Micropatterned Magneto-Rheological Elastomers to Drive Changes in Cardiomyocyte Alignment

Summary

We present a dynamic and reversible stiffness assay platform fabricated using an ultra-soft silicone elastomer (polydimethylsiloxane, PDMS) with embedded iron particles. This novel assay is suitable for studying emergent time-dependent physical changes in cell phenotypes, including contact guidance. Here, we measure neonatal rat cardiomyocyte alignment upon matrix stiffening using magnetics.

Abstract

Substrate-associated cues, such as mechanical and topographic, profoundly influence cellular response. However, much of the foundational research employs static or isolated effects. The direction and timescale of these mechanical effects on emergent cellular responses remain largely unexplored. Tools to examine how time-varying substrate-associated stimuli drive physiological and pathological processes can unlock the next level of mechanobiological insight. Here, we use micro-patterned magnetorheological elastomers (MREs) that can rapidly stiffen and soften in response to an external magnetic field, allowing for a more rigorous investigation of the effects of mechanical (stiffness) and contact-guided (topographic) stimulation on neonatal rat cardiomyocyte orientation and alignment. By integrating dynamic control of mechanical stiffness that can be temporally tuned and reversed, we can rigorously test the effects of load by (1) pre-conditioning under identical conditions and (2) acutely changing in vitro biomechanics to mimic clinically relevant phenomenology, such as myocardial infarction properly. This approach allows us to study the impact of load on cellular responses in a more realistic and controlled manner.

Introduction

The heart's ability to pump blood efficiently relies on the architecture and mechanical properties (stiffness) of its myocardium, where the precise organization of cells and tissue is essential for coordinated contraction and overall function. The myocardium is primarily composed of cardiomyocytes (CMs), which, in adults, exhibit an elongated, almost rod-like morphology with sarcomeres (the contractile unit of the CMs) aligned along the length of the cell. In contrast, fetal or induced pluripotent stem cell (iPSC)-derived CMs differ significantly from adult CMs. They tend to have a more rounded shape, a single nucleus, a disorganized or unaligned sarcomeric structure, and an irregular beating frequency. The structural resemblance of the in vivo organization of the myocardium allows for synchrony of contraction and force transmission; this is critical and essential to recapitulating natural myocardium through an in vitro model. To generate CMs with structural characteristics similar to mature myocardium, previous studies have demonstrated successful strategies for enhancing maturation, including manipulating substrate stiffness^1,2, applying mechanical strain³, utilizing contact guidance^4,5, or employing electrical pacing^6,7.

It has long been understood that the stiffness of the extracellular environment impacts cells. A vast diversity of stiffness exists throughout the human body, ranging from hundreds of pascals (Pa) in the brain and up to tens of gigapascals (GPa) in bone^8,9,10. It has been found that during development, tissues change stiffness, and this alteration regulates cellular level differentiation and maturation into the adult phenotypes that are needed for normal function^11,12,13,14. Changes in stiffness have also been linked to disease states and the progression of specific pathologies^15,16,17,18. Specifically, the stiffness of the myocardium changes with the development and progression of diseases; for instance, a healthy myocardium has an elastic modulus of ~10 kPa in contrast to a failing heart, which has an elastic modulus of 35-70 kPa or more¹⁹. Additionally, numerous in vitro studies have reported that cardiomyocytes cultured on a substrate similar to the native myocardium stiffness had better sarcomere organization²⁰, generated optimal contraction^1,19, and had the longest action potential duration²¹.

Additionally, it has become more well-recognized that cells in vivo are exposed to a complex variety of topographical cues within their microenvironment that drive form and function. These topographical cues come in various sizes and geometries, from the random arrangement of integrins at a molecular level to the micron-level alignment of cells in muscle tissues^22,23. Similar to other mechanical regulators, topography is important to cell morphology, such as spreading, elongation, alignment, motility, differentiation, and apoptosis^24,25,26. With the introduction of synthetic culture substrates, methods have been developed to create controlled surface topography for cell and tissue culture^27,28,29. For instance, micropatterned nano- and micro-grooves have been used to provide contact guidance for mammalian cells so that cells orient and remodel their cytoskeleton to follow the topographical structures of the culture substrate.

