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This work was supported by funds from CIHR Research Excellence, Diversity, and Independence Early Career Transition Award 202305ED8-509143-244657 (to F.B.K.) and the National Institute of Health Cancer Institute R35 CA242447-01A1 to V.M.W. K.J. is the Lois A. Cinelli Fellow of the Damon Runyon Cancer Research Foundation (DRG-2547-25).

1. Generation of PA-based ECM hydrogels
<ol>
	<li>Functionalization of glass surfaces for PA gel attachment 
	NOTE: Perform all steps at room temperature on an orbital shaker. Rinse thoroughly with high-quality water between steps.
	<ol>
		<li>Clean the glass surfaces with 10% bleach for 3 h in a chemical-resistant dish (e.g., polystyrene). Use sufficient volume to fully cover the glass and ensure there are no trapped bubbles or dry areas. 
		CAUTION: Bleach is a highly corrosive and reactive chemical. Follow the manufacturer&#39;s safety instructions.</li>
		<li>Incubate the coverslips in 0.2 M hydrochloric acid (HCl) for 16 h with gentle agitation on a shaker. 
		CAUTION: HCl is highly corrosive and can cause skin burns. Wear appropriate PPE.</li>
		<li>Add 0.1 M NaOH and incubate for 1 h. 
		CAUTION: NaOH solutions are corrosive and can cause skin burns. Wear appropriate PPE.</li>
		<li>Incubate the coverslips in 0.5% APTES in high-quality water for 30 min. 
		CAUTION: APTES is toxic. Work with APTES in a fume hood with appropriate PPE.</li>
		<li>Rinse coverslips with a large volume of water to remove any residual APTES. 
		NOTE: Incomplete washing will cause a reaction between APTES and glutaraldehyde in the next step and lead to white precipitates that will compromise glass surface activation.</li>
		<li>Incubate the coverslips in 0.5% glutaraldehyde in PBS for 1 h. 
		CAUTION: Glutaraldehyde is toxic. Work with glutaraldehyde in a fume hood with appropriate PPE.</li>
		<li>Dry the coverslips on absorbent material (e.g., paper towels) overnight. Store the coverslips in a clean, sealed container until use.</li>
	</ol>
	</li>
	<li>Preparation of PA hydrogel solution and gel casting 
	NOTE: The formulations needed to obtain hydrogels with the specified elastic modulus are provided in Table 1. Recommended gel volumes and glass dimensions for different experimental applications are presented in Table 3.
	<ol>
		<li>Mix PA hydrogel components in a 1.7 mL microcentrifuge tube, excluding ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). 
		NOTE: Do not prepare large volumes because higher stiffness gels polymerize rapidly and may harden before casting is complete.</li>
		<li>Degas the PA gel mixture, APS, and TEMED tubes in a vacuum chamber at 25 mmHg for 30 min.</li>
		<li>Prepare the hydrophobic top coverslips by applying water-repellent coating to clean coverslips using a cotton swab. Ensure coverslips are free of smudges to maintain consistent gel polymerization.</li>
		<li>Add APS and TEMED to the degassed solution, then gently pipette the mixture onto the activated coverslips.</li>
		<li>Place the hydrophobic coverslips on top of the solution to create a uniform thin hydrogel layer. Allow the gels to polymerize for 35 min at room temperature. Ensure that no bubbles are trapped between the coverslips and that the solution does not spill out over the edges. 
		NOTE: Follow recommended volumes for gel casting to ensure proper thickness; excessively thin gels allow cells to sense the underlying glass15. Before disassembling the gel sandwich, check the residual mixture in the microcentrifuge tube to confirm polymerization.</li>
		<li>Submerge the hydrogel sandwich in PBS to separate the layers. Ensure that the hydrogel remains attached to the activated bottom coverslip, and the hydrophobic top coverslip detaches easily.</li>
	</ol>
	</li>
	<li>Functionalization of hydrogels with ECM ligands 
	NOTE: All steps in this section must be performed under sterile conditions inside a biosafety cabinet.
	<ol>
		<li>Sterilize the hydrogels by immersing them in 70% ethanol, then transfer them to tissue culture plasticware with sterile PBS. 
		NOTE: Do not leave hydrogels in ethanol for an extended period as this will dehydrate the gels and compromise ECM conjugation.</li>
		<li>Prepare a master mix functionalization solution composed of water, ethanol, HEPES, and bisacrylamide (Table 4). 
		CAUTION: Bisacrylamide is toxic upon inhalation or ingestion.</li>
		<li>Prepare diluted solutions of UV photoinitiator and tetraacrylate. Both reagents are dissolved in ethanol (Table 4). 
		NOTE: Perform serial dilutions of tetraacrylate each time as excessive amounts may lead to over-polymerization and visible black surface deposits (see Figure 3A).</li>
		<li>Weigh the required amount of NHS-acrylate using an analytical balance. Resuspend NHS-acrylate in 50% ethanol/50% water immediately prior to use. 
		NOTE: Excessive NHS-acrylate amount will result in the formation of visible black surface deposits and may lead to uneven ECM coating (Figure 3B).</li>
