{"paper_id":"0aaa37ad-b819-46e1-b40e-ba75b4158ac5","body_text":"Adenocarcinoma cell mechanobiology is altered by the loss \nmodulus of the surrounding extracellular matrix \nAriell M. Smith1, Brandon M. Pardi2, Isaac Sousa2, Arvind Gopinath3,4,*, and Roberto Andresen \nEguiluz2,4,* \n1Materials and Biomaterials Science and Engineering Graduate Program, University of \nCalifornia, Merced, Merced, California 95344, United States of America \n2Department of Chemical and Materials Engineering, University of California, Merced, Merced, \nCalifornia 95344, United States of America \n3Department of Bioengineering, University of California, Merced, Merced, California 95344, \nUnited States of America \n4Health Sciences Research Institute, University of California, Merced, Merced, California 95344, \nUnited States of America \nCorresponding authors: \n*randreseneguiluz@ucmerced.edu \n*agopinath@ucmerced.edu \nAbstract  \nElastic and viscoelastic properties of extracellular matrices (ECM) are known to regulate cellular \nbehavior and mechanosensation differently, with implications for morphogenesis, wound \nhealing, and pathophysiology. Most in vitro  cellular processes, including cell migration, are \nstudied on linear-elastic substrates to mimic extracellular matrices. However, most tissues are \nviscoelastic and display a loss modulus ( G/i1/i1 ) that may be 10-20% of their storage modulus \n(G/i1 ) under biophysically relevant conditions. Recent research has shown that cells can \ndistinguish between elastic and viscoelastic ECM, leading to alterations in their cellular \nmorphology, migration rates, and contractility. Here, we present a protocol for creating PAH-\nbased model ECMs that enables the fabrication of viscoelastic substrates with storage moduli \nsimilar to those of their elastic counterparts. To explore how G\n/i1/i1 influences epithelial cell \nmechanobiology, we fabricated tunable viscoelastic model ECMs with G/i1  of 3 kPa, 8 kPa, and \n12 kPa, and for each, independently tuned G/i1/i1  values to approximately 300 Pa, 500 Pa, and \n700 Pa, respectively. We found that A549 cells cultured on stiff elastic model ECMs migrated \n~30% slower and formed larger focal adhesions compared to their viscoelastic counterparts. \nConversely, A549 cells on intermediate viscoelastic model ECMs exhibited a ~54% reduction in \nmigration speed, with no significant difference in focal adhesion size relative to their elastic \ncounterparts. These findings highlight the complex interplay between substrate (ECM) elastic \nand viscoelastic properties in regulating epithelial cell mechanobiology and emphasize the \nimportance of time-dependent matrix mechanics in governing epithelial responses. \n \n \n \nKeywords: Polyacrylamide; Viscoelasticity; Mechanobiology; Storage and loss moduli; \nExtracellular matrix; Cell migration; Focal adhesions. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\n \n1. Introduction  \nThe extracellular matrix (ECM) is a macromolecular scaffold that provides mechanical support \nand structure to cells. 1–4 The mechanical properties of the ECM depend on the concentration \nand ratio of proteins, 1,2 fiber orientation,4 molecular conformation,3 among other factors.5 This is \nevidenced during aging or pathophysiology, conditions in which ECM protein ratios, orientation, \nand confirmation of the molecular constituents change. 3,5,6 These changes lead to significant \nalterations in the mechanical properties of the ECM, 7 including elastic and viscoelastic \nproperties, such as stiffness or relaxation times. 8 Decades of research have established that \nelasticity alone can regulate cellular behavior. For example, adherent cells, such as fibroblasts \n(3T3-Swiss) and epithelial (normal rat kidney) cells, on rigid model EMCs exhibit larger, longer, \nand more mature focal adhesions than on compliant ECMs, leading to larger cytoskeletal size, \nincreased cell spreading area, and increased cell proliferation.\n9–11 Substrate stiffness also \nmediates cell differentiation. For example, mesenchymal stem cells on softer ECMs (stiffness \n0.1-1 kPa, corresponding to, for instance, brain-like tissue) become neurogenic, while those on \nrigid ECMs (stiffness 25-40 kPa, seen in bone-like tissue) turn osteogenic. 