Here, we introduce an in vitro culture method that integrates both structural (contact cue) and mechanical (stiffness) guidance using a patterned magnetorheological elastomer (MRE) substrate, a tunable material that can be stiffened or softened by adjusting the strength of the magnetic field^17,30,31,32. By positioning magnets near the culture substrate, stiffness can be increased, with the degree of stiffening modifiable by varying the distance between the sample and the magnet. Conversely, stiffness can be reduced by removing the magnet altogether. This approach provides a robust, dynamic, and controllable method to replicate the in vivo-like mechanical conditions, while the surface micropatterning introduces anisotropy, enabling the creation of more biomimetic 2D cell culture substrates.

Protocol

1. Preparation of patterned PDMS stamp

NOTE: A PDMS stamp is essential for its flexibility, allowing easy manipulation and precise contact when preparing the patterned MRE surface. The stamp's flexible nature also enables effortless removal from the MRE PDMS layer without damaging the pattern, ensuring both accurate pattern transfer and preservation of the MRE surface.

1. Prepare 10 g of 10:1 (base: curing agent) 184 PDMS.
2. Pour 5 g of the 184 PDMS into a 35 mm Petri dish and degas in a desiccator for ~5-10 min, until all bubbles have dissipated.
3. Allow 184 PDMS to partially cure in an oven at 60 °C for 30 min.
4. When 5 min remains for the 184 PDMS to partially cure, add a thin layer of the extra 184 PDMS to the diffraction grating and degas in the desiccator for ~5 min.
5. Remove the 35 mm dish with the partially cured 184 PDMS from the oven.
6. Flip the diffraction grating with PDMS onto the partially cured 184 PDMS in a 35 mm dish.
   NOTE: Ensure that the diffraction grating is facing down into the partially cured 184 PDMS in the 35 mm dish.
7. Lightly press the diffraction grating until a small amount of 184 PDMS surrounds the grating.
8. Use the remaining uncured 184 PDMS to backfill the dish surrounding the grating to ensure it stays in place while curing.
9. Place the dish containing the diffraction grating into the oven set at 60 °C for 1.5 h.
10. After the 184 PDMS is fully cured, remove the 35 mm dish with the diffraction grating from the oven.
11. Score around the diffraction grating with a scalpel.
12. Apply a small amount of isopropyl alcohol (IPA) to penetrate underneath the diffraction grating.
13. Pull the grating off the 184 PDMS in the direction parallel to the patterns.
14. Remove excess 184 PDMS, leaving only the patterned section.
15. Cut the 184 PDMS stamp to the size needed.
    NOTE: This study uses a 1 cm × 1 cm stamp for the devices.

2. Surface coating of PDMS stamp

NOTE: A silane treatment creates a surface coating that prevents unwanted adhesion between PDMS layers during stamping, ensuring smooth separation and preserving the quality of the pattern transfer.

1. Silane treatment (preferred method)
   1. Place the fabricated 184 PDMS stamp in an O₂ plasma cleaner with the patterned surface facing up. Treat the PDMS surface with 45 W of O₂ plasma for 30 s.
      NOTE: Once removed from the plasma cleaner, place a lid (or cover) over the stamps immediately to ensure no debris settles on the cleaned stamps.
   2. Place 184 PDMS stamps into a desiccator in a fume hood.
   3. Tear off the lid of a microcentrifuge tube.
   4. Place the microcentrifuge tube lid next to the 184 PDMS stamps in the desiccator.
   5. Add 20 µL of trichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane to the microcentrifuge tube lid.
      CAUTION: This chemical is hazardous for health and thus must only be used in a chemical fume hood.
   6. Close the desiccator and pull a vacuum.
   7. Allow the silane to coat the stamps from 1 h to overnight.

