Micropatterned Magneto-Rheological Elastomers to Drive Changes in Cardiomyocyte Alignment

We're investigating how cardiomyocytes respond to dynamic reversible changes in their mechanical environment, mimicking the fluctuations in stiffness and structure they experience in vivo. Since the introduction of the MRE, we've published several important studies in cardiology. Using these materials, we found that stiffness-driven hypertrophy in adult cardiomyocytes depends on the microtubule networks.

More recently, we uncovered more information about mechanical memory in both cardiac fibroblasts and iPSC-derived cardiomyocytes. The best part of using magnetorheological elastomers is their simplicity. Our protocols is straightforward as it sounds, just a combination of off-the-shelf products working together to create something powerful.

It's an easy-to-use approach that makes cutting-edge science accessible to everyone. This paved the way for more realistic disease models, especially for condition like heart failure, where the mechanics of the tissue are always shifting. It also help researchers design smarter biomaterials that are highly tuneable, dynamic, and reversible.

Our team has mainly focused on heart disease, but recently we expanded the dynamic stiffest range of our MREs from as low as 5 kilopascals to as high as 400 kilopascals. That breakthrough opens the door for us to extend our research and collaborate on other organs beyond the heart. To begin, mix the base and curing agent in a 10-to-1 ratio to prepare 10 grams of 184 polydimethylsiloxane, or PDMS.

Pour five grams of the 184 PDMS into a 35-millimeter Petri dish. Then place the dish in a desiccator and degas for approximately five to 10 minutes until all bubbles have dissipated. Allow 184 PDMS to partially cure for 30 minutes in an oven at 60 degrees Celsius.

When five minutes remain for curing, apply a thin layer of additional 184 PDMS onto the surface of the diffraction grating. Place it in a desiccator to degas for approximately five minutes. Now, remove the 35-millimeter Petri dish containing the partially cured 184 PDMS from the oven.

Flip the diffraction grating with PDMS onto the partially cured 184 PDMS in the dish. Lightly press the diffraction grating until a small amount of PDMS surrounds it. Use the remaining uncured 184 PDMS to backfill the dish surrounding the grating to ensure it stays in place while curing.

Then place the dish into an oven set at 60 degrees Celsius for 1.5 hours. After curing, remove the dish with the grating from the oven. Use a scalpel to score around the diffraction grating.

Apply a small amount of isopropyl alcohol to penetrate underneath the diffraction grating. Pull the grating off the PDMS in the direction parallel to the patterns. Remove any excess PDMS, leaving only the patterned section intact.

Then cut the PDMS stamp to the desired size. Mark the direction of the pattern on the MRE dish. To coat the stamp with silane, place the fabricated 184 PDMS stamp inside an oxygen plasma cleaner with the patterned surface facing up.

Treat the surface with 45 watts of oxygen plasma for 30 seconds. Place the treated 184 PDMS stamps inside a desiccator positioned in a fume hood. Now, tear off the lid from a microcentrifuge tube.

Place the microcentrifuge tube lid next to the PDMS stamps inside the desiccator. Pipette 20 microliters of silane to the microcentrifuge tube lid. Close the desiccator and pull a vacuum to initiate vapor coating.

For the MRE preparation, measure the desired amount of silicone thinner and ECO elastomer part B.Mix them at 2, 500 revolutions per minute in a speed mixer for one minute. Add ECO elastomer part A and carbonyl iron particles to the mixture and mix again at 2, 500 revolutions per minute for one minute. Using a transfer pipette with the tip cutoff, dispense five grams of the MRE mixture into a new 35-millimeter Petri dish.

Degas in a desiccator for approximately five minutes, and then partially cure for 10 minutes in an oven at 60 degrees Celsius. When five minutes remain for curing, use a transfer pipette to add uncured MRE to just coat the patterned surface of the PDMS stamp. Place the coated stamp in a desiccator to degas for five minutes.

Remove the partially cured MRE dish from the oven. Using forceps, flip the coated stamp face down onto the MRE surface and press lightly. Mark the direction of the pattern on the MRE dish.

Place the assembly back into the oven at 60 degrees Celsius for an additional 25 minutes. Once the material is fully cured, use a scalpel to score the MRE at the bottom edge of the stamp in a direction perpendicular to the pattern lines. Apply a small amount of isopropyl alcohol onto the cut region around the stamp.

Then use forceps to pull the PDMS stamp off the MRE in the direction parallel to the pattern's. Sterilize the surface of the MRE by washing with 70%ethanol three times and allow the third wash to sit for 20 minutes. Then wash the MRE surface three times with sterile PBS.

Coat the surface of the MRE with 10 micrograms per milliliter fibronectin in PBS containing magnesium and calcium for one hour at 37 degrees Celsius to promote cell adhesion to the device. Finally, wash the MRE surface three times with PBS containing magnesium and calcium, and seed the neonatal rat cardiomyocytes at a density of 20, 000 cells per square centimeter. The fidelity of micropattern transfer from the master mold to the MRE substrate is demonstrated in this figure through normalized height profiles and quantitative measurements of feature pitch and height.

The X profile comparison showed that the normalized feature height profile of the MRE closely followed that of the master mold, though with slightly reduced peak heights and broader feature shapes. The average pitch of the MRE substrate was approximately 10 micrometers and was not significantly different from the pitch of the master mold. The average feature height on the MRE substrate was significantly lower than that of the master mold, with mean values of 3.81 micrometers and 5.49 micrometers, respectively.