		<li>Add UV photoinitiator, tetraacrylate, and NHS-acrylate solutions to the master mix prepared in Step 1.3.2. Aspirate the PBS and add the master mix to the gels, ensuring that they are fully covered.</li>
		<li>Expose the hydrogels (without tissue culture dish lids) to UV light for 400 s (UV light source with a wavelength less than 365 nm). 
		CAUTION: Wear appropriate eye protection when working with UV light.</li>
		<li>Remove the gels and place them on ice. Aspirate the functionalization solution, then wash the gels three times with prechilled sterile 25 mM HEPES (pH 6.0), followed by three washes with prechilled sterile 0.9% NaCl.</li>
		<li>Incubate the hydrogels with the ECM ligand of choice in prechilled sterile 100 mM HEPES (pH 8.0) with 100 mM NaCl overnight at 4 &#176;C on an orbital shaker. 
		NOTE: The concentration of ECM ligands may be selected at the user&#8217;s discretion; however, it is critical to maintain identical ligand concentrations across all substrates of varying stiffness within the same batch.</li>
		<li>Carefully aspirate the ECM ligand solution 16 h later. Wash the hydrogels three times&#160;with PBS and add serum-free base media (or PBS) to keep them hydrated. Incubate the gels overnight in a tissue culture incubator to verify sterility prior to cell seeding. 
		NOTE: The media will appear cloudy after overnight incubation at 37 &#176;C in base culture media if contamination is present.</li>
		<li>Use the gels immediately for cell seeding or store them at 4 &#176;C in base medium for no more than 3 weeks.</li>
	</ol>
	</li>
</ol>
2. Generation of mechanically tunable silicone-based ECM substrates
<ol>
	<li>Preparation of glass coverslips for silicone gel coating
	<ol>
		<li>Clean glass coverslips with 3 M NaOH for 20 min.</li>
		<li>Wash thoroughly with high-quality water five times and aspirate the water at the end. Allow them to air dry and use a nitrogen air jet to remove any remaining droplets or debris.</li>
	</ol>
	</li>
	<li>Preparation of silicone gel
	<ol>
		<li>Weigh Component A and Component B of the two-component silicone elastomer according to the weight ratio specified in Table 2, using an analytical balance. Use a cut pipette tip to aspirate the viscous solutions.</li>
		<li>Mix Component A and Component B thoroughly using a pipette tip to ensure complete incorporation.</li>
		<li>Degas the mixture in a vacuum chamber at 25 mmHg for 10 min.</li>
	</ol>
	</li>
	<li>Gel casting onto glass coverslips
	<ol>
		<li>Place a coverslip onto a rubber mat positioned on the spin coater, ensuring it is well-centered. 
		NOTE: The rubber mat ensures a proper vacuum seal. Without an adequate seal, the coverslip may be displaced and break. Improper centering may result in non-uniform coating.</li>
		<li>Engage the vacuum to secure the coverslip.</li>
		<li>Add 100 &#181;L of the gel mixture to the center of the coverslip. Spin coat at 500 rpm for 10 s, followed by 8,000 rpm for 60 s.</li>
		<li>Bake the coated coverslip at 80 &#176;C for 2 h. Allow it to cool to room temperature. 
		NOTE: Store at room temperature for up to one month before use.</li>
	</ol>
	</li>
	<li>Functionalization of the silicone gel with ECM ligands
	<ol>
		<li>Expose the silicone gel-coated coverslips to oxygen plasma for 5 min at 11 W power. 
		NOTE: Oxygen plasma exposure for a longer time or a higher power may lead to cracking of the gels. The optimal combination of power and time may need to be tested depending on the equipment used.</li>
		<li>Prepare a silanization solution containing 95% ethanol, 0.5% APTES, and 4.5% dH2O. 
		NOTE: Perform all subsequent steps under sterile conditions inside a biosafety cabinet. 
		CAUTION: APTES is toxic. Work with APTES in a fume hood with appropriate PPE.</li>
		<li>Incubate the gel-coated coverslip with the silanization solution for 10 min.</li>
		<li>Wash thoroughly with PBS five times.</li>
		<li>Prepare the ECM solution containing 100 &#181;g/mL EDC and the ECM of choice at the desired concentration in PBS. 
		NOTE: EDC is unstable in solution and sensitive to moisture. Prepare it fresh prior to use and store it tightly closed in a cool, dry place, protected from moisture and direct sunlight. 
		CAUTION: EDC is harmful if swallowed and toxic if inhaled. Wear appropriate PPE and work in a fume hood.</li>
		<li>Incubate the coverslip with the ECM solution for 1 h at 37 &#176;C or overnight at 4 &#176;C.</li>
		<li>Wash the coverslip with PBS five times. Rinse with base media and add cells. 
		NOTE: Optimal cell density depends on the specific cell line and experimental goals (e.g., subconfluent for morphology or monolayer for cell-cell adhesion). For example, a density of 0.1 x 106 cells/cm2 is commonly used for fibroblasts to achieve 50% confluency within 48 h.</li>
	</ol>
	</li>
</ol>
3. Preparation of ECM substrate for immunostaining and imaging
<ol>
	<li>Immunostaining and imaging on PA-based substrates 
	NOTE: Eighteen-millimeter PA hydrogels generally provide sufficient imageable area for quantitative analysis. For imaging applications, cast the hydrogels and perform immunostaining in &#8220;orange chambers&#8221; prepared from 15 mL tubes. Reuse the orange chambers for multiple rounds of gel casting and staining, provided it retains structural integrity.