3. Preparation of magnetorheological elastomers

NOTE: To create tunable and reversible MREs, a precise ratio of carbonyl iron particles and elastomer, combined with the application of a magnetic field, enables the adjustment of stiffness across a range of values. By varying the particle fraction, base elastomer modulus, and magnetic field strength, the material's stiffness can be finely tuned to meet specific requirements.

1. Softer dynamic range MRE preparation (see Table 1 for example weights)
   1. Measure the desired amount of silicone thinner and Eco elastomer part B and mix at 2500 rpm in a speed mixer for 1 min.
   2. Add Eco elastomer part A and carbonyl iron particles to the solution and mix at 2500 rpm in a speed mixer for 1 min.
      NOTE: The order of mixing is critical to ensure the desired polymerization timing.
   3. Using a transfer pipette with the tip cut off, add 5 g of the MRE to a new 35 mm Petri dish and degas in a desiccator for ~5 min, then allow to partially cure in the oven at 60 °C for ~10 min.
      NOTE: Ensuring that the oven and desiccator are properly leveled is essential for achieving a flat sample, which is critical for the ease of imaging.
   4. Once 5 min remain, use a transfer pipette to add extra uncured MRE to just coat the surface of the 184 PDMS stamp. Place the coated stamp in the desiccator to degas for 5 min.
      NOTE: Be careful not to use too much MRE because if it covers the edges of the stamp, it will make it more difficult to remove the stamp once the MRE is fully cured. Also, make sure to denote the direction of the pattern through every step so that when removing the stamp from the MRE, it can be pulled in the direction parallel to the patterns.
   5. Remove the MRE from the oven, and use a pair of forceps to carefully move the coated stamp. Flip it face down onto the partially cured MRE and lightly press. Put the MRE and coated stamp back into the oven at 60 °C for another 25 min.
   6. Once fully cured, use a scalpel to score the MRE at the bottom of the stamp, perpendicular to the patterns.
   7. Apply a small amount of IPA onto the cut area around the stamp.
      NOTE: The IPA should seep into the cut and start to move underneath the stamp.
   8. Using forceps, pull the stamp off the MRE in the direction parallel to the patterns.
2. Stiffer dynamic range MRE preparation (see table for example weights)
   1. Prepare 30:1 184 PDMS.
      NOTE: Make extra because some will remain stuck to the mixing container.
   2. Add the desired amount of 527 PDMS part A and part B, then carbonyl iron particles and 30:1 184 PDMS in ratios based on the example in the table. 
   3. Mix it at 2500 rpm in a speed mixer for 1 min.
   4. Using a transfer pipette with the tip cut off, add 5 g of the MRE to a new 35 mm petri dish and degas in a desiccator for ~5 min, then allow to partially cure in the oven at 60 °C for ~40 min.
   5. Once 5 min remain, use a transfer pipette to add extra uncured MRE to just coat the surface of the PDMS stamp. Place the coated stamp in the desiccator to degas for 5 min.
      NOTE: Be careful not to use too much MRE because if it covers the edges of the stamp, it will make it more difficult to remove the stamp once the MRE is fully cured. Also, make sure to denote the direction of the pattern through every step so that when removing the stamp from the MRE, it can be pulled in the direction parallel to the patterns.
   6. Remove the MRE from the oven.
   7. Use a pair of forceps to carefully move the coated stamp, flip it face down onto the partially cured MRE, and lightly press.
   8. Put the MRE with the coated stamp on the top rack into the oven at 60 °C overnight.
   9. Once fully cured, use a scalpel to cut the MRE at the bottom of the stamp, perpendicular to the patterns.
   10. Apply IPA onto the cut area around the stamp.
       NOTE: The IPA should seep into the cut and start to move underneath the stamp.
   11. Using forceps, pull the stamp off the MRE in the direction parallel to the patterns.

