Here, we show how to build an electrical stimulation chamber using common six wall culture plates, and how to use it in order to stimulate osteogenic differentiation in mesenchymal stem cells. This chamber is easy and not expensive to build, and can be reused in multiple experiments. After being pre-treated with electrical stimulation in our chamber, mesenchymal stem cells can be used to improve outcomes in bone tissue engineering treatments.
This chamber can also be used to investigate other cell types and electro-sensitive cell behaviors like migration, proliferation, changes in the membrane potential, apoptosis, and careful attachment. In a six well plate lid, mark and then drill two one-millimeter diameter holes, 25 millimeters apart, near the outer edge of each of the six wells. Next, cut a platinum wire into 12 five-centimeters long sections.
Manually bend each five-centimeter section into an L"shape, leaving one end three centimeters long, and the other two centimeters long. Then, cut the silver-coated wire into two 35-centimeter lengths. Insert the longer bent end of a platinum wire into the drilled hole, leaving one to two millimeters protruding from the outside of the lid.
Bend it using forceps. Afterward, secure the platinum wires in the lid holes with super-conductive glue and leave them to dry for approximately six hours. To prepare the cathodes, solder the tips of all six platinum wires protruding from the lid to one of the silver-coated wires.
To prepare the anodes, solder the remaining six platinum wires to other silver-coated wires. Subsequently, add LEDs in the circuit between each of the six anode-cathode platinum-electrode pairs to confirm the functionality. Place a piece of black insulation tape under each LED to prevent exposing the cells in the culture plates to LED light.
Next, glue the wire terminal block connector to the top left corner of the six-well plate lid, and connect both silver-coated wires to the input terminals. Then, cut out a 20-millimeter by 20-millimeter square section from the top left corner of a second six-well plate lid, to accommodate the terminal block connector on the first lid. Cover the first lid, equipped with the electrodes, with the second lid, and tape them together with adhesive tape.
Connect one end of the two standard insulated copper wires to the output terminals of the wire connector, and the other ends to banana male connectors. Turn on the power supply by pressing the ON-OFF button on the front panel. Activate channel one by pressing button one.
Afterward, press button four to set the voltage. Set the load output at 2.5 volt. Then press Enter.
On the day the cells are treated with E stem, sterilize the electrodes in 70%ethanol solution for 30 minutes. Then, dry them under UV light in a safety cabinet for an additional 30 minutes. In a laminar flow hood, cover the six-well plate containing the cultured MSCs with the lid equipped with the electrodes, making sure that the electrodes are completely submerged in the medium.
Then, transfer the covered six-well plate with cells to the incubator and connect its wires to the power supply. Next, set the power supply to 2.5 volt load output and treat the cells with E stem for one hour. After stimulation, disconnect the power supply and remove the E stem chamber from the incubator.
Under sterile conditions, replace the lid, equipped with electrodes, with a standard six-well plate lid. Subsequently, return the cells to the incubator, and leave them overnight. Clean the electrodes, first with PBS, and then with 70%ethanol solution.
Then, clean the accumulated corrosion products from the electrode surface with fine sandpaper. Repeat the E stem treatment for six consecutive days. On day four, prior to applying electrical stimulation, and under sterile conditions, change the culture medium by aspirating 1.5 milliliters of medium and replacing it with 1.5 milliliters of pre-warmed, fresh osteogenic differentiation medium.
After applying electrical stimulation for seven consecutive days, maintain the cells in culture for an additional seven days, exchanging the medium every three to four days. When the treatment is completed, analyze the cell morphology changes under a microscope. To assess the effect of electrical stimulation on MSC osteogenic differentiation, measure calcium deposition, alkaline phosphatase activity, and osteogenic marker gene expression, as described elsewhere.
To evaluate the effect of 100 millivolts per millimeter of electrical stimulation on the MSC osteogenic differentiation, cells treated with electrical stimulation for three, seven, and 14 days and non-treated cells, were analyzed at day 14 of culture by assessing morphological changes and calcium deposition. Significant changes in cell morphology and calcium deposits were visible in cells treated with electrical stimulation for seven and 14 days. A detailed analysis of osteogenic marker gene expression changes was performed at days three, seven, and 14 of culture.
At day seven of culture, RunX2 expression was significantly higher in cells treated with electrical stimulation for seven days. Whereas Colla1 expression was significantly higher in cells treated for seven days, measured at day 14 of culture. The expression of osteopontin was significantly increased in electrically stimulated cells at days three, seven, and 14 of culture.
Osterix expression was absent in control cells at all time points, and was seen only at seven and 14 days of culture in cells exposed to electrical stimulation. The electrical stimulation chamber is relatively easy to build. However, special care must be taken when handling the platinum wires, and also when cleaning and sterilizing the electrodes.
Cells pre-treated to electricity in our chamber, alone or seated on a scaffold material, could be implanted into animal models, to study how the positive effect of electrical stimulation persists in vitro. This technique provides a simple yet powerful tool to study the mechanism by which electrical stimulation regulates cell functions. This knowledge can be available in regenerative medicine cells.