	<ol>
		<li>Punch rings from polycarbonate film and silicone rubber film using 18 mm outer diameter and 14 mm inner hole punches to prepare spacers and gaskets.</li>
		<li>Cut an opening in the center of a 15 mL centrifuge tube cap using scissors to create a chamber for gel casting and staining.</li>
		<li>Saw the centrifuge tube at the 14 mL mark. Trim uneven edges with a scalpel and file the rim of the cap to ensure it fits into a standard 12-well plate.</li>
		<li>Assemble the hydrogel-coverslip sandwich (as described in Section 1), placing the spacer ring onto the activated coverslip.</li>
		<li>Disassemble the sandwich once the gels are fully polymerized.</li>
		<li>Sterilize the 18-mm hydrogels and transfer them to a 12-well tissue culture plate with sterile PBS.</li>
		<li>Perform ECM ligand functionalization as described in section&#160;1.</li>
		<li>Seed cells and fix them with 4% formaldehyde. 
		NOTE: Avoid alcohol-based fixatives, as they dehydrate the gels and compromise structural integrity. 
		CAUTION: Formaldehyde is toxic and must always be used inside a fume hood.</li>
		<li>(Optional) Reassemble the samples onto the centrifuge tube cap chamber. Use a silicone gasket to seal the chamber. 
		NOTE: Perform a leak test with PBS for several hours before staining. Although this method may appear more laborious, it promotes uniform staining and reduces antibody consumption.</li>
		<li>Proceed with immunofluorescence staining according to user-preferred protocols.</li>
		<li>After staining, store samples in PBS supplemented with 50% glycerol. Image the samples as soon as possible. 
		NOTE: Avoid standard mounting media that harden, as they can crack hydrogels over time and damage samples. Fluorescence intensity diminishes over time; image within 1 week for optimal signal quality.</li>
		<li>Place a small drop of PBS (or mounting medium) onto the glass-bottom surface of a tissue culture dish. Carefully invert the stained hydrogels onto the drop so that the cells face the cover glass and proceed with imaging.</li>
	</ol>
	</li>
	<li>Immunostaining and imaging on silicone-based ECM substrates
	<ol>
		<li>Fix the samples using 4% formaldehyde and stain them directly on a glass-bottom dish using a standard immunofluorescence protocol. Avoid alcohol-based fixatives as they will crack the ECM substrates.</li>
		<li>Image samples directly using a traditional fluorescence microscope.</li>
	</ol>
	</li>
</ol>
4. Harvesting proteins and RNA from cells plated on ECM substrates
<ol>
	<li>Using forceps, carefully remove the gels from the culture dish and submerge them in sterile PBS to rinse away residual culture media and serum. 
	NOTE: Handle the coverslips gently, as they are extremely fragile and can break easily.</li>
	<li>Gently dab the edge of the hydrogel on low-lint wipes to remove excess PBS.</li>
	<li>Add an appropriate volume of RNA extraction buffer to the lid of a tissue culture dish. 
	NOTE: Phenol-guanidine-based reagents are compatible with RNA extraction from PA gels. Protein lysis buffers may be used according to experimental requirements. Visualize samples under a microscope following lysis buffer incubation to ensure complete cell disruption.</li>
	<li>Place the coverslip (gel side down) onto the lysis solution. Carefully lift and lower the hydrogel several times to ensure uniform lysis of all cells.</li>
	<li>Gently scrape the gel surface to release the lysate. 
	&#8203;NOTE: Do not use commercial plastic cell scrapers, as the rigid plastic may damage the soft hydrogels and contaminate the lysate with gel chunks. The soft plastic used to make spacers (see Step 3.1.1) is suitable for this purpose and can be repurposed to make custom scrapers.</li>
	<li>Store the RNA or protein lysates at -80 &#176;C until further processing using conventional biochemical methods or use them immediately.</li>
</ol>
5. Generation of single-plane spheroid culture
<ol>
	<li>Prepare the 75 Pa PA hydrogel on 18-mm coverslips and functionalize the surface with basement membrane extract (BME) or another appropriate ECM ligand to facilitate cell attachment.</li>
	<li>Seed cells onto the ECM-coated hydrogels or silicone gels at the desired density.</li>
	<li>After 2 h, gently remove unattached cells and add a BME overlay diluted in prechilled basal medium. 
	NOTE: BME polymerizes as it warms up. Prechill tips prior to pipetting. The percentage of BME incorporated is determined by the desired mechanical integrity and stability of the resulting 3D scaffold.</li>
	<li>Incubate the cultures at 37 &#176;C in a tissue-culture incubator for 1.5 h to allow BME polymerization.</li>
	<li>Gently add cell culture medium to fully cover the 3D gels.</li>
	<li>Maintain cultures under standard conditions. Cells will continue to grow and self-assemble into 3D spheroids over time until the desired experimental endpoint is reached.</li>
</ol>