12,13 Yet another \nexample is the organization of the human umbilical vascular endothelial (HUVECs) network. In \nthis instance, cells communicate mechanically by applying strain fields to the substrates, \nfacilitating and coordinating the formation of a network. This mechanical signaling has been \ndemonstrated for cells on substrates that are neither too stiff nor too compliant.\n9,14,15 Therefore, \nit is clear that if the mechanical environment is altered, the cues sensed by the cells modify \nmechanotransductive signaling pathways. This is the case for the mechanosensitive signaling \npathway YAP/TAZ, which activates integrins, focal adhesions, the cytoskeleton, and transmits \nsignals to the Hippo pathway, thereby altering cell proliferation, growth,\n16 and fibrosis.17,18 \nSeveral studies in mechanobiology have focused mainly on the elastic properties of the ECM; \nhowever, ECMs are intrinsically viscoelastic. 19,20 Viscoelastic materials exhibit a complex \nresponse to stress or strain, encompassing instantaneous as well as time-dependent \nresponses.21,22 Temporal responses are commonly characterized by relaxation time constants, \nthe loss modulus (encapsulating the non-elastic, dissipative time-dependent responses), and \nthe storage modulus (quantifying elastic, instantaneous responses). Native tissue elastic moduli \nrange from extremely soft (~0.5 kPa, fat tissue)\n23 to very rigid (1-5 GPa, bone tissue); 24 loss \nmoduli meanwhile, depending on tissue type, can vary from 10-20% of the associated elastic \nmoduli under biophysically relevant conditions. 21 It has been shown that model ECMs with \nsimilar elastic moduli but distinct (different) loss moduli affect cells in a cell-line-specific \nmanner.25,26 These include differences in cell spreading, 27–29 cell migration,21,30 polarization, and \ndifferentiation,21,31,32 among others. Typically, alginate or alginate-based gels have been used to \nfabricate a broad range of tunable elastic and viscoelastic model ECMs to investigate cell \nmechanobiology.\n29,33–36 3T3 mouse fibroblast cells seeded on alginate model ECMs with \nYoung’s modulus of 1.4 kPa and varying loss moduli, exhibited larger cell-spreading areas and \nstress fiber formation on viscoelastic model ECMs than on linear elastic model ECMs.\n29 \nConversely, human mesenchymal stem cells seeded on alginate-PEG hydrogels with a Young's \nmodulus of approximately 3 kPa and varying viscoelasticity showed a larger spread area on \nelastic (faster stress relaxation) compared to viscoelastic (slower stress relaxation) model \nECMs.\n28 Cell-specific responses are not unique to cells on alginate substrates. Differences in \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\ncellular responses have been illustrated using elastic and viscoelastic polyacrylamide-based \n(PAH-based) model ECMs. For example, Huh7 and primary human hepatocytes were explored \non elastic ( G/i1  = 5 kPa, G/i1/i1  = 0 Pa) and viscoelastic ( G/i1  = 5 kPa, G /i1/i1  = 600 Pa) \npolyacrylamide hydrogel (PAH) substrates and exhibited opposite mechanobiology responses. \nHuh7 displayed increased cell area, speed, and longer protrusion lengths on viscoelastic model \nECMs. Conversely, hepatocytes displayed decreased cell area and speed on viscoelastic model \nECMs.\n37 \n \nPAHs are another commonly used platform for cell mechanobiology studies, as model ECMs \ncan be readily fabricated across a wide range of physiologically relevant elasticities. To \nintroduce dissipative effects and increase the loss moduli of these elastic model ECMs, linear \nacrylamide chains can be embedded into the elastic PAH network.\n21,32 Using a PAH platform of \ntunable viscoelasticity, it is reported that human mammary epithelial cells (MCF10A) seeded on \nlow elastic modulus (0.3 kPa) viscoelastic model ECMs showed an increase in cell migration \nrate and displayed larger cell area, as opposed to those seeded on higher elastic modulus \nviscoelastic model ECMs.