate cells from the microdevice outlet and replenish the outlet with recovery media. Iterate to the next electroporation pulse condition and repeat until all electroporation pulse conditions are tested. Then, determine the electroporation pulse parameters for high throughput population-based feedback.

For population-based feedback controlled electroporation, set the syringe pump flow rate to one to three microliters per minute and set the pulse amplitude to the optimized condition. Then turn off the trigger mode and set the pulse width to match the cell transit time. After the desired number of cells have been electroporated turn off both the syringe pump and function generator.

Then transfer the cells from the outlet reservoir into an appropriately sized cell culture flask or plate filled with pre-warmed recovery media and transfer the culture flask or plate into an incubator. For data analysis, Load the data into an analysis software and generate a plot of current versus time for each pulsing condition. Then determine the degree of cell membrane permeabilization, generate the cell membrane permeabilization map over all tested pulse conditions, and verify the optimal pulsing condition.

After the incubation, capture epi-fluorescence images using FITC and far red filters, and analyze the image sets manually or via an algorithm. Operating principles behind the single cell level permeabilization detection for a single pulse amplitude are highlighted here. After each electroporation pulse parameter, the next highest energy electroporation pulse is tested.

The cell membrane permeabilization map for HEK293 cells showed a distinct correlation between applied electrical energy and the degree of cell membrane permeabilization. In this experiment, an electric field strength of 1.8 kilovolts per centimeter and a 670 microsecond pulse was determined as optimal. At these values, a 70%electro-transfection efficiency was achieved.

In contrast with an electric field strength of 0.4 kilovolts per centimeter and a three millisecond pulse duration, the electro-transfection efficiency at 24 hours was less than 5%The term recipe, which is often used when describing the specific of the micro fabrication process insert the importance of following or optimizing each step to successfully fabricate a functioning device. This electroporation technology can be utilized in additional biomedical research projects such as the generation and testing of CAR T-cells or the optimization and testing of CRISPR-Cas9 gene editing techniques. This micro-electroporation technology allows for the electrical interrogation of different cell types'responses to apply the electrical pulsing conditions and correlating that information to the delivery of clinically relevant molecular cargo.