<ol>
	<li>Guimar&#227;es CF, Gasperini L, Marques AP, Reis RL. The stiffness of living tissues and its implications for tissue engineering. Nat Rev Mater. 2020;5(5):351-70.</li>
	<li>Butcher DT, Alliston T, Weaver VM. A tense situation: forcing tumour progression. Nat Rev Cancer. 2009;9(2):108-22.</li>
	<li>Barnes JM, Przybyla L, Weaver VM. Tissue mechanics regulate brain development, homeostasis and disease. J Cell Sci. 2017;130(1):71-82.</li>
	<li>Kai FB, Laklai H, Weaver VM. Force matters: biomechanical regulation of cell invasion and migration in disease. Trends Cell Biol. 2016;26(7):486-97.</li>
	<li>Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science. 2005;310(5751):1139-43.</li>
	<li>Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677-89.</li>
	<li>Miroshnikova YA, et al. Tissue mechanics promote IDH1-dependent HIF1&#945;-tenascin C feedback to regulate glioblastoma aggression. Nat Cell Biol. 2016;18(12):1336-45.</li>
	<li>Mekhdjian AH, et al. Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix. Mol Biol Cell. 2017;28(11):1467-88.</li>
	<li>Northey JJ, et al. Mechanosensitive hormone signaling promotes mammary progenitor expansion and breast cancer risk. Cell Stem Cell. 2024;31(1):106-26.e13.</li>
	<li>Kai FB, et al. ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells. EMBO J. 2022;41(17):e109205.</li>
	<li>Kai FB, Leidal AM, Weaver VM. Tension-induced organelle stress: an emerging target in fibrosis. Trends Pharmacol Sci. 2025;46(2):117-31.</li>
	<li>Mei Y, et al. Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells. Nat Mater. 2010;9(9):768-78.</li>
	<li>Lakins JN, Chin AR, Weaver VM. Exploring the link between human embryonic stem cell organization and fate using tension-calibrated extracellular matrix functionalized polyacrylamide gels. Methods Mol Biol. 2012;916:317-50.</li>
	<li>Ou G, et al. Visualizing mechanical modulation of nanoscale organization of cell-matrix adhesions. Integr Biol (Camb). 2016;8(7):795-804.</li>
	<li>Buxboim A, Rajagopal K, Brown AE, Discher DE. How deeply cells feel: methods for thin gels. J Phys Condens Matter. 2010;22(19):194116.</li>
	<li>Liu F, et al. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression. J Cell Biol. 2010;190(4):693-706.</li>
	<li>Paszek MJ, et al. Tensional homeostasis and the malignant phenotype. Cancer Cell. 2005;8(3):241-54.</li>
	<li>Cosgrove BD, et al. Mechanosensitive genomic enhancers potentiate the cellular response to matrix stiffness. Science. 2025;390(6778):eadl1988.</li>
	<li>Qin Z, et al. Matrix stiffness of GelMA hydrogels regulates lymphatic endothelial cells toward enhanced lymphangiogenesis. ACS Appl Mater Interfaces. 2024;16(41):55130-41.</li>
	<li>Greaney AJ, et al. A comprehensive protocol for PDMS fabrication for use in cell culture. PLoS One. 2025;20(5):e0323283.</li>
	<li>Muncie JM, et al. Patterning the geometry of human embryonic stem cell colonies on compliant substrates to control tissue-level mechanics. J Vis Exp. 2019;(151):e60334.</li>
	<li>Kai FB, Drain AP, Weaver VM. The extracellular matrix modulates the metastatic journey. Dev Cell. 2019;49(3):332-46.</li>
	<li>Bonneh-Barkay D, Wiley CA. Brain extracellular matrix in neurodegeneration. Brain Pathol. 2009;19(4):573-85.</li>
</ol>