	Stiffness Range	Materials	Example Weight
Softer	10–80 kPa	Eco (A+B)	11.25 g (A)	11.25 g (B)
		Silicone Thinner	22.5 g
		Iron Particles	45 g
Stiffer	60–120 kPa	527 PDMS (A+B)	18.75 g (A)	18.75 g (B)
		Iron Particles	37.5 g
		184 PDMS (Base+Cure)	14.5 g (Base)	0.5 g (Cure)

Table 1: Example weights of all materials for fabricating both softer and stiffer MREs. The weights in the table are for making a total of 90 g of MRE material. Preparing less can be accomplished by mixing all constituents in the same ratios as the example weights in the table.

4. Cell culture preparation

NOTE: Cell culture preparation involves selecting the appropriate cell line, preparing culture media with necessary nutrients and growth factors, and ensuring sterile conditions to prevent contamination. Cells are plated in culture vessels and incubated at optimal conditions (usually 37 °C with controlled CO₂). Regular monitoring, medium changes, and passaging are performed to maintain healthy cell growth.

1. Sterilize the surface of the MRE by washing it with 70% ethanol 3 times and allow the third wash to sit for 20 min.
2. Wash the MRE surface 3 times with sterile phosphate-buffered saline (PBS) by adding enough volume to cover the dish completely, followed by aspirating off all liquid.
3. Coat the surface of the MRE with fibronectin (10 µg/mL) in PBS for 1 h at 37 °C to promote cell adhesion to the device.
4. Wash the MRE surface 3 times with PBS, similar to step 4.1.2.
5. Seed the neonatal rat cardiomyocytes at a density of 20,000 cells/cm².
6. Add magnets to the devices temporally to stiffen the matrix during the cell culture.
   NOTE: Be careful when placing devices with magnets in the incubator. The devices need to be placed far apart to guarantee that the magnets do not interact. This study recommends the usage of stainless steel-lined incubators over copper-lined incubators as there is less interaction.

5. Staining and imaging

1. Fixed cell imaging
   NOTE: Fixed cell imaging involves several key steps to visualize specific cellular components. First, cells are fixed to preserve their structure. Next, the cell membrane is permeabilized to allow stain penetration, followed by a blocking step to prevent the non-specific binding of antibodies or stains to unintended sites, such as other proteins or cellular debris. Cells are then stained with dyes or antibodies targeting specific molecules, and any excess stain is removed through washing. Finally, the stained cells are imaged using fluorescence microscopy for analysis of cellular structures.
   1. Wash once with 1x PBS by adding enough volume to cover the dish completely, followed by aspirating off all liquid.
   2. Fix cells by using 4% (v/v) paraformaldehyde for 10 min.
   3. Wash three times with 1x PBS (similar to step 5.1.1).
   4. Add 0.1% Triton X (in 1x PBS) and incubate for 5 min to permeabilize.
   5. Wash three times with 1x PBS (similar to step 5.1.1).
   6. Add NH₄Cl (50 mM in 1x PBS) and incubate for 15 min to quench.
   7. Wash three times with 1x PBS (similar to step 5.1.1).
   8. Add blocking buffer (5% BSA in 1x PBS) and incubate for 1 h at room temperature (RT).
   9. Combine the primary Anti-α-Actinin primary antibody at 1:200 into 5% BSA (in 1x PBS).
      NOTE: Mix the antibody thoroughly.
   10. Add the primary antibody solution to the sample.
       NOTE: Add enough to cover the entire sample.
   11. Incubate overnight at 4 °C.
       NOTE: Consider placing the sample on the rocker at a slow speed to help the solutions mix.
   12. Wash three times with 1x PBS (similar to step 5.1.1).
   13. Dilute the secondary antibody (555 Alexa Fluor goat anti-mouse) in 1x PBS (1:2000).
       NOTE: The secondary antibodies are light-sensitive (fluorophores). When not directly in use, make sure they are covered and out of the light.
   14. Add the secondary antibody solution to the sample.
   15. Incubate at RT for 2 h.
   16. Wash three times with 1x PBS (similar to step 5.1.1).
   17. Add DAPI (1 µg/mL in sterilized de-ionized water) and incubate for 5-10 min in the dark.
   18. Image the cells with an upright microscope with a 20x dip-in lens.
   19. Take 50 images per device.
   20. Process the images using an ImageJ plugin called Directionality.
       NOTE: Directionality options: This study used n = 45 bins, ranging from 0° to 176° to prevent a wrapping effect. The method employed was local gradient orientation, and the display table was selected. The direction output corresponds to the mean vector angle (indicating the angle of alignment), while the goodness output represents the mean vector length (indicating the degree of alignment).
2. Live cell imaging
   NOTE: Live cell imaging enables the tracking of dynamic cellular processes in real-time while keeping the cells in their natural, living state. This technique involves using a fluorescence microscope equipped with an incubation chamber to capture high-resolution images or videos of cells as they grow, divide, or respond to stimuli. Enabling the study of cellular behavior and interactions over time and providing valuable insights into cellular function and dynamics.
   1. Incubate the cells with SiR-actin 1:1000 ratio with culture media for 1 h at 37 °C.
   2. Aspirate the media and replace it with 1:2000 (SiR-actin: Media).
      NOTE: This dilution is to reduce cell toxicity for extended live imaging.
   3. Collect immunofluorescence images every 15 min for 5 h on an upright microscope with an incubation chamber, automated stage, and 20x dip-in lens.
      NOTE: For the live cell imaging experiment, the magnet was added as soon as the MRE was placed in the live imaging system.