\n30 This contradicts observations on purely elastic model PAH ECMs, \nwhere higher elastic modulus model ECMs lead to a higher cell migration rate and a larger \nprojected cell area compared to their more compliant elastic counterparts. Mechanobiology \nexperiments have also investigated fibroblasts. Fibroblasts (MF3) seeded on substrates of \nsimilar elasticity, but different loss modulus, displayed significant differences in YAP activation \nand subsequent proliferation.\n40 YAP nuclear translocation was higher on elastic substrates in \ncomparison to viscoelastic substrates, and was accompanied  by higher migration speeds on \nviscoelastic as opposed to elastic substrates.\n41 \n \nTaken together, the examples above clearly illustrate  that cells respond differently to elastic and \nviscoelastic model ECMs, including PAH-based substrates. However, most studies in the \nliterature use PAH substrates with relatively low elastic moduli.\n21,30,31,37 Here, we present a \nprotocol for fabricating model ECMs based on PAH, targeting a wider range of viscoelastic \nsubstrates with a fixed storage modulus and tunable loss modulus. Our reported library of \ntunable viscoelastic model ECMs consists of substrates with storage moduli G\n/i1  of 3 kPa (E ≃ 8 \nkPa, referred to as soft), 8 kPa ( E ≃ 25 kPa, referred to as intermediate), and 12 kPa ( E ≃ 32 \nkPa, referred to as stiff). For the viscoelastic substrates, the loss moduli G /i1/i1  were tuned to \nvalues  of ≃  300 Pa, 500 Pa, and 700 Pa, for soft, intermediate, and stiff substrates, \nrespectively.39 Targeted loss moduli values were achieved by embedding 1.8 %  of linear \npolymer polyacrylamide chains into the soft, intermediate, or stiff elastic PAH networks, \nadapting previously published protocols. 21,32 PAHs surfaces were functionalized with collagen \ntype-I to promote cell adhesion. Human lung carcinoma epithelial cells (A549s) were then used \nto investigate how properties such as cell migration, cell area, and cell adhesion differed \nbetween elastic and viscoelastic model ECMs. Overall, we found that the most significant \ndifferences exhibited by A549 cells were between stiff elastic and stiff viscoelastic PAH model \nECMs. Specifically, cells migrated ~30% more slowly on elastic model ECMs than on their \nviscoelastic counterparts. This correlated with larger focal adhesion areas on elastic than on \nviscoelastic model ECMs. On the other hand, cells migrated at similar rates on soft elastic and \nsoft viscoelastic model ECMs, but focal adhesion areas were around 63% smaller on soft elastic \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\ncompared to soft viscoelastic. Overall, our findings elucidate the intricate interplay between \nelastic and viscoelastic properties in regulating epithelial cell mechanobiology, underscoring the \nsignificance of time-dependent matrix mechanics in governing epithelial responses. More \nspecifically, our results reveal that epithelial cells distinguish between elasticity and \nviscoelasticity independent of the storage modulus.  \n \n2. Materials and methods \n \n2.1 Linear elastic polyacrylamide gel preparation \nElastic polyacrylamide hydrogels (PAH) were fabricated following previously reported \nprotocols.\n21,32,38,39,41 Linearly elastic polyacrylamide hydrogels were created by mixing different \nconcentrations of 40% (v/v) acrylamide (Sigma Aldrich, catalog #1610149), and 2% (v/v) bis-\nacrylamide (Sigma Aldrich, catalog #1610142), leading to 3 different values of the elastic \nmodulus stiffnesses\n (stiff, intermediate, and soft; see Table 1). Polymerization was initiated by \nadding 5 μ L of 10% w/v ammonium persulfate (APS) (Invitrogen, ref. HC2005), and 0.5 μ L of \n0.1% (final concentration) of N,N,N,N-tetramethylethylene (TEMED) (Thermo Scientific, Lot # \nWJ334964), of indicated amounts.