The authors have no conflicts of interest to disclose.

The outlined ECM fabrication protocol provides a reproducible, scalable, and cost-effective workflow and can easily be implemented in most labs. Although traditional glass and plastic culture ware is convenient and enables rapid data collection, the synthetic substrates fail to replicate physiologically relevant tissue stiffness. Alternative mechanically tunable substrates such as polydimethylsiloxane (PDMS) or gelatin methacryloyl (GelMA) are easier to fabricate, but they generally cannot achieve stiffness values in the...

The extracellular matrix (ECM) is a fundamental structural and biochemical scaffold that defines the mechanical properties of tissues and supports their biological functions. For instance, elastic tissues such as the lungs and arteries are enriched in collagen- and elastin-based ECM that provides the mechanical resilience required for repeated stretch-recoil cycles during respiration. In contrast, the central nervous system contains glycosaminoglycan-rich ECM that creates a highly compliant environment optimized for neuronal differentiation and signaling1. Across diverse tissues within the body, tissue stiffness spans several orders of magnitude, ranging from hundreds of pascals in soft tissues like the brain and lungs to megapascal and gigapascal levels of stiffness in tendons and bone, respectively2,3. These mechanical differences are not merely structural but also play a critical role in regulating cell behavior4,5. For example, mesenchymal stem cells undergo stiffness-dependent lineage specification, adopting neuronal fates on soft substrates, myogenic fates on intermediate substrates, and osteogenic fates on stiff substrates6. Pathological alterations in ECM stiffness can also drive aberrant cellular responses and contribute to disease initiation and progression7,8,9. Similarly, changes in ECM properties around cells also alter organelle function and various cell behaviors10,11. Despite the recognized importance of ECM in guiding signaling, organelle function, and disease pathogenesis, the mechanisms that cells sense and transduce ECM cues into biochemical signals remain incompletely understood.
Progress in understanding cell-ECM crosstalk has been hindered in part by the limited availability of experimental platforms that offer precise control over ECM properties while remaining fully compatible with standard biochemical assays and imaging workflows. In addition, studying cell-ECM interactions is often perceived as technically challenging, as it requires cross-disciplinary expertise spanning materials science, cell biology, and bioengineering. This method aims to address these challenges by providing step-by-step protocols for generating PA- and silicone-based ECM substrates with defined mechanical properties, which can be readily prepared in most wet-lab settings.
PA hydrogels with tunable stiffness are staples of mechanobiology; however, their utility is often hampered by high cost of reagents for ECM conjugation, inconsistent surface functionalization, and poor compatibility with large-scale biochemical workflows. In addition, practical and standardized approaches for sample harvesting and imaging across substrates of varying stiffness are not well established. These bottlenecks can be addressed using our described PA gel platform that is characterized by four key innovations: (i) substantial cost reduction, (ii) reproducible functionalization chemistry, (iii) scalable production through reusable hardware, and (iv) seamless integration with many conventional cell biology and biochemical assays. This approach utilizes different ratios of acrylamide and bisacrylamide to generate hydrogels with distinct mechanical properties (Table 1). PA gels are inert and fail to directly conjugate to ECM proteins. Therefore, a UV-initiated free radical polymerization reaction is used to conjugate a thin, crosslinked network of di(trimethylolpropane) tetraacrylate, acrylic acid N-hydroxysuccinimide ester (NHS-acrylate), and bisacrylamide onto the surface of PA gels (Figure 1). This network creates a dual-action adhesive surface for ECM proteins: the NHS-acrylate provides stable covalent anchoring for ECM proteins, while the tetraacrylate facilitates uniform ECM protein adsorption12. In addition, we provide methods demonstrating how to harvest proteins and nucleic acids such as RNA from these substrates in a manner compatible with downstream cell biology analyses such as western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, the resulting compliant PA substrates also support spheroid formation and position spheroids within a consistent focal plane, enabling systematic analysis of 3D cellular behaviors13.
In contrast, the optical limitations of PA hydrogels restrict their use in advanced imaging modalities, particularly total internal reflection fluorescence (TIRF) microscopy14. To address this constraint, we developed mechanically tunable silicone-based ECM substrates optimized for TIRF imaging. These materials exhibit a refractive index closely matched to glass while maintaining tunable stiffness across a broad range (Figure 2A). A thin coat of silicone gel, with varying part ratios, is spin-coated onto a cover glass prior to ECM conjugation (Figure 2B&#160;and Table 2). However, the intrinsic hydrophobicity and chemical inertness of silicone preclude direct ECM conjugation. We therefore implement a surface modification strategy to enable covalent attachment of ECM ligands. Oxygen plasma treatment introduces hydroxyl groups onto the silicone surface (Figure 2C). Subsequently, (3-aminopropyl)triethoxysilane (APTES) converts these hydroxyl groups into reactive amine. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), a zero-length carbodiimide crosslinker, then facilitates the formation of covalent amide bonds between the amine groups on the gel surface and the carboxyl groups on ECM proteins.
Collectively, these ECM platforms provide cost-effective, reproducible, and scalable systems for probing ECM-stiffness-dependent signaling mechanisms. Their flexibility permits seamless integration with biochemical assays, high-resolution imaging, and high-throughput workflows, enabling the investigation of how ECM stiffness influences cellular behavior and drives disease progression.