Results

Ultra-soft silicone-based magnetorheological elastomers (MREs) have elastic moduli that can be precisely tuned by applying a magnetic field. This allows for rapid, bi-directional adjustments within a physiological range, with precise control over both the rate and magnitude of changes (Figure 1). Gel mechanics were evaluated by measuring the elastic modulus as previously described, and the average elastic modulus was found to be 36.3 ± 5.2 kPa with magnet and 9.3 ± 1.2 kPa without magnet for...

Discussion

This technique has been rigorously tested, but careful attention to several steps is essential for a successful experiment.

In this protocol, we use a commercial diffraction grating as the master mold for patterning. However, alternative patterned molds-such as silicon or SU8 micro-manufactured molds-can also be employed to create a wide variety of patterns in different sizes^33,35. Some commercially available PDMS materials come pre-pa...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
555 Alexa Fluor goat anti-mouse secondary antibody	ThermoFisher Scientific	A-21422	https://www.thermofisher.com/antibody/product/Goat-anti-Mouse-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-21422
20x dip-in lens	Zeiss	421452-9800-000	https://www.micro-shop.zeiss.com/zh/us/shop/objectives/421452-9800-000/Objective-W-Plan-Apochromat-20x-1.0-DIC-M27-75mm
35 mm Dishes	ThermoFisher Scientific	130180	https://www.thermofisher.com/search/results?query=130180&focusarea=Search%20All
Anti-alpha actinin primary antibody	abcam	EP2528Y	https://www.abcam.com/en-us/products/primary-antibodies/alpha-actinin-actn1-antibody-ep2528y-ab81265?srsltid=AfmBOoq4HwHixqCheQwwq VVbjJ3ogKUmZxa4GtG 3SyllFzWvb5XPnwgB
Carbonyl Iron Powder Microspheres	chemicalstore.com	CIPMS	https://ironpowders.com/types-of-iron-powder/cipms-carbonyl-iron-powder/
DAPI stain	ThermoFisher Scientific	D1306	https://www.thermofisher.com/order/catalog/product/D1306
Desiccator	Fisher Scientific	08-642-7	https://www.fishersci.com/shop/products/nalgene-transparent-polycarbonate-classic-design-desiccator/086427#?keyword=
Diffraction Grating	Thor Labs	GR2550-45031	https://www.thorlabs.com/thorproduct.cfm?partnumber=GR2550-45031
Ecoflex Gel part A & B	Smooth On	Ecoflex GEL	https://www.smooth-on.com/products/ecoflex-gel/
Expanded tabletop plasma cleaner	Sigma Aldrich	Z561657-1EA	https://www.sigmaaldrich.com/US/en/product/aldrich/z561657
Fibronectin from bovine plasma	Sigma Aldrich	F1141	https://www.sigmaaldrich.com/US/en/product/sigma/f1141
Flacktek DAC150 SpeedMixer	Flacktek	DAC150	https://flacktek.com/products/small-flacktek/
ImageJ Software	NIH		https://imagej.net/ij/download.html
Isopropyl Alcohol	Amazon		https://www.amazon.com/Isopropyl-Alcohol-Grade-Anhydrous-gallon/dp/B01M6YK5I4/ref=sr_1_1_sspa?dib=eyJ2IjoiMSJ9.xiBzaAiE5lSJqXz ZhS_JhDh8zEHgkMzrbsyosvyBpS Jjufk90Gt79vk1jOZxq_mG3GKeN8 KT1ulag3ZuMShbWoEOCQUy6txx uGz4709PyUY5YWyeR2GS0PBNx dbmEdWvgmCYiDBLf8E2prhImZW l98MoWlVDU4lg1BtcHTTSOwdg87 5l6Ia3YLPs_0alILBXFsizwwnJodR 8D60ZggK5AHBfCfPadXgfJ-9hUE7 uIto.V3Vf62Lnprtq2u5PRO SbUFHnPOj1FvD7np7uBge6NuU& dib_tag=se&hvadid=598724400239 &hvdev=c&hvlocphy=9007460&hvn etw=g&hvqmt=e&hvrand=1609157 209668794739&hvtargid=kwd-3203 62094308&hydadcr=5273_13 227703&keywords=isopropyl+alcohol+ amazon&mcid=9f9d3252906531 3ea89ab7766d9c244c&qid=1740 675756&sr=8-1-spons&sp_csd=d2l kZ2V0TmFtZT1zcF9hdGY&psc=1
Magnet	CMS Magnetics	ND05578-45N	https://www.magnet4sale.com/n45-neodymium-disc-magnet-1-1-2x1-4-35-lb-pull-strong-magnet/
Nikon Eclipse 80i upright microscope	Nikon	Eclipse 80i
Oven	Fisher Scientific	15-103-0503	https://www.fishersci.com/shop/products/fisher-scientific-isotemp-general-purpose-heating-drying-ovens/p-4935382
Silicone Thinner	Smooth On	Silicone Thinner	https://www.smooth-on.com/products/silicone-thinner/
SiR Actin Stain	Cytoskeleton	CY-SC001	https://www.cytoskeleton.com/sir-actin
Sylgard 184 PDMS (Base + Curing agent)	Ellsworth Adhesives	2065622	https://www.ellsworth.com/products/by-market/consumer-products/encapsulants/silicone/dow-sylgard-184-silicone-encapsulant-clear-3.9-kg-kit/
Sylgard 527 PDMS part A & B	Ellsworth Adhesives	1696742	https://www.ellsworth.com/products/by-market/consumer-products/encapsulants/silicone/dow-sylgard-527-silicone-dielectric-gel-clear-0.9-kg-kit/
Transfer Pipettes	Fisher Scientific	13-711-5AMMD	https://www.fishersci.com/shop/products/transfer-pipette-32/137115AMMD?searchHijack=true&searchTerm=13 -711-5AMMD&searchType= RAPID&matchedCatNo=13 -711-5AMMD
Trichloro -(1H, 1H, 2H, 2H-perfluorooctyl) silane	Sigma Aldrich	448931	https://www.sigmaaldrich.com/US/en/product/aldrich/448931