\n31,38,42 \nTable 1: Formulations for elastic and viscoelastic polyacrylamide hydrogels (PAH). \n Acrylamide \n(%) \nBis-\nacrylamide \n(%) \nLinear \nAcrylamide \n(%) \nAcrylamide \nfrom 40% \nstock \nsolution \n(mL) \nBis-\nacrylamide \nfrom 2% \nstock \nsolution \n(mL) \nLinear \nAcrylamide \n(mL) \nWater \n(mL) \nSoft  \nE \n5 0.30 0 0.125 0.150 0 0.725 \nSoft  \nVE \n5 0.30 1.8 0.125 0.150 0.450 0.275 \nIntermediate \nE \n8 0.25 0 0.200 0.100 0 0.700 \nIntermediate  \nVE \n8 0.25 1.8 0.200 0.100 0.450 0.250 \nStiff  \nE \n8 0.48 0 0.200 0.240 0 0.560 \nStiff \nVE \n8 0.48 1.84 0.200 0.240 0.460 0.100 \n \n2.2 Linear acrylamide \nLinear acrylamide was fabricated by mixing different amounts of 40% acrylamide, milliQ water, \nAPS, and TEMED as summarized in Table 2. Samp les were polymerized overnight at 37°C in \nthe dark. 21,31,32,42 The shear viscosity of the resulting polymer solution was measured with \nsteady shear experimentson an Anton Paar 302e rheometer using a parallel-plate attachment \n(PP-25 mm). The average zero-shear viscosity, η 0, measured regularly every week over a 5-\nweek period, was 15420 /g3399  386 mPas, Figure S1. The consistency of the measured values of η 0 \nsuggests that the linear acrylamide chains remained stable over the five-week period, with no \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nsigns of degradation. Based on these observations, linear acrylamide solutions were stored for \nup to 5 weeks and used to fabricate viscoelastic polyacrylamide hydrogels (PAH), as described \nbelow.  \nTable 2: Recipe for Linear Acrylamide \n40% Acrylamide (mL) Water (mL) TEMED (mL) APS (mL) NHS 4% \n1.25 8.72 0.005 0.024 0 \n \n2.3 Fabrication of viscoelastic polyacrylamide gels \nViscoelastic PAHs were fabricated by modifying previously described linearly elastic PAH \nprotocols,21,31,32,42 by adding the linear acrylamide solution and adjusting the water content as \nsummarized in Table 1.   To remove bubbles and minimize dissolved oxygen content, the \nsolution was degassed for 10 minutes, after which TEMED and APS were added. After \npolymerization, PAHs were fully immersed in PBS and allowed to swell overnight at 4 °C.  \n2.4 Treatment of glass-bottom dishes \nGlass-bottom dishes were used as substrates for fabricating elastic and viscoelastic PAHs. The \nglass portion of the glass-bottom dishes was cleaned with 0.1 M NaOH and allowed to dry \novernight. Clean dishes were then treated with (3-aminopropyl)trimethoxysilane, 97% (APTMS) \n(ThermoFisher Scientific, A11284) for 6 minutes, washed three times with milliQ water (18.2 \nMΩ ·cm at 25  /g1271C) . Next, glass-bottom dishes were treated with 200 /i1 L of 0.5% glutaraldehyde in \nPBS (ThermoFisher Scientific, A17876) for 30 mi nutes, followed by three washes with milliQ \nwater. Samples were dried before further addition of 30 /i1 L of premixed PAH solution. The \ndroplet was subsequently sandwiched between the activated glass well and a clean coverslip \n(MercedesScientific, 12 mm round #1, #MER R0012), flipped immediately to ensure a horizontal \nsubstrate, and allowed to polymerize for 15 minutes. Finally, gels were fully swollen by adding \nPBS overnight, resulting in PAHs of ~150 /i1m in thickness. This protocol has been described in \ndetail elsewhere.4,21,31 \n2.5 Preparation of PAH surfaces for cell attachment \nTo promote cell attachment to both PAHs types (elastic and viscoelastic), the surfaces were \nfunctionalized with an adhesive ligand, collagen type I (Col-I). First, the coverslips used in the \nprevious step to create a uniform surface were carefully removed. The surfaces of both elastic \nand viscoelastic PAH substrates were first activated with Sulfo-SANPAH (ThermoFisher \nScientific, A35395). For that purpose, 1 mg of Sulfo-SANPAH was initially dissolved in 200 /i1 L \nDMSO, yielding a 10 mM solution. Next, Sulfo-SANPAH was further diluted by adding 50 µL of \nstock solution to 950 µL of HEPES buffer. The diluted Sulfo-SANPAH solution was added to \nthe PAH sample and exposed to UV light for 15 minutes. Samples were rinsed three times with \nHEPES buffer. The process was repeated once more. Then, 100 /i1 L of Col-I at 100 /i1 g/mL was \nadded to the Sulfo-SANPAH-treated PAH and incubated overnight at 4  /g1271C. To conclude, PAH \nsamples were rinsed with PBS and UV-sterilized for 10 minutes before cells were seeded as \ndescribed below.