<table><tbody><tr><td>(3-Aminopropyl)triethoxysilane (APTES)</td><td>919-30-2</td><td>-</td><td>Thermo Fisher Scientific</td></tr><tr><td>1.7 mL microcentrifuge tubes</td><td>-</td><td>LMCT1.7B</td><td>FroggaBio</td></tr><tr><td>12-well tissue culture-treated plates</td><td>-</td><td>83.3921</td><td>Sarstedt</td></tr><tr><td>15 mL centrifuge tubes (for orange chamber)</td><td>-</td><td>CLS430790</td><td>Corning</td></tr><tr><td>2-Hydroxy-4&amp;prime;-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure)</td><td>106797-53-9</td><td>-</td><td>Thermo Fisher Scientific</td></tr><tr><td>2% Bisacrylamide solution</td><td>110-26-9</td><td>-</td><td>Bio-Rad</td></tr><tr><td>3-(3-Dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDC)</td><td>25952-53-8</td><td>-</td><td>Chem Impex International</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride</td><td>28718-90-3</td><td>-</td><td>Sigma</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>-</td><td>D1306</td><td>Invitrogen</td></tr><tr><td>40% Acrylamide solution</td><td>01/06/1979</td><td>-</td><td>Bio-Rad</td></tr><tr><td>50% Glutaraldehyde</td><td>111-30-8</td><td>-</td><td>Thermo Fisher Scientific</td></tr><tr><td>6 cm tissue culture-treated dishes</td><td>-</td><td>83.3901</td><td>Sarstedt</td></tr><tr><td>A549 cells</td><td>-</td><td>CCL-185</td><td>ATCC</td></tr><tr><td>Acrylic acid N-hydroxysuccinimide ester</td><td>38862-24-7</td><td>-</td><td>Thermo Fisher Scientific</td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit antibody</td><td>-</td><td>A11008</td><td>Invitrogen</td></tr><tr><td>Alexa Fluor 594 NHS ester (tris(triethylammonium salt))</td><td>-</td><td>A37572</td><td>Invitrogen</td></tr><tr><td>Ammonium persulfate</td><td>7727-54-0</td><td>-</td><td>Bio-Rad</td></tr><tr><td>Clear impact-resistant polycarbonate film (0.005 in.; spacer)</td><td>-</td><td>85585K72</td><td>McMaster-Carr</td></tr><tr><td>CY 52-276 silicone gel, Components A and B</td><td>2624028</td><td>-</td><td>Dow</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>-</td><td>11995-065</td><td>Thermo Fisher Scientific</td></tr><tr><td>Ethanol, anhydrous</td><td>64-17-5</td><td>-</td><td>Greenfield Canada</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>-</td><td>4385612</td><td>Thermo Fisher Scientific</td></tr><tr><td>Fetal bovine serum</td><td>-</td><td>A52567-01</td><td>Thermo Fisher Scientific</td></tr><tr><td>Fibronectin</td><td>-</td><td>356008</td><td>Corning</td></tr><tr><td>Formaldehyde (16%)</td><td>-</td><td>28908</td><td>Thermo Fisher Scientific</td></tr><tr><td>HEPES</td><td>7365-45-9</td><td>-</td><td>BioShop</td></tr><tr><td>High-temperature silicone rubber sheet (0.020 