The overall goal of the following experiment is to achieve site-specific transfection in adherent cells. This is achieved by culturing a monolayer of cells on a micro electrode array. As a second step, the application of an electric pulse to select electrodes leads to electroporation of the targeted cells adhered on top of the electrodes.
This leads to the creation of pores in the cell membrane that allow the entry of exogenous molecules into the cell. The targeted cells can then be screened to detect the resultant phenotype using an appropriate assay. The microscale transfect technique we described here has several key advantages over conventional transfect techniques such as bulk electroporation or chemical transfect.
A, it allows us to achieve exquisite spatial control over the transfect process itself and helps us to transfect single cells or cluster of cells with high efficiency even in difficult to transfect cell lines. B, it is an approach that scales up readily and helps us to realize high throughput molecular assays. One of the key advantages of using a micro electrode area for site-specific transfection is that it enables simultaneous functional assessment of targeted cells such as quantitative electrophysiology and morphological changes.
To begin the procedure, sterilize the MEA in an autoclave, then transfer the autoclave MEA to a laminar flow hood, and place it in a standard sterile polystyrene Petri dish prior to seeding the cells on the MEA, sterilize the laminar flow hood by turning on the UV light for 20 minutes. If the MEA is sensitive to UV light, cover the Petri dish containing the MEA with sterile aluminum foil while the flow hood is under UV illumination. Next, treat the MEA surface with 10 micrograms per milliliter of fibronectin for one to two hours for helo cells.
Perform a wash with DI water and let the MEA air dry in the laminar flow hood. After that, harvest the cells from a running cell line of adherent mammalian cells using trypsin ETTA and raise them to the concentration of 100 to 200 cells per microliter in one milliliter of cell media for plating. Then place a 20 to 30 microliter drop of media with cells on the MEA.
Make sure that it is large enough to cover all the electrodes on the MEA. Subsequently transfer the Petri dish with the MEA to a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. Two to four hours.
After seeding the cells, transfer the Petri dish with the MEA from the incubator to the laminar flow hood. After that, gently add 400 to 500 microliters of 37 degrees Celsius pre warmed media to the MEA well next check under the microscope to ensure that the cells are still attached and evenly spread on the MEA surface. Then transfer the MEA back to the incubator eight to 12 hours after seeding the cells, perform one to two washes with PBS to remove any dead cells.
In a typical experiment, the cells are usually ready for transfection 24 to 48 hours after seating. To maintain consistency in electroporation efficiencies, it is recommended to wait until the culture is near co fluency before electroporation. In order to achieve highly efficient sarna transfection, it is necessary to first determine the optimal electroporation voltage pulse parameters for a given MEA.
Here we describe a sequence of experimental steps involving the use of propidium iodide to determine the optimal electroporation parameters prior to the electroporation. Prepare a 300 to 500 microliter solution of 30 micrograms per milliliter PI in ice cold electroporation buffer, and 400 microliters of four micromolar calcium AM solution in PBS. Then transfer the MEA from the incubator to the laminar flow hood.
Remove the cell media from the MEA and perform a wash with PBS. Then at 300 to 500 microliters of the ice cold electroporation buffer with pi. Next place a one millimeter high spacer in the MEA.
Well, it is extremely important to ensure that it does not touch the cells in the center of the MEA. Afterwards connect the MEA with the electrical interconnect and select the desired electrodes for electroporation. Subsequently, apply the electric pulses with the desired pulse amplitude and duration to the targeted electrodes for the electroporation step.
Lower the steel cathode covered in aluminum foil in the MEA well until it makes contact with the one millimeter spacer. Immediately afterwards, apply electric pulses to the targeted electrodes. Next, repeat the step with a different set of electrodes depending upon the experimental design.
In our experiments with helo cells, we observed that a single voltage pulse of an amplitude from three volts to nine volts and a duration from one millisecond to 10 milliseconds. For an electrode the size of 100 micrometers led to successful electroporation with minimal cell death. Immediately after applying the voltage pulses, transfer the MEA to the incubator for five minutes.
After a five to 10 minute incubation period, gently remove the spacer from the MEA. Well then remove 75%of the electroporation buffer from the MEA well and replace it with warm cell media. Then transfer the MEA back to the incubator.
After an incubation period of two to four hours, replace the cell media in the MEA well with 300 to 500 microliters of four micromolar calcium AM solution in PBS. Transfer the MEA back to the incubator for another 30 minutes following the incubation of the cells with calcium am. Replace the calcium AM in the well with PBS after a wash step, then obtain the fluorescent images of cells using an optical microscope with filters for pi.
The uptake of PI due to electroporation causes the cells to fluoresce red, and the calcium am causes the cells that are alive to fluoresce green. The cell viability for each electrode can be estimated by this equation, and the electroporation efficiency for each electrode can be estimated by this equation. Once the optimal electroporation parameters are determined, they can be used for site-specific delivery of an a molecule, and the electroporation procedure for siRNA molecules is the same as the electroporation procedure for pi.
Shown here is the site specific delivery of propidium iodide in helo cells on a 200 micrometer electrode. The calcium-based live assay was performed two hours post electroporation. The live cells are indicated by green fluorescence here, and the red fluorescence here demonstrates the uptake of pi by cells on the electrode.
Here is the superimposed image. These cells are electroporated and alive while these cells are dead. This figure shows the transfection of rumine tagged irna on a 200 micrometer diameter electrode, and this figure shows the transfection of hela cells with Alexa 4 88 tagged irna on a 100 micrometer diameter electrode.
The white arrows indicate the cells transfected with irna while the black arrows indicate the dead cells. The technique we have demonstrated here can be extended to different cell lines, including hard to transfect primary cell lines, and to transfect them with larger molecules such as plasmids and proteins.
