Preparation of a Micropatterned Substrate to Study Schwann Cell Phenotypes
Transkript
To prepare a micropatterned substrate, place a patterned silicon wafer inside a circular 150-millimeter diameter by 15-millimeter height Petri dish and pour degassed PDMS onto the wafer. Solidify the PDMS in an oven at 60 degrees Celsius overnight. Allow the PDMS to cool to room temperature, then, use a surgical scalpel to cut stamps of 30-by-30 millimeters squares containing the correct patterns, taking care to not damage the silicone wafer. Sterilize the PDMS stamps and tunable coverslips by immersing them in 70% ethanol for 30 minutes.
To confirm the efficacy of the micropattern, dry the surface of the PDMS stamps using a filtered airstream, and pipette 50 micrograms per milliliter BSA solution to cover the entire patterned side of the stamp. Incubate the stamps with the BSA solution for 1 hour at room temperature to allow for protein adsorption, then air-dry the stamps to remove the BSA solution. Use the filtered airstream to dry the surface of the tunable coverslips.
Bring the patterned side of the stamp into conformal contact with the tunable coverslip to allow for BSA absorption on the coverslip surface, and gently press the stamp against the coverslip for 5 minutes. Examine the micropattern using a fluorescence microscope with a FITC filter. To print cell-adhesive areas rather than fluorescent patterns, substitute laminin for BSA protein.
Remove the stamps from the coverslips, and transfer the coverslips into a sterilized 6-well plate. Add 2 milliliters of 0.2% Pluronic F127 solution into each well to cover the surface of the coverslip and incubate the plate for 1 hour at room temperature. Aspirate the F127 solution, and wash the coverslips 5 times with PBS, once with the cell culture medium, then seed the Schwann cells at a seeding density of 1,000 cells per centimeter squared. 45 minutes after cell seeding, remove the cell culture medium and wash the coverslips twice with PBS to prevent multiple cells from adhering to the same pattern. Maintain cells in the desired cell culture environment for 48 hours before quantification.