in.; gasket)</td><td>-</td><td>86435K452</td><td>McMaster-Carr</td></tr><tr><td>Hydrochloric acid</td><td>7647-01-0</td><td>-</td><td>BioShop</td></tr><tr><td>Large hollow hole punch tool set</td><td>-</td><td>66000</td><td>Mayhew Pro</td></tr><tr><td>Matrigel</td><td>-</td><td>356234</td><td>Corning</td></tr><tr><td>Media storage borosilicate glass bottle, 250 mL</td><td>-</td><td>CLS1395250</td><td>Corning</td></tr><tr><td>Media storage borosilicate glass bottle, 500 mL</td><td>-</td><td>CLS1395500</td><td>Corning</td></tr><tr><td>MicroAmp Fast Optical 96-well reaction plate</td><td>-</td><td>4346906</td><td>Thermo Fisher Scientific</td></tr><tr><td>Microscope slide coverslips, 18 mm diameter</td><td>-</td><td>72222-01</td><td>Electron Microscopy Sciences</td></tr><tr><td>Microscope slide coverslips, 50 mm diameter</td><td>-</td><td>1.84E+16</td><td>eBay</td></tr><tr><td>PBS (1&amp;times;)</td><td>-</td><td>14190144</td><td>Thermo Fisher Scientific</td></tr><tr><td>PBS (10&amp;times;)</td><td>-</td><td>70011044</td><td>Thermo Fisher Scientific</td></tr><tr><td>Phalloidin</td><td>17466-45-4</td><td>-</td><td>Biotium</td></tr><tr><td>Phospho-FAK (Tyr397) antibody</td><td>-</td><td>85567</td><td>Cell Signaling Technology</td></tr><tr><td>Phospho-MLC (Ser19) antibody</td><td>-</td><td>3671</td><td>Cell Signaling Technology</td></tr><tr><td>Plasma cleaner</td><td>-</td><td>PDC-32G</td><td>Harrick Plasma</td></tr><tr><td>qPCR primers (forward and reverse)</td><td>-</td><td>N/A</td><td>Integrated DNA Technologies</td></tr><tr><td>qScript cDNA Synthesis Kit</td><td>-</td><td>95047</td><td>QuantaBio</td></tr><tr><td>Rain-X</td><td>-</td><td>-</td><td>Rain-X</td></tr><tr><td>Screw-cap tubes, conical base, 15 mL</td><td>-</td><td>62.554.101</td><td>Sarstedt</td></tr><tr><td>Screw-cap tubes, conical base, 50 mL</td><td>-</td><td>62.547.205</td><td>Sarstedt</td></tr><tr><td>Serological pipette, 10 mL</td><td>-</td><td>170356N</td><td>Thermo Fisher Scientific</td></tr><tr><td>Serological pipette, 25 mL</td><td>-</td><td>170357N</td><td>Thermo Fisher Scientific</td></tr><tr><td>Serological pipette, 5 mL</td><td>-</td><td>170355N</td><td>Thermo Fisher Scientific</td></tr><tr><td>Serological pipette, 50 mL</td><td>-</td><td>170358N</td><td>Thermo Fisher Scientific</td></tr><tr><td>Sodium hydroxide</td><td>1310-73-2</td><td>-</td><td>BioShop</td></tr><tr><td>SYPRO Ruby protein gel stain</td><td>-</td><td>S12000</td><td>Invitrogen</td></tr><tr><td>TEMED</td><td>110-18-9</td><td>-</td><td>Bio-Rad</td></tr><tr><td>TRIzol reagent</td><td>-</td><td>15596026</td><td>Thermo Fisher Scientific</td></tr><tr><td>UV chamber</td><td>-</td><td>165-5031</td><td>Bio-Rad</td></tr></tbody></table>

