This protocol allows for the consistent creation of high-fidelity protein micro patterns of whatever shape is desired, notably isolated islands of pattern for study of cell clusters. This technique can be used to make isolated island micro patterns of any shape and size in only one step, whereas previously making such patterns required two separate steps. Begin by mixing PDMS in the correct curing agent to base ratio as described in the manufacturer's instructions.
Incubate at room temperature and pressure for 15 minutes, then degas the mixture under vacuum for 15 minutes. Pour the PDMS into the master mold and transfer it to an incubator set to 37 degrees Celsius to cure overnight. Remove the master mold from the incubator and allow it to cool down to room temperature.
While the master mold is cooling down, sonicate 25 millimeter coverslips in ethanol for 10 minutes. Use as many coverslips as the number of stamps being prepared. Thoroughly rinse the coverslips with DI water and dry them using a filtered air gun.
Treat the coverslips with plasma for one minute using the plasma cleaner under a high vacuum. Slowly release the vacuum after treatment to prevent the coverslips from moving inside the chamber. Coat each coverslip with 100 microliters of the fluorescent labeled protein solution in a room devoid of direct sunlight or overhead lighting.
Cover the samples for extra protection from light and incubate them for 20 minutes at room temperature. Thoroughly rinse each coverslip with DI water. Remove excess water from the surface by gently tapping the sides of each coverslip onto a paper towel or a similar absorbent material.
Allow the coverslips to dry completely by leaving them uncovered in the dark for at least 30 minutes. While the protein coated coverslips dry, remove the PDMS stamp from the master mold by cutting it with a scalpel or any sharp blade. Treat the PDMS stamps with plasma for two minutes under a high vacuum.
Place the stamps in a container with a lid under the fume hood, then coat each stamp with a thin layer of 10%3-APTMS diluted in 100%ethanol. Cover the container with the stamps and incubate at room temperature for five minutes. Thoroughly rinse each stamp on both sides with DI water.
Place the stamps in a clean container and coat them with 2.5%glutaraldehyde prepared in DI water. Cover the stamps and incubate them at room temperature for 30 minutes. Rinse the stamps thoroughly with DI water.
Remove excess water from the surface as previously described and allow the stamps to dry uncovered for 30 minutes. If after 30 minutes the protein-coated coverslips or the stamps are not completely dry, use a filtered air gun to dry them completely. Push the pattern side of the stamps onto the coverslips with enough pressure for them to make complete contact.
Leave it undisturbed for 15 minutes. Then carefully peel the PDMS stamps off the coverslips. Using a fluorescent microscope with the appropriate filter, check the fidelity of the patterned coverslips.
Use the patterned coverslips immediately or store them away from direct light until further use. Before preparing the PAA hydrogel precursor, remove the acrylic acid NHS ester from the refrigerator to reach room temperature before being opened. Prepare an interchangeable coverslip dish set by sterilizing it with 70%ethanol, then incubate it under UV light in a biosafety cabinet for at least 30 minutes before use.
Under the fume hood, add 1.25 milliliters of 40%acrylamide prepared in DI water to a 15 milliliter conical tube. To the same tube, add 175 microliters of bis-acrylamide solution prepared in DI water. Then add 500 microliters of 10X PBS, followed by 2.915 milliliters of DI water.
Pipette 969 microliters of this precursor into a 1.5 milliliter microcentrifuge tube and store the rest at four degrees Celsius for up to two weeks. Measure 50 to 100 milligrams of APS in a fresh microcentrifuge tube and dilute it to 100 milligrams per milliliter in DI water. Open the NHS ester in the hood and carefully measure up to three milligrams of NHS in a microcentrifuge tube.
Dilute the NHS ester to one milligram per milliliter in 1X PBS. Under the fume hood, add two microliters of TEMED to the microcentrifuge tube containing the 969 microliter aliquot of PAA precursor. Add 15 microliters of one molar HCL to decrease the pH of the hydrogel solution and avoid hydrolysis of the NHS ester.
Add 10 microliters of the NHS ester solution to the tube. Perform the following steps in a biosafety cabinet. Carefully place the 30 millimeter coverslip within the middle part of the coverslip dish set with the 3-APTMS and glutaraldehyde treated side up and screw the plastic ring on top.
Set up the patterned coverslip with minimum light exposure to prepare for the next step. Pipette five microliters of APS solution into the PAA precursor aliquot tube, then invert and mix. Immediately pipette 35 microliters of this solution onto the 30 millimeter coverslip.
Place the patterned coverslip on the solution with the protein side down. Be careful not to create air bubbles in the hydrogel. Protect the hydrogel from light and allow it to polymerize for 90 minutes.
Once the hydrogel has polymerized, use a razor blade or scalpel to remove the upper coverslip, ensure that the coverslip does not fall back or slide off the gel once removed to avoid ruining the pattern on the surface of the gel. To passivate any remaining NHS ester in the hydrogel, add two milliliters of sterile PBS to the gel and incubate at 37 degrees Celsius for 45 minutes. Immediately prepare the gels for experiments or store them overnight in sterile PBS at four degrees Celsius until further use.
The hydrogels were indirectly patterned with fluorescent fibronectin to visualize and measure cellular traction forces within clusters and create island patterns of predetermined size and shape. The pattern quality was directly related to the fidelity of the master mold from which the PDMS stamp was cast. This method could fabricate isolated, well-defined island patterns of predetermined shape.
The fibronectin adhesion dots were present only within the desired area of the island. These isolated islands of adhesion dots further allowed better control of cluster shape. Coating PDMS stamps with two little 3-APTMS is critical because it will limit the effectiveness of the removal method, while too much will create an orange residue on the stamp surface.
The patterns created here can be used to determine cellular traction forces in both individual cells and clusters, which gives insight into their ability to maintain mechanical homeostasis.
