This is a method to apply electromechanical stimulation on cells. It has multiple applications to study its effects on a cell population, pre-training for in vivo delivery, and cell maturation. The advantage is that electrical and mechanical stimuli can be applied with the same device individually or simultaneously while keeping sterile viral intact.
This technique is an indirect approach to our therapy. Cell therapy is considered electromechanically stimulated cells may be an interesting cell population to treat intro-myocardium. This method generally belongs to the cardiovascular field, but it could also be applied to the nervous system.
This is an easy technique that requires patience. The most difficult part will be working with the small pieces, and keep the sterility throughout. There are little details of manipulation which are difficult to explain, so the visual demonstration is really helpful.
Begin by transferring 12 cleaned PDMS constructs to sterile 10 centimeter plates. To insure complete sterilization, expose the plates to UV light for five minutes. Then transfer each construct to a separate 35 millimeter cell culture plate, or 6-well plates for immediate cell seeding.
To start cell seeding, first wash a confluent T75 flask of cardiac ATDPCs with five milliliters of 1x PBS. To detach the cells, add one milliliter of 0.05%trypsin-EDTA. And incubate at 37 degrees Celsius for five minutes.
Then, add five milliliters of complete medium to inactivate the trypsin-EDTA. Collect all detached cells in a 15 milliliter tube. Wash the cell glass twice with five milliliters of PBS to collect any remaining cells, and add them to the 15 milliliter tube.
Centrifuge at 230 g for five minutes at 22 degrees Celsius. Remove the supernatant, and re-suspend the cells in two milliliters of complete medium, and count them with the hemocytometer. Seed 200 microliters of cardiac ATDPCs into each plate with the PDMS constructs to obtain about 80%of the seeding surface covered by cells the following day.
Incubate at 37 degrees Celsius, and 5%CO2. Gently add 2 milliliters of pre-warmed complete medium per plate. Incubate the cells with the constructs at 37 degrees Celsius, and 5%CO2 overnight.
Before starting this procedure, assign six out of 12 constructs for electromechanical stimulation, and keep six for non-stimulated controls. Use 70%ethanol to clean the stimulation unit. Place the stimulation unit, sterile electrodes, and tweezers inside the flow cabinet.
In order to easily manipulate the electrodes and constructs, remove 90%of the medium from each cell culture plate. Place the PDMS constructs in the right position toward the magnet to insure magnetic attraction between both fixed and mobile magnets. These two previous steps in which you manipulate the cell's seeded PDMS constructs and the electrodes while keeping the sterility throughout the process are the most critical.
Then, connect the platinum wire to the electrode connectors. Orient the PTFE part of the electrodes in their designated spaces in the PDMS constructs. Add 2.5 milliliters of fresh, pre-warmed complete medium to each construct.
Once all PDMS constructs are placed and electrically connected to the platform, place the platform back into the 37 degrees Celsius, and 5%CO2 incubator. Then connect the electrical and mechanical source. To configure the stimulation program, specify electrical and mechanical stimulation regimes through the user interfaces of the electrical stimulator and the application, which controls the mechanical stimulation.
To set the synchronism, switch on the electrical stimulator, and wait for the main menu to appear on the display. Then select option two, edit sequence, plus Enter. To edit the sequence menu, use the mode tab to select current"by clicking plus"and pressing Enter"For the period, select 1000"with plus/minus and press Enter"And for trigger mode tab, select external by software and press Enter"For the amplitude tab, select 1"with plus/minus and press Enter"Then, back in the main menu, select option 4, and then generate sequence, and press Enter"In the mechanical stimulation section of the control application panel, first write 1000"in the plus period text control.
Then write 500"in the ON time text control, to set the mechanical pulse duration. Finally, write 2000"in the excursion text control to deliver a 10%construct elongation. Change the cell media twice a week, by first removing the old media, and then adding warm, fresh media on the sides of the PDMS support.
On day seven, at the end of the experiment, collect the samples as described in the manuscript. Electromechanically stimulated cardiac ATDPCs enhanced their cardiomyogenic potential. This was indicated by real-time PCR showing increased expression of early and late cardiac genes specifically the cardiac transcription factor GATA-4, the structural marker beta-myosin heavy chain, and the calcium related gene Connexin43.
Phalloidin staining against actin fibers showed that the majority of cells aligned according to the vertical pattern. Connexin43 distribution was mostly in the cytoplasm, and at the plasma membrane, contributing to intracellular communication through gap junctions. MEF2 and GATA-4 transcription factors were located at the nuclei in cardiac ATDPCs.
But GATA-4 was not detected in subcutaneous ATDPCs. The cytoplasmic markers SERCA2, and sarcomeric alpha-actinin, did not show a mature sarcomere organization typical for cardiomyocytes. And beating was not observed in control and stimulated cell populations.
A healthy cell monolayer at the beginning of this protocol and keeping the sterility throughout the procedure are crucial. After sample collection, the standard genome protein expression techniques can be performed. This technique helps to better understand the effect of cell electrical and/or mechanical stimuli.
Until recently, it was impossible to use the same device for both stimulation, which made results harder to compare.