generation of polyacrylamide and silicone extracellular matrix substrates with defined stiffness for cell biology applications

Generation of Polyacrylamide and Silicone Extracellular Matrix Substrates with Defined Stiffness for Cell Biology Applications

The extracellular matrix (ECM) is a critical regulator of cell behavior, with ECM stiffness serving as a key mechanical cue that governs cellular signaling, morphology, and fate. However, experimental investigation of stiffness-dependent cellular responses is often limited by the availability of reproducible, cost-effective, and accessible culture platforms with precisely tunable mechanical properties. This protocol describes polyacrylamide (PA)- and silicone-based methods for fabricating ECM substrates with tunable stiffness. The procedures outline substrate preparation, surface functionalization, and ECM protein conjugation to ensure consistent cell adhesion across a physiologically relevant stiffness range. PA-based substrates are compatible with downstream biochemical assays, including protein and RNA extraction, as well as high-resolution fluorescence imaging. Silicone-based substrates are optimized for total internal reflection fluorescence (TIRF) microscopy, enabling visualization of cell-ECM interactions at the basal membrane. In addition, soft substrates can be adapted to support spheroid cultures positioned within a consistent imaging plane, facilitating consistent image acquisition. These methods provide robust, cost-effective, and reproducible platforms for systematically probing how ECM stiffness regulates cellular processes, advancing mechanistic insight into cell-ECM crosstalk in both physiological and disease contexts.

PA gel stiffness generated using the formulations in Table 1 was experimentally tested using a rheometer. Gels with nominal stiffness values of 0.4 kPa, 6 kPa, and 60 kPa, spanning three orders of magnitude, were selected. Measured stiffness values were 363 ± 76 Pa for 400 Pa gels, 6.41 ± 0.50 kPa for 6 kPa gels, and 36 ± 8 kPa for 60 kPa gels (Figure 4A). To assess uniform ECM attachment to the PA gels, fluorescently labeled fibronectin was conjugated to the gel surface. Five random fie...

Although extracellular matrix (ECM) stiffness is a key regulator of cellular behavior, the underlying sensing and transduction mechanisms remain unclear. To address this, we provide protocols for creating polyacrylamide- and silicone-based substrates with tunable stiffness to advance our understanding of cell-ECM crosstalk.

The extracellular matrix (ECM) is a critical regulator of cell behavior, with ECM stiffness serving as a key mechanical cue that governs cellular ...

generation-polyacrylamide-silicone-extracellular-matrix-substrates

Research

JoVE Journal

Biology

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

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

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

A protocol for the manufacturing process of polymeric thin film composite structures possessing either different Young's moduli or thicknesses is presented. Films are produced for advanced cell culture studies or as skin adhesives.

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in ...

BioMEMS and Cellular Biology: Perspectives and Applications

University of Calgary.  Although extracellular matrix (ECM) stiffness is a key regulator of cellular behavior, the underlying sensing and transduction mechanisms remain unclear. To address this, we provide protocols for creating polyacrylamide- and silicone-based substrates with tunable stiffness to advance our understanding of cell-ECM crosstalk.

Generation of Polyacrylamide and Silicone Extracellular Matrix Substrates with Defined Stiffness for Cell Biology Applications

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