Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. Acoustofluidic method can realize fast and non-destructive measurement in suspended states. This technique can be used for mirroring the change of self compressibility during epithelial to mesenchymal transition and separating circulating tumor cells, showing its application prospects in cancer diagnosis.
To begin, bind the PDMS block to the chip by punching holes in the PDMS block for inlet and outlet ports with a one millimeter diameter hollow needle. Then put the PDMS blocks and chip with the backside up in a plasma cleaner for one minute. After aligning the holes on the PDMS blocks with the chip inlet and outlet, gently press the PDMS blocks to the chip for 15 seconds, resulting in bonding to occur between the PDMS blocks and the surface of the chip.
To connect the PTFE catheter to the chip, cut two pieces of catheter with an inner diameter of 0.8 millimeter and a length of 10 centimeter. Bend a stainless steel needle with an inner diameter of 0.7 millimeter and a length of 1.5 centimeter by 90 degrees into an L shape and connect it to one end of the catheter. Insert the stainless steel needles to the holes of the PDMS blocks.
For the inlet, connect a 19 gauge dispensing needle to the other end of the catheter as a connector for a syringe. Afterward, inject deionized water to test the tightness of the overall channel. For a piezoelectric ceramic assembly, select precut piezoelectric ceramic sheets with a width of five millimeter.
Then weld wires on both sides of the piezoelectric ceramic at one end. Glue the piezoelectric ceramic to the middle of the back of the chip by placing a drop of cyanoacrylate glue on the piezoelectric ceramic. Smooth the glue with the toothpick and remove the excess glue.
Then quickly press it on the chip and continue to press for about one minute. To mount the micro device, cut a piece of PDMS as the base of the micro device and stick one side of the base to the inlet and outlet PDMS blocks and the other side to a transparent glass slide using double-sided tape. Fix the whole microdevice to the microscope stage to keep the chip in one focal plane.
To prepare the polystyrene standard particle solutions add 0.05 milliliters of polystyrene particle solution to 10 milliliter of PBS and mix well. Then prepare the cell suspensions by washing the adherent cells at 90%confluency with PBS. Add 500 microliters of 0.25 Trypsin for one to two minutes at room temperature.
After removing the Trypsin, add one milliliter complete medium and form a cell suspension by pipetting. Next centrifuge the cell suspension at 100 G for five minutes. Remove the supernatant and resuspend in 0.5 to one milliliter of PBS in order to obtain a cell suspension.
Then cells were counted with a hemocytometer and the concentration was about 300 to 500, 000 cells per milliliter. After a preparing the cell nucleus suspension as demonstrated previously, remove the supernatant and add 200 microliters of cytoplasmic protein extraction reagent A per 20 microliters cell palette and mix well. Then vortex the above mixture at 220 G for five seconds and place on ice bath for 10 minutes.
Next, add 10 microliters of cytoplasmic protein extraction reagent B to the solution after incubation. After vortexing, place on ice bath for one minute and vortex again at 220 G for five seconds. Then, finally centrifuge at 1000 G for five minutes at four degrees Celsius.
Remove the supernatant and resuspend the palette in one milliliter of PBS. Then centrifuge at 1000 G at four degrees Celsius for four minutes. After removing the supernatant, resuspend in 100 microliters of PBS as cell nucleus suspension.
Add trypan blue to the above cell nucleus suspension and stain at room temperature for four minutes. Next, count the number of nuclei under the inverted microscope with a 10x objective. Dilute the above cell nucleus suspension with PBS buffer to a concentration of 200 to 300, 000 nucleus per milliliter.
Then filter the cell nucleus suspension through a 70 micrometer sieve. To set up the measurement system, turn on the light source of the microscope and open the camera software. Use the 4x objective to find the middle position of the microchannel, that is, the position of the piezoelectric ceramic.
After connecting the wires, weld them to the positive and negative terminals of the signal generator output of the piezoelectric ceramic respectively. Then place the syringe on the microinjection pump and connect it to the inlet catheter. To collect the fluid from the microchannel, place a small container at the end of the outlet catheter.
To determine measurement parameters, aspirate the polystyrene particle solution with the syringe. Then inject the solution into the chip microchannel while avoiding air bubbles and in chip microchannel to ensure accurate measurement. Set the output of the signal generator to a sign signal with a frequency of one megahertz and peak-to-peak voltage of 10 volts until it's observed that the particles move toward the midline of the microchannel and remain in forward motion along the midline after reaching the midline.
After mixing one milliliter cell or nucleus suspension with the standard particle solution at the ratio of one to one, inject it into the microchannel with a syringe to measure cells and nuclei. Start recording with CCD camera when the cells or nuclei enter the field of view and turn on the signal generator. Then rinse the microchannel with deionized water, 75%alcohol, and deionized water in sequence for later use.
The movement of the cells and particles was observed under the action of the acoustic field force towards the midline of the microchannel at different time intervals. The calculation of energy, density, and cell compressibility were performed and acoustic energy density and the measured motion trajectory for the particle was obtained from the best fitting result. The high purity and intact nuclei were isolated from cells and nucleus compressibility measurement was achieved by obtaining the motion trajectory of the nucleus.
The compressibility of different types of cancer cells and cells after drug-induced EMT or x-ray irradiation with different doses was measured and compared. The measured cells were collected and it was found that there was no significant difference in cell proliferation and viability compared to the untreated group after 48 hours of culture. Indicating that the compressibility measurement has no effect on cell proliferation and survival.
The most important thing is to fine tune the frequency of the signal until it's observed that particles move towards the midline of the microchannel.
