Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The overall goal of this microfluidic experiment is to evaluate the effects of kinase inhibitors on tumor cell migration under a direct electric field. So this method can help answer key questions in cellular electro Texas studies such as what are the determinant pathways in electric field guided migration of cancer cells. So the main advantage of this technique is that it is a highly reproducible and time-saving electro Texas study that can be easily implemented.

The implication of this technique extend toward the diagnosis of metastatic potency of cancer. As the mobility of magnetic cancer cells can be enhanced by an electro field. So this method can provide the insider ular, electro testis of tumor cells.

It can also be applied to skin regeneration processis, such as wound healing. Begin by using a drafting and design program to draw an individual acrylic layer pattern. Save the pattern, and then turn on the laser scriber.

Connect the scriber to the controlling laptop and click on designed pattern file to open the acrylic layer pattern. Next, place a piece of blank acrylic sheet on the x, Y, Z stage of the laser scriber and use the appropriate corresponding laser scriber auto alignment stick. To set the focus of the laser beam on the surface of the sheet.

Send the designed pattern to the laser scriber for direct machining of the acrylic sheet. Then use forceps to remove the protective paper from the acrylic sheets and blow the surfaces clean with nitrogen gas. Now stack the sheets and bond them together under a pressure of two kilograms per square centimeter in a thermal bonder for 45 minutes at 110 degrees Celsius to form the flow electrical stimulation channel assembly.

After preparing double-sided tape in the same manner, tape the clean cover glass to the flow electrical stimulation channel assembly. Then use super glue to adhere 13 pieces of acrylic adapters to the individual openings in layer one of the multi-channel dual electric field or MDF chip assembly to set up the MDF chip salt bridge network. Begin by connecting floor plastic tubes to the MDF chip assembly via the medium inlet and outlet adapters.

Next, connect the lure taper of the medium inlet and outlet plastic tubes to three-way stop Cox A three milliliter syringe containing 2.5 milliliters of carbon dioxide equilibrated PBS to the three-way stop cock of the inlet plastic tube and an empty three milliliter syringe to the three-way stop cock of the outlet plastic tube. Use white solid finger tight nuts to seal the openings of the blue and green adapters on the layer one acrylic sheet. Then fill the salt bridge channels and culture chambers with the carbon dioxide equilibrated PBS taking care to avoid bubbles.

Transfer the chip to a 37 degree Celsius 5%carbon dioxide cell culture incubator overnight to allow the dissolved air in the double-sided tape to form bubbles within the chambers the next morning. Use the two syringes to generate a rapid PBS flow to flush away the bubbles pumping the PBS back and forth as necessary. When all of the bubbles have been removed, use the three-way stopcock connected to the medium outlet tube to drain the PBS from the channels.

Then replace the syringe connected to the inlet with a three milliliter syringe containing 2.5 milliliters of carbon dioxide a equated DM EM and refill the chambers with the medium when the chambers are full. Remove the solid nuts from the green adapters and inject 3%aros heated to at least 70 degrees Celsius into the Salt bridge Channel. Through the openings in the adapters, The hot A prepare salt bridge for the injection, ensuring blockage of the median row between cultural chambers.

Stop injecting the agros when the compound fills three quarters of the length of the salt bridge channel and reseal the pores with the solid nuts. Now replace the solid nuts on the blue adapters with translucent tubular finger tight nuts and load aros into the tubular nuts. Then embed silver, silver chloride electrodes into the tubular nuts before the aro solidifies.

To set up an electro tactic experiment, inject 0.3 milliliters of cells into the MDF chip through the outlet tubing, and return the chip to the cell culture incubator. After two to four hours, install the MDF microfluidic system onto a transparent indium tin oxide glass heater and attach a K type thermocouple between the MDF chip and the indium tin oxide glass. To measure the temperature of the chip, use a proportional integral derivative controller to set the MDF microfluidic system incubation temperature to 37 degree Celsius and mount the temperature controlled MDF chip assembly onto the computer controlled XYZ motorized stage of an inverted microscope.

Then using a four channel syringe pump, dispense complete medium into the culture chambers through the inlets at a flow rate of 20 microliters per hour. Collecting the culture waste from the outlets in microcenter fuge tubes. Incubate the cells at 37 degrees Celsius for another 16 to 18 hours.

The next day, replace the medium in each chamber with fresh, medium supplemented with the appropriate concentration of kinase inhibitor for 60 minutes of treatment. Then connect a DC power supply to the MDF chip via the silver, silver chloride electrodes and serially. Connect an ammeter to the electric circuit to monitor the electric current in the MDF chip.

Finally turn on the power supply and adjust the voltage to between 15 and 19 volts. To set the ammeter current to 86.94 microamps. In this representative MDF Microfluidic system experiment lung cancer cells treated with various concentrations of the kinase inhibitor.

Y 2 7 6 3 2 exhibited no changes in cell migration speed with or without electrical stimulation. However, Y 2 7 6 3 2 treatment under the addition of an electric field significantly reduced the nautic of the cancer cells migration. Indeed, at a 50 micromolar concentration, the rock inhibitor eliminated the nautic movement of the cells completely without affecting their migration speed.

Moreover, there was a dose dependent correlation between the applied chemical concentrations and the directedness index confirming the reliability and efficiency of the MDF microfluidic system as a method for studying axxis. Once master, this technique can be completed in seven to eight hours if it is performed properly after its development. This technique takes away for researchers in the field of electro Texas to explode with hitting in the skin.

Don't forget that working with a laser scraper can be extremely hazardous and that precautions should always be taken while performing this procedure.