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\n2.7 Rheology \nElastic and viscoelastic PAHs were characterized using shear rheology, and both strain and \nfrequency dependence were probed. Measurements were performed using an MCR-302e \nrheometer (Anton Paar) at 25 °C with a sandblasted parallel-plate geometry (PP-25/S, 25 mm \ndiameter). Pre-mixed polyacrylamide solutions were prepared using the monomer acrylamide \nand the cross-linker bis-acrylamide, as shown in Table 1. The bottom plate was loaded with 510 \nμ L of pre-mixed solution. Next, the sandblasted top plate was slowly lowered until a 1 mm gap \nwas achieved, ensur ing that the droplet contacted the top plate and filled the gap. Water was \nthen added to the Peltier-controlled temperature hood (H-PTD220) to prevent the sample from \ndrying. Gels were left to polymerize for 30 minutes. After polymerization, the gap size was \nreduced slightly to 0.990 mm, resulting in a 1% compressive strain. The storage ( G\n/i1 ) and loss \n(G/i1/i1 ) moduli were then systematically measured as a function of angular frequency ( ω ) and as \na function of shear strain ( γ ). For the angular frequency sweep tests, the frequency ω  was \nvaried between 0.1-200 rad/s (or equivalently 0.016-31.8 Hz) at constant shear strain ( γ  = 1%), \nwithin the linear regime of previously reported elastic PAH measurements. 39 For shear strain \nsweep experiments, the shear γ  was varied between 0.1-50% at constant angular frequency ( ω  \n= 6.28 rad/s, or 1 Hz). All reported data are presented as the mean ± standard error of the mean \ncalculated from 3 independent tests, each with a freshly prepared and loaded sample. \n2.9 Cell culture \nAdenocarcinoma human alveolar basal epithelial cells (A549) were purchased from Berkeley \nBiosciences Divisional Services, UC Berkeley. Cells were cultured in growth media composed \nof Dulbecco's Modified Eagle Medium- high glucose (DMEM 1X) (ThermoFisher Scientific, \n2906246) supplemented with 10% v/v fetal bovine serum (FBS) (Corning, 35-015-CV) in a 100 \nmm petri dish. No antibiotics were used, as these have been shown to induce metabolic \nchanges.\n43 \n2.10 Single-cell migration time-lapse studies \nA549s between passages 2-20 were seeded at 1000 cells/cm 2 onto sterilized PAH elastic or \nviscoelastic samples and cultured for 12 hours before imaging, as described below, was \ninitiated.  Images were taken every 15 minutes for a total period of 24 hours. We used an \ninverted epifluorescence microscope (Olympus IX 83 P2ZF) equipped with autofocus and a \nsterile environmental chamber with temperature (37 °C), humidity, and CO\n2 (5%) control. An \nOlympus LWD UPLAN FLUOR 20X PH  air objective with a numerical aperture (NA) of 0.45 and \na working distance of 2.10 mm was used to acquire images in *.tiff format. The static images \nobtained were then stacked to generate time-lapse movies using ImageJ (Fiji). As soon as the \nimaging period ended, samples were immediately fixed as described below. Fixed samples \nwere further used for immunostaining and immunofluorescence imaging to quantify focal \nadhesion sizes.  \nTo analyze the time-lapse movies and to characterize cell migration quantitatively, we curated \nthe data as follows. Cell trajectories that fulfilled the following criteria were used in the \nevaluation of time dependent displacements: (1) only imaged cells that migrated at least a \ndistance typical of its diameter (~30 - 40 µm) within the first hour were considered; (2) cell \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\ntrajectories of cells that contacted other cells were not taken into account;  (3) cells that exited \nthe field of view during the imaging process (24 hours) were not considered, and (4) cells \nthat underwent division within the 24-hour period were not considered. \n2.11 Immunofluorescence imaging \nCells were fixed immediately after the 24-hour time-lapse imaging process was completed. To \nfix the cells, 200 /i1 L of 4% paraformaldehyde (PFA) (Spectrum, P1010) in PBS was applied for \n10 minutes at room temperature (25 °C), followed by three thorough washes with PBS. Fixed \ncells were then permeabilized with 200 \n/i1 L of 0.1% Triton X-100 (Sigma, X100) in PBS for 15 \nminutes and incubated in blocking  buffer (2% bovine serum albumi n [BSA; Fisher Scientific, \nBP671-10] in PBS). 4',6-diamidino-2-phenylindol e, dihydrochloride (DAPI) (ThermoFisher \nScientific, 62247), Alexa Fluor 488 phalloidin (ThermoFisher Scientific, A12379), and Alexa \nFluor 647 anti-paxillin (Santa Cruz Biotechnology, sc-365379) st aining solutions were prepared \nfollowing the manufacturer's instructions. First, cells were immersed in DAPI solution for 10 \nminutes, followed by a thorough PBS wash. Then, the cells were immersed in 488 phalloidin for \n30 minutes, followed by a thorough wash in PBS. T he final staining consisted of immersing cells \nin 647 anti-paxillin and left overnight at 4°C, followed by a thorough PBS wash. Samples were \ndried, Prolong Live Antifade Reagent (ThermoFisher Scientific, P36975) was added, and \ncapped with a coverslip to increase fluorescence signal stability. Immunofluorescence images \nwere taken on an LSM 880 upright confocal microscope using a Plan-Apochromat 63x/1.4 Oil \nDIC M27 objective, resulting in a pixel-to-micron ratio of 0.132. All images were taken at the \nbasal cellular plane and were analyzed with ImageJ FIJI software\n45 following previously reported \nprotocols.46 \n2.12 Quantification of focal adhesion areas \nFocal adhesion areas were quantified from cell images using ImageJ FIJI. 46 All images were \nfirst converted to 8-bit images. Background subtraction was performed by setting the rolling ball \nradius parameter to 50 pixels and selecting the sliding paraboloid function of FIJI. Next, the \ncontrast-limited adaptive histogram equalization (CLAHE) function with a block size of 19 was \napplied to the adjusted images; we set the histogram to 256 and used a maximum slope of 6. \nThe image processing protocol ended with the application of the exponential operator to further \nminimize background noise effects and adjust brightness. Each image was then manually \ncropped to exclude everything except the focal adhesions of the cell being analyzed. To quantify \nfocal adhesion areas, the particle plug-in was used, setting a particle size threshold ranging \nfrom 0.10 \n/i1 m² to 15 /i1 m² and a circularity between 0.00 and 0.99. \n2.13 Statistical Analysis \nAll statistical analyses were performed using IBM SPSS Statistics and GraphPad Prism. A \nStudent’s t-test was conducted to determine whether differences in elastic and viscoelastic \nbiophysical parameters among the soft, intermediate, and stiff means, with standard errors of \nthe means (SEMs), were statistically significant. (NS = non-significant, * p < 0.05, and *** p < \n0.001). \n2.14 Cell tracer tool \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nAreas of migratory cells were also quantified using Marker Tracker. Details of the in-house \nsoftware are available at https://github.com/MECHANO3B-I-OLOGY/Marker-Tracker. A custom \nGUI-based application enabled manual delineation of cell boundaries. Users traced contours \nframe by frame using a brush interface, with optional preprocessing to enhance contrast. From \neach mask, the software computed the area, perimeter, and centroid using standard contour-\nbased image analysis. Results were exported as structured *.csv files, with optional conversion \nto physical units based on user-defined pixel scaling. Visual overlays and mask stacks (TIFF \nformat) supported trace validation and reproducibility. \n2.15 Cell migration quantification via Marker Tracker \nCell migration data analysis was performed using in-house Python-based software: Marker \nTracker XYZ (https://github.com/MECHANO3B-I-OLOGY). The Marker Tracker tool recorded \nthe x- and y -coordinates of selected objects over time. Cells were tracked using an adjustable \nbounding box (bbox) to ensure the tracker captured the appropriate region of interest (ROI) \naround the cell. Marker Tracker used the Channel and Spatial Reliability Tracker (CSRT) Python \npackage, which is optimized for tracking deformable objects, such as cells. The centroid \ncoordinates, (c\nx, cy), in the imaging (xy) plane for each cell (with identified area) at each point in \ntime were computed using image moments as follows: \n/g1855 /g3051/g3404\n/g3014/g3291/g3116\n/g3014/g3116/g3116\n,/g1855 /g3052/g3404 \n/g3014/g3116/g3292\n/g3014/g3116/g3116\n      ( 1 )  \nwhere M00 represents the zero-order moment: \n/g1839 /g2868/g2868/g3404 ∑ 1/g4666 /g3299,/g3300 /g4667  /g3354 /g3252       ( 2 )  \nand spatial moments Mpq are defined by: \n/g1839 /g3043/g3044/g3404 ∑ /g1876 /g3043\n/g4666/g3299,/g3300/g4667 /g3354 /g3252 /g1877 /g3044     (3) \n      \nIn equations (2) and (3), C  denotes the contour of the cell. Variables x, y within the summation \nin equation (3) are considered only for locations (points) within the cell contour. This protocol \nenabled accurate spatial tracking of the cell centroid, even as the moving cell changed shape. \nCell speed was then calculated from the estimated centroid positions by calculating cell \n(centroid) displacements between image frames. The time increments were determined by the \nacquisition frame rate. The change in cell centroid position between successive frames was \nthen  used to calculate the cell speed. Specifically, the instantaneous speed at time t was \ncomputed using the equation \n/g1874 /g4666 /g1872 /g4667 /g3404 /g3495 /g4672\n/g3031/g3030/g3299\n/g3031/g3047/g4673\n/g2870\n/g3397/g4672\n/g3031/g3030/g3300\n/g3031/g3047/g4673\n/g2870\n     ( 4 )  \nwhere cx and cy represent the tracked (x,y) coordinates of the cell centroid. The identification of \nthe cell contour C, and the cell centroid position (shown in red) is illustrated in Figure S2.  \n2.16 Calculations of the mean square displacement (MSD) \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nCells that satisfied the criteria listed above were considered for analysis. For each tracked cell, \nthe location of the cell-centroid in the imaging plane (defined as the x-y plane) was calculated \nfrom the images. The mean square displacement (MSD) was then obtained by using the time-\nsequence of centroid coordinates\n44: \n/g1839/g1845/g1830 /g4666 /g2028 /g4667 /g3404 /g1731/g4670 /g1876 /g4666 /g1846/g3397/g2028 /g4667 /g3398/g1876 /g4666 /g1846 /g4667/g4671 /g2870/g3397 /g4670 /g1877 /g4666 /g1846/g3397 /g2028 /g4667 /g3398/g1877 /g4666 /g1846 /g4667/g4671 /g2870/g1732     ( 5 )  \nwhere x and y represent the centroid coordinates at experimental time /g2028 , and /g1846  is the initial time.  \nTo estimate if calculated cell MSDs followed diffusive, sub- or super-diffusive behavior, we \ncalculated the power-law exponent \nα , by fitting MSD data to the following relationship: \n/g1839/g1845/g1830 /g4666 /g2028 /g4667 /g34044 /g1830 /g2028 /g3080        ( 6 )  \nThe constant D can be identified with the cell diffusion coefficient when α  = 1. More generally, \nwe treated D as a prefactor and used the power-law exponent α  to quantify the type of migratory \nbehavior. When α  < 1, cell migration was sub-diffusive; α  > 1 suggested super-diffusive \nmigration, while α  = 1 was indicative of Brownian-like diffusion. 44 The mean square \ndisplacement was calculated for each cell based on the trajectory of its centroid. Averages were \ncalculated by combining results from an ensemble of cell trajectories.  \n3. Results \n3.1 Rheology revealed elastic and viscoelastic tunability of polyacrylamide-based model \nECMs.  \nWe conducted shear rheology measurements to characterize the shear modulus G´ and loss \nmodulus G´´ of the linear elastic and viscoelastic model ECMs fabricated. This characterization \nwas done as a function of angular frequency, as well as a function of shear strain to get a \ncomplete characterization of the linear viscoelastic properties of the ECM models. \nViscoelastic model ECMs were fabricated by adding linear polyacrylamide chains to the elastic \nnetworks, as described in the Materials and Methods section. Figure 1a shows a schematic of \nthe PAH network for elastic and viscoelastic ECM models and illustrates how the addition of \nlinear polyacrylamide chains enhances dissipative effects, thereby allowing tunability of the loss \nmodulus. Following the rheological characterization of the model ECM’s, we evaluated the low-\nstrain and low-frequency values of the storage (G´) and loss (G´´) moduli.  \nThe storage modulus is controlled by the elasticity of the cross-linked network. In our case, the \nstorage modulus curves attained a non-zero, constant strain-dependent plateau at low \nfrequencies, as is expected from the elasticity of the permanently networked structure. The \nresting elastic modulus was calculated as the zero-strain limit of this plateau regime. For ideal \nnetworks, the loss modulus is expected to vanish under static conditions since there is no \nviscous dissipation in the absence of flow. Therefore, strictly speaking, at zero frequency, ideal \ncross-linked viscoelastic substrates respond as purely elastic materials with \n/g1833 /g4593/g4593/g13720 . Typically, \nhydrogels may exhibit viscous losses at very low frequencies (with G´´ > 0) due to the effects of \ndangling chains, chain friction, and dissipative effects as water flows through the background \nnetwork. Here, to enable consistent quantification of linear viscoelastic values, we estimated the \nzero-strain limit of the storage modulus via extrapolation. These were then compared to the \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nvalue of the storage modulus obtained from frequency-sweep measurements extrapolated to \nzero frequency. For the loss modulus, the zero-strain estimate was compared with the low \nangular frequency (~0.1 rad/s) value extrapolated from the frequency-sweep measurements.  \nFigure 1b shows the log-log plots of G´ and G´´ of elastic soft, intermediate, and stiff model \nECMs as a function of angular frequency ω . The zero-angular frequency G0/i1  values were \nestimated to be G0/i1 = 2.67 ± 0.10 kPa, G0/i1 = 6.59 ± 0.61, and G0/i1 = 10.65 ± 1.00 kPa for soft, \nintermediate, and stiff elastic model ECMs, respectively. The low-angular frequency G0/i1/i1  \nvalues were G0/i1/i1  = 0.00 ± 0.00 kPa, G0/i1/i1  = 0.00 ± 0.00 kPa, and G0/i1/i1  = 0.01 ± 0.01 kPa for \nsoft, intermediate, and stiff elastic model ECMs, respectively. Results revealed that within the \ninvestigated angular frequency range of 0.1-200 rad/s (or 0.159-31.8 Hz), the elastic model \nECMs responded linearly. This is consistent with previous reports in which a combination of \ntensile testing and shear rheology demonstrated linear-elastic responses in similar model \nECMs.\n21,32,38,39,41 Therefore, we concluded that the low G0/i1/i1  values measured do not contribute \nsignificantly to the mechanical response, and G0/i1/i1  will be considered negligible in this study. \nFigure 1c shows the log-log plots of G´ and G´´ of viscoelastic soft, intermediate, and stiff model \nECMs as a function of angular frequency ω . The zero-angular frequency G0/i1  values were \nestimated to be G0/i1 = 1.96 ± 0.77 kPa, G0/i1 = 7.46 ± 3.84 kPa, and G0/i1 = 8.23 ± 0.36 kPa for \nsoft, intermediate, and stiff viscoelastic model ECMs, respectively. The low angular frequency \nG\n0/i1/i1  values were meanwhile estimated to be G 0/i1/i1 = 0.06 ± 0.006 kPa, G0/i1/i1 = 0.20 ± 0.040 \nkPa, and G 0/i1/i1 = 0.246 ± 0.011 kPa for soft, intermediate, and stiff viscoelastic model ECMs, \nrespectively. Results revealed that within the investigated angular frequency range of 0.1-200 \nrad/s (or 0.159 - 31.8 Hz), the viscoelastic model ECMs G´ response was linear; G´´ values \ngradually increased with increasing angular frequency.  \nFigure 1d shows the log-log plots of G´ and G´´ for the elastic soft, intermediate, and stiff model \nECMs as a function of shear strain \nγ . The estimated values of G0/i1  at zero-shear strain were \nG0/i1 = 2.09 ± 0.18 kPa, G0/i1 = 7.2 ± 0.78 kPa, and G0/i1 = 10.24 ± 1.82 kPa for soft, intermediate, \nand stiff elastic model ECMs, respectively. The zero-shear strain G 0/i1/i1  values (examined at a \nfrequency of ω  = 6.28 rad/s, or 1 Hz) were estimated as G0/i1/i1  = 0.001 ± 0.001 kPa, G0/i1/i1  = \n0.01 ± 0.004, and G0/i1/i1  = 0.005 ± 0.005 kPa for soft, intermediate, and stiff elastic model \nECMs, respectively. These results revealed that within the investigated shear strain range of \n0.01-100%, soft elastic model ECMs responded linearly. Similarly, we found that between 0.01 \nto 10%, intermediate and stiff ECM exhibited a linear response and began to soften above \n~10% shear strain. The sudden decrