Name: Micropatterned Magneto-Rheological Elastomers to Drive Changes in Cardiomyocyte Alignment (Video) | JoVE Journal
Uploaded: 2025-06-10T12:40:00.000+00:00
Duration: 8 min 10 s
Description: Learn how micropatterned magneto-rheological elastomers drive cardiomyocyte alignment changes in response to dynamic stiffness. This method enables researchers to study mechanical memory in cardiac cells.

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5 minutes

This research was partly supported by the Delaware Center for Musculoskeletal Research COBRE (P20 GM139760), with grants from the National Institute of General Medical Science from the National Institutes of Health and the State of Delaware. We would like to express our sincere gratitude to Sagar Doshi from the Center for Composite Materials (CCM) and Ashika Singh from the Dhong lab at the University of Delaware for their invaluable support in allowing us to use their laser confocal microscopes and for providing the expertise necessary for the surface characterization of our materials. Their contribution significantly enhanced the quality of our work.

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&#39;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.
<ol>
	<li>Prepare 10 g of 10:1 (base: curing agent) 184 PDMS.</li>
	<li>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.</li>
	<li>Allow 184 PDMS to partially cure in an oven at 60 &#176;C for 30 min.</li>
	<li>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.</li>
	<li>Remove the 35 mm dish with the partially cured 184 PDMS from the oven.</li>
	<li>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.</li>
	<li>Lightly press the diffraction grating until a small amount of 184 PDMS surrounds the grating.</li>
	<li>Use the remaining uncured 184 PDMS to backfill the dish surrounding the grating to ensure it stays in place while curing.</li>
	<li>Place the dish containing the diffraction grating into the oven set at 60 &#176;C for 1.5 h.</li>
	<li>After the 184 PDMS is fully cured, remove the 35 mm dish with the diffraction grating from the oven.</li>
	<li>Score around the diffraction grating with a scalpel.</li>
	<li>Apply a small amount of isopropyl alcohol (IPA) to penetrate underneath the diffraction grating.</li>
	<li>Pull the grating off the 184 PDMS in the direction parallel to the patterns.</li>
	<li>Remove excess 184 PDMS, leaving only the patterned section.</li>
	<li>Cut the 184 PDMS stamp to the size needed. 
	NOTE: This study uses a 1 cm&#160;&#215; 1 cm stamp for the devices.</li>
</ol>
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.
<ol>
	<li>Silane treatment (preferred method)
	<ol>
		<li>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.</li>
		<li>Place 184 PDMS stamps into a desiccator in a fume hood.</li>
		<li>Tear off the lid of a microcentrifuge tube.</li>
		<li>Place the microcentrifuge tube lid next to the 184 PDMS stamps in the desiccator.</li>
		<li>Add 20 &#181;L 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.</li>
		<li>Close the desiccator and pull a vacuum.</li>
		<li>Allow the silane to coat the stamps from 1 h to overnight.</li>
	</ol>
	</li>
</ol>
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&#39;s stiffness can be finely tuned to meet specific requirements.
<ol>
	<li>Softer dynamic range MRE preparation (see Table 1 for example weights)
	<ol>
		<li>Measure the desired amount of silicone thinner and Eco elastomer part B and mix at 2500 rpm in a speed mixer for 1 min.</li>
		<li>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.</li>
		<li>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 &#176;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.</li>
		<li>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.</li>
		<li>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 &#176;C for another 25 min.</li>
		<li>Once fully cured, use a scalpel to score the MRE at the bottom of the stamp, perpendicular to the patterns.</li>
		<li>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.</li>
		<li>Using forceps, pull the stamp off the MRE in the direction parallel to the patterns.</li>
	</ol>
	</li>
	<li>Stiffer dynamic range MRE preparation (see table for example weights)
	<ol>
		<li>Prepare 30:1 184 PDMS. 
		NOTE: Make extra because some will remain stuck to the mixing container.</li>
		<li>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.&#160;</li>
		<li>Mix it at 2500 rpm in a speed mixer for 1 min.</li>
		<li>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 &#176;C for ~40 min.</li>
		<li>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.</li>
		<li>Remove the MRE from the oven.</li>
		<li>Use a pair of forceps to carefully move the coated stamp, flip it face down onto the partially cured MRE, and lightly press.</li>
		<li>Put the MRE with the coated stamp on the top rack into the oven at 60 &#176;C overnight.</li>
		<li>Once fully cured, use a scalpel to cut the MRE at the bottom of the stamp, perpendicular to the patterns.</li>
		<li>Apply IPA onto the cut area around the stamp. 
		NOTE: The IPA should seep into the cut and start to move underneath the stamp.</li>
		<li>Using forceps, pull the stamp off the MRE in the direction parallel to the patterns.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Stiffness Range</td>
			<td>Materials</td>
			<td colspan="2">Example Weight</td>
		</tr>
		<tr>
			<td rowspan="3">Softer</td>
			<td rowspan="3">10&#8211;80 kPa</td>
			<td>Eco (A+B)</td>
			<td>11.25 g (A)</td>
			<td>11.25 g (B)</td>
		</tr>
		<tr>
			<td>Silicone Thinner</td>
			<td colspan="2">22.5 g</td>
		</tr>
		<tr>
			<td>Iron Particles</td>
			<td colspan="2">45 g</td>
		</tr>
		<tr>
			<td rowspan="3">Stiffer</td>
			<td rowspan="3">60&#8211;120 kPa</td>
			<td>527 PDMS (A+B)</td>
			<td>18.75 g (A)</td>
			<td>18.75 g (B)</td>
		</tr>
		<tr>
			<td>Iron Particles</td>
			<td colspan="2">37.5 g</td>
		</tr>
		<tr>
			<td>184 PDMS (Base+Cure)</td>
			<td>14.5 g (Base)</td>
			<td>0.5 g (Cure)</td>
		</tr>
	</tbody>
</table>
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 &#176;C with controlled CO2). Regular monitoring, medium changes, and passaging are performed to maintain healthy cell growth.
<ol>
	<li>Sterilize the surface of the MRE by washing it with 70% ethanol 3 times and allow the third wash to sit for 20 min.</li>
	<li>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.</li>
	<li>Coat the surface of the MRE with fibronectin (10 &#181;g/mL) in PBS for 1 h at 37 &#176;C to promote cell adhesion to the device.</li>
	<li>Wash the MRE surface 3 times with PBS, similar to step 4.1.2.</li>
	<li>Seed the neonatal rat cardiomyocytes at a density of 20,000 cells/cm2.</li>
	<li>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.</li>
</ol>
5. Staining and imaging
<ol>
	<li>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.
	<ol>
		<li>Wash once with 1x PBS by adding enough volume to cover the dish completely, followed by aspirating off all liquid.</li>
		<li>Fix cells by using 4% (v/v) paraformaldehyde for 10 min.</li>
		<li>Wash three times with 1x PBS (similar to step 5.1.1).</li>
		<li>Add 0.1% Triton X (in 1x PBS) and incubate for 5 min to permeabilize.</li>
		<li>Wash three times with 1x PBS (similar to step 5.1.1).</li>
		<li>Add NH4Cl (50 mM in 1x PBS) and incubate for 15 min to quench.</li>
		<li>Wash three times with 1x PBS (similar to step 5.1.1).</li>
		<li>Add blocking buffer (5% BSA in 1x PBS) and incubate for 1 h at room temperature (RT).</li>
		<li>Combine the primary Anti-&#945;-Actinin primary antibody at 1:200 into 5% BSA (in 1x PBS). 
		NOTE: Mix the antibody thoroughly.</li>
		<li>Add the primary antibody solution to the sample. 
		NOTE: Add enough to cover the entire sample.</li>
		<li>Incubate overnight at 4 &#176;C. 
		NOTE: Consider placing the sample on the rocker at a slow speed to help the solutions mix.</li>
		<li>Wash three times with 1x PBS (similar to step 5.1.1).</li>
		<li>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.</li>
		<li>Add the secondary antibody solution to the sample.</li>
		<li>Incubate at RT for 2 h.</li>
		<li>Wash three times with 1x PBS (similar to step 5.1.1).</li>
		<li>Add DAPI (1 &#181;g/mL in sterilized de-ionized water) and incubate for 5-10 min in the dark.</li>
		<li>Image the cells with an upright microscope with a 20x dip-in lens.</li>
		<li>Take 50 images per device.</li>
		<li>Process the images using an ImageJ plugin called Directionality. 
		NOTE: Directionality options: This study used n = 45 bins, ranging from 0&#176; to 176&#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).</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Incubate the cells with SiR-actin 1:1000 ratio with culture media for 1 h at 37 &#176;C.</li>
		<li>Aspirate the media and replace it with 1:2000 (SiR-actin: Media). 
		NOTE: This dilution is to reduce cell toxicity for extended live imaging.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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<li>Nguyen, A. T., Sathe, S. R., Yim, E. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+nano+to+micro%3A+topographical+scale+and+its+impact+on+cell+adhesion%2C+morphology+and+contact+guidance%5BTitle%5D)+AND+%22J+Phys+Condens+Matter%22%5BJournal%5D)">From nano to micro: topographical scale and its impact on cell adhesion, morphology and contact guidance.</a> J Phys Condens Matter. 28 (18), 183001(2016).</li>
<li>DowellMesfin, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topographically+modified+surfaces+affect+orientation+and+growth+of+hippocampal+neurons%5BTitle%5D)+AND+%22J+Neural+Eng%22%5BJournal%5D)">Topographically modified surfaces affect orientation and growth of hippocampal neurons.</a> J Neural Eng. 1 (2), 78-90 (2004).</li>
<li>Loesberg, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+threshold+at+which+substrate+nanogroove+dimensions+may+influence+fibroblast+alignment+and+adhesion%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion.</a> Biomaterials. 28 (27), 3944-3951 (2007).</li>
<li>Corbin, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tunable+and+reversible+substrate+stiffness+reveals+a+dynamic+mechanosensitivity+of+cardiomyocytes%5BTitle%5D)+AND+%22ACS+Appl+Mater+Interfaces%22%5BJournal%5D)">Tunable and reversible substrate stiffness reveals a dynamic mechanosensitivity of cardiomyocytes.</a> ACS Appl Mater Interfaces. 11 (23), 20603-20614 (2019).</li>
<li>Bouhrira, N., Vite, A., Margulies, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+cytoskeletal+regulators+of+mechanical+memory+in+cardiac+fibroblasts+and+cardiomyocytes%5BTitle%5D)+AND+%22Basic+Res+Cardiol%22%5BJournal%5D)">Distinct cytoskeletal regulators of mechanical memory in cardiac fibroblasts and cardiomyocytes.</a> Basic Res Cardiol. 119 (2), 277-289 (2024).</li>
<li>Lee, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+human+cardiomyocyte+mechanics+in+osteogenesis+imperfecta%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">Adult human cardiomyocyte mechanics in osteogenesis imperfecta.</a> Am J Physiol Heart Circ Physiol. 325 (4), H814-H821 (2023).</li>
<li>Cao, Z., Ball, J. K., Lateef, A. H., Virgile, C. P., Corbin, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomimetic+substrate+to+probe+dynamic+interplay+of+topography+and+stiffness+on+cardiac+fibroblast+activation%5BTitle%5D)+AND+%22ACS+Omega%22%5BJournal%5D)">Biomimetic substrate to probe dynamic interplay of topography and stiffness on cardiac fibroblast activation.</a> ACS Omega. 8 (6), 5406-5414 (2023).</li>
<li>Clark, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetic+field+tuning+of+mechanical+properties+of+ultrasoft+PDMSbased+magnetorheological+elastomers+for+biological+applications%5BTitle%5D)+AND+%22Multifunct+Mater%22%5BJournal%5D)">Magnetic field tuning of mechanical properties of ultrasoft PDMSbased magnetorheological elastomers for biological applications.</a> Multifunct Mater. 4 (3), 035001(2021).</li>
<li>Kim, Y., Kwon, C., Jeon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+engineered+phage+induced+selective+H9c2+cardiomyocytes+patterning+in+PDMS+microgrooves%5BTitle%5D)+AND+%22Materials%22%5BJournal%5D)">Genetically engineered phage induced selective H9c2 cardiomyocytes patterning in PDMS microgrooves.</a> Materials. 10 (8), 973(2017).</li>
<li>Fujiwara, Y., Deguchi, K., Miki, K., Nishimoto, T., Yoshida, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+contraction+force+measurement+of+hiPSCderived+engineered+cardiac+tissues%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">A method for contraction force measurement of hiPSCderived engineered cardiac tissues.</a> Methods Mol Biol. 2320, 171-180 (2021).</li>
<li>Dirar, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+and+degranulation+of+CART+cells+using+engineered+antigenpresenting+cell+surfaces%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Activation and degranulation of CART cells using engineered antigenpresenting cell surfaces.</a> PLoS One. 15 (9), e0238819(2020).</li>
<li>Kim, C., Kim, H., Park, H., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlling+the+porous+structure+of+alginate+ferrogel+for+anticancer+drug+delivery+under+magnetic+stimulation%5BTitle%5D)+AND+%22Carbohydr+Polym%22%5BJournal%5D)">Controlling the porous structure of alginate ferrogel for anticancer drug delivery under magnetic stimulation.</a> Carbohydr Polym. 223, 115045(2019).</li>
<li>Fan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+of+magnetic+nanomaterials+in+bone+tissue+repair%5BTitle%5D)+AND+%22Front+Chem%22%5BJournal%5D)">Recent advances of magnetic nanomaterials in bone tissue repair.</a> Front Chem. 8, 745(2020).</li>
<li>GonzalezRico, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tuning+the+cell+and+biological+tissue+environment+through+magnetoactive+materials%5BTitle%5D)+AND+%22Appl+Sci%22%5BJournal%5D)">Tuning the cell and biological tissue environment through magnetoactive materials.</a> Appl Sci. 11 (18), 8746(2021).</li>
<li>AntmanPassig, M., Shefi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remote+magnetic+orientation+of+3D+collagen+hydrogels+for+directed+neuronal+regeneration%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Remote magnetic orientation of 3D collagen hydrogels for directed neuronal regeneration.</a> Nano Lett. 16 (4), 2567-2573 (2016).</li>
</ol>

The authors declare no conflicts of interest.

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 sizes33,35. Some commercially available PDMS materials come pre-pa...

The heart&#39;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 stiffness1,2, applying mechanical strain3, utilizing contact guidance4,5, or employing electrical pacing6,7.
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 bone8,9,10. 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 function11,12,13,14. Changes in stiffness have also been linked to disease states and the progression of specific pathologies15,16,17,18. 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 contraction1,19, 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 tissues22,23. Similar to other mechanical regulators, topography is important to cell morphology, such as spreading, elongation, alignment, motility, differentiation, and apoptosis24,25,26. With the introduction of synthetic culture substrates, methods have been developed to create controlled surface topography for cell and tissue culture27,28,29. 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 field17,30,31,32. 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.

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

micropatterned magneto-rheological elastomers to drive changes in cardiomyocyte alignment

Micropatterned Magneto-Rheological Elastomers to Drive Changes in Cardiomyocyte Alignment

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.

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...

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.

Substrate-associated cues, such as mechanical and topographic, profoundly influence cellular response. However, much of the foundational research employs ...

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.

micropatterned-magneto-rheological-elastomers-to-drive-changes

Research

JoVE Journal

Bioengineering

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography.  The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capillary Force Lithography for Cardiac Tissue Engineering

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Bioinspired Soft Robot with Incorporated Microelectrodes

This protocol provides detailed methods describing the fabrication and implementation of a magnetics-based afterload tuning platform for engineered heart tissues.

Magnetic Adjustment of Afterload in Engineered Heart Tissues

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Learn how micropatterned magneto-rheological elastomers drive cardiomyocyte alignment changes in response to dynamic stiffness. This method enables researchers to study mechanical memory in cardiac cells.

Micropatterned Magneto-Rheological Elastomers to Drive Changes in Cardiomyocyte Alignment

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