This light-induced dielectrophoresis protocol or LiDEP provides a step-by-step method for the characterization of human mesenchymal stem cells. This method can help provide answers about the heterogeneity of stem cell samples. The advantage of LiDEP is the ability to make real time changes to the virtual electrode configuration in response to stem cell heterogeneity which is not possible for traditional DEP methods.
A new user of this technique might struggle with condensing the projector light enough onto the micro fluidic device to make the virtual electrode. We would advise reconfiguring the setup until it works. Helping demonstrate the procedure will be Zuri Rashad, a graduate student from my laboratory.
To begin prepare the microchannel by punching four millimeter diameter holes on a double-sided tape about five to six millimeters from the edge of the shorter dimension and centered between the longer sides of the tape. Using a scalpel, cut two straight lines three millimeters apart across the holes ensuring the protective sheets on both faces of the tape are on during the entire microchannel cutting. Mark the whole location with a washable marker ensuring the drilled holes align with the holes punched in the double-sided tape.
These two holes will be used as the inlet and outlet holes of the microfluidic device. Drill two three millimeter diameter holes in the top ITO glass slide. Next, on the top of the drilled ITO coated glass, place the long edge of the tape aligning with the long edge of the glass.
Remove one side of the protective film on the double-sided tape. Align the holes of the tape in the top ITO glass slide and gently press them together removing air pockets, especially near the microchannel. Remove the other protective film from the double-sided tape.
Press onto the molybdenum and amorphous silicon coated ITO glass slide matching the edge of the photo conductive slide opposite to the two millimeter clearance side with the edge of the double-sided tape toward the center of the top ITO glass slide. This way, a hangover exists between the ITO and the photo conductive ITO slide. Press on a flat surface to ensure good adhesion and cut off the excess tape on the sides.
To connect the function generator, apply copper tape to the edges of the ITO glass layer and photo conductive coated ITO glass layer. Wrap the tape on the side of the ITO or photo conductive material from the edge of the double-sided tape to about three centimeters onto the uncoated side of the glass substrate. To ensure successful device fabrication, use a altimeter to test the resistance reading between the coated slides of both glass substrates and the copper tape that was attached to the glass.
Add 25 milliliters of ultrapure water to a 50 milliliter conical tube containing 4.25 grams of sucrose and 0.15 grams of glucose. Mix well until half of the sucrose and glucose dissolves and make up with ultrapure water up to the 50 milliliter mark. Then vigorously mix this DEP buffer until completely dissolved.
Place 20 milliliters of the prepared sucrose and glucose solution into a 50 milliliter conical tube. Add 0.1 grams of bovine serum albumin, BSA, into the tube. Vortex well until all the BSA dissolves to get a final concentration of 0.5%BSA in the DEP buffer.
Place one times 10 to the six human mesenchymal stem cells or HMSCs or human embryonic kidney, HEK293 suspended in one milliliter of growth medium into a 10 milliliter centrifuge tube. Centrifuge, the HEK293 cells at 201 G for five minutes and the HMSCs at 290 G for 10 minutes. After centrifugation, aspirate the supernatant.
01 And gently resuspend the cells in one milliliter DEP buffer solution with 0.5%BSA. Repeat centrifugation two more times and resuspend the cells in the DEP buffer with 0.5%BSA. Prepare the experimental setup items such as a laptop, projector, objective lens, digital microscope, and a function generator for quantifying the cellular responses to LiDEP.
Position the 10x objective lens, LiDEP chip and holder on top of the projector lens. Use the laptop to design light projections such as star, diamond, three lines and oval and connect the laptop to the projector. Connect the LiDEP chip to the function generator to apply the AC electric field.
To observe the cells experiencing the LiDEP force, use a digital microscope for imaging NVIDIA recording. After completing the setup, flush the microchannel with 70%ethanol then flush was 0.5%BSA solution to remove the ethanol in previous cells. Repeat the flush three times to ensure complete wash away as previously exposed cells to the DEP field will respond differently than fresh cells and may disrupt the data collection.
Remove the 0.5%BSA solution with a pipette. Next, set the function generator to the desired voltage and frequency. Carefully add 70 microliters of the prepared cell suspension into the device microchannel using a smaller pipette tip, tilting it slightly in the hole toward the microchannel to avoid spilling due to the thinness of the microchannel.
Wipe away the access solution with single use paper wipes and discard them into biohazard waste. Then, project the desired virtual electro geometry circles, diamonds, stars, or parallel lines onto the LiDEP chip. In the digital microscope software, set the video length to three minutes.
Set a lab timer to two minutes and 30 seconds. Once the cells are stationary in the microchannel of the LiDEP chip, press start in the digital microscope software to begin the video recording process. After 10 seconds, press the On button of the function generator channel output to apply the AC electric field and press start for the timer.
Monitor the cell DEP behavior through the digital microscope and prevent shaking or movement around the setup. Once the timer goes off, press the On button of the function generator channel output which turns the channel output off and the AC electric field is no longer supplied through the electrodes. Stop the video recording after three minutes and save it for future analysis.
Pipette the cells out of the outlet end of the LiDEP chip by slowly pushing 60 microliters of DEP buffer with 0.5%BSA into the microchannel and simultaneously collecting out the outlet. Continue until there are little to no cells in the microchannel and repeat the DEP with all frequencies as demonstrated. The DEP responses in terms of velocity for the HMSCs and their viability percentage were determined.
The measurements of the positive DEP responses at five, 10, amd 20 voltage peak to peak or VPP showed that the cells moved on an average velocity of 0.025, 0.036, and 0.051 micrometers per second respectively. The viability findings of the HMSCs after experiencing the DEP force showed that the higher voltages generally resulted in lower cell viability with 57%of the cells viable at 20 VPP. White, yellow, red and blue colored electrodes were chosen for this study, but the illumination through the LiDEP depth chip was affected by the photo conductive layer, which had a red orange color.
Hence, the projected white electrode appeared yellow with a white interior, the red electrode appeared orange with a red outline, and the blue electrode appeared light green. This phenomenon suggests that yellow and white had the strongest DEP field while blue and red were weaker. The DEP responses of HMCs were measured with LiDEP in three depth analyzer at 10 VPP.
With LiDEP, there was a decay in the positive DEP response from 30 kilohertz to 20 megahertz. From the three analyzer, the cells increased in the positive DEP from 37 to 255 kilohertz and decreased in positive DEP from 1, 772 kilohertz to 20 megahertz. Two important components in this method are a strong light source and a solid electrical connection to ensure the production of the electric field and cell manipulation.
We observed cell spinning in response to virtual electrodes. Thus, electro rotation is another cell analysis method that can be performed. Electro rotation would provide information about HMSCs heterogeneity and rare subpopulations.
