Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

The overall goal of this procedure is to fabricate micro-pattern hydrogels for 2D traction force microscopy studies. This is accomplished by first making a methacrylated glass cover slip and forming the first layer of a polyacrylamide hydrogel on it. The second step is to create another hydrogel layer containing fluorescent beads for traction force microscopy.

The hydrogel is sandwiched with a micro-pattern cover slip on top of it, resulting in the transfer of the micro-pattern matrix proteins on the hydrogel surface. Next, the polyacrylamide hydrogel is left to polymerize at room temperature for 45 minutes. The top cover slip is then carefully removed.

Fluorescence microscopy imaging is used to show the presence of the beads and the successful transfer of micro-patterned extracellular matrix proteins. This method helps to efficiently and precisely measure cellular traction forces by using the light induced release of cells. Making possible to obtain a higher number of experimental observations from the same sample.

So demonstrating the procedure will be Joel Christian, a graduate student from my laboratory. Add 10 milliliters of double distilled water into a beaker. After, add 18.75 milliliters of acetic acid, 18.75 milliliters of 3-(Trimethoxysilyl)propyl methacrylate and 252.5 milliliters of ethanol to the solution.

When the solution is complete, transfer it to a crystallizing dish. Take 24 millimeter round cover slips, clean them with a precision wipe, and put them into a custom made teflon rack. Then immerse it into the solution and incubate for 15 minutes.

Take the rack and rinse it with ethanol. Dry the cover slips under airflow. Methacrylated cover slips can be stored up to one month at room temperature.

Take a 15-millimeter round cover slip and set it on a Petri dish. Use a diamond pen to mark the upper side of the cover slip. Then proceed to cleaning via oxygen plasma treatment.

The cover slip is clean at 0.4 millibar and 200 Watts for two minutes. Pipette 100 microliters of 0.01%of Poly-L-Lysine solution onto the surface of the cover slip and incubate for 30 minutes at room temperature. After, wash the cover slip with 10-millimolar HEPES pH of 8.5.

Take out the excess liquid, but keep the surface wet. Pipette 100 microliters of 50 milligram per milliliter of mPEG-SVA in 10-millimolar HEPES pH 8.5 solution onto the surface of the cover slip and incubate for one hour at room temperature. After, rinse the cover slips with 10-millimolar HEPES pH 8.5.

Then add two microliters of PLPP gel followed by 40 microliters of ethanol onto the surface. Gently tilt the Petri dish to homogenize the solution. Wait for five minutes for the solution to polimerise.

Place the cover slip inside a 35-millimeter bottom hole Petri dish. Put the Petri dish containing the cover slip on the microscope stage. Adjust the focus on the surface of the glass.

Load and lock the pre-drawn pattern. Start the patterning by UV dose of 30 millijoules per millimeter squared. After completing the patterning step, take out the pattern cover slip and rinse the surface with PBS.

Incubate the sample with a 100-microliter mixture of 25 microgram per milliliter fibronectin and 25 microgram per milliliter fibrinogen conjugated with Alexa 488 dissolved in PBS for one hour at room temperature. To prepare the hydrogels substrate, start with putting the methacrylated cover slip into a Petri dish. To start, prepare fresh oxidized HEA solution by adding 9.55 milliliters of double distilled water into a 15-milliliter Falcon tube.

Then add 0.5 milliliters of HEA. Then add 42 milligrams of sodium meta pyruvate to the solution. Set the solution on the shaker for four hours.

Then prepare the stock solution by mixing acrylamide, bis-acrylamide, and double distilled water according to Table 1. The stock solution can be kept for up to a year at four degrees Celsius. Prepare the working solution for the bottom layer by mixing 99.3 microliters of stock solution with 0.5 microliters of ammonium persulfate 1%and 0.2 microliters of TEMED.

Take 10 microliters from the solution and pipette drop-wise onto the methacrylated cover slip. Carefully place a 15-millimeter round cover slip on the droplet and wait 45 minutes for it to polymerize. Next, detach the top cover slip with the scalpel.

Then prepare the working solution for the top layer by mixing 93.3 microliters of stock solution with one microliter oxidized HEA, five microliters fluorescent beads, 0.5 microliters of ammonium persulfate 1%and 0.2 microliters of TEMED. Take five microliters from the solution and pipette it drop-wise onto the bottom layer hydrogel. Carefully place a micro-pattern cover slip on the droplet.

Wait 45 minutes for it to polymerize. Gently detach the cover slip with a scalpel. And glue the cover slip onto the bottom of a custom drilled six-well plate.

Add PBS to the wells. To see the cells, aspirate the PBS from the six-well plate. For fibroblasts, add high glucose DMEM containing L-glutamine and supplement it with 10%FBS and 1%penicillin and streptomycin.

Incubate the cells overnight at 37 degrees Celsius and 5%CO2. Turn on the CO2 and the heating for the microscopy stage. Then turn on the microscope and place the well plate on the stage.

Set up the illumination pattern and mark cells of interest. Refocus on the surface of the hydrogel. Acquire the image of the cells in the brightfield channel.

To take the image of the beads in the deformed state, switch to Cy5 channel. Then switch to the laser channel and illuminate the cell according to the pattern for three minutes. This corresponds to a dose of 6, 000 millijoules per millimeter squared.

After, switch the channel and acquire the image of the beads in the undeformed state. To calculate the traction forces, open the fluorescent bead images that already corrected for the lateral drift. The complete analysis of traction forces is detailed in the attached script.

To verify the selective adhesion of cells to the micro-patterned regions on the hydrogels surface, samples were imaged with fluorescent microscopy using pre-labeled matrix proteins and with wide field microscopy to visualize cells. The mechanical properties of polyacrylamide hydrogels were varied by mixing different amounts of acrylamide and bis-acrylamide. The hydrogel's stiffness was evaluated with nanoindentation experiments by atomic force microscopy.

Traction force measurements were performed after applying a spatial, temporally controlled, ultraviolet laser beam illumination to selectively illuminate micron-sized regions of the polyacrylamide hydrogel. The traction forces were reconstructed by using regularized FTTC with a regularization parameter chosen by generalized cross validation. Our model modular approach based on traction force microscopy combined with light induced release of micro-pattern cells is versatile and could be further exploited with other microscopy and imaging setup to improve its resolution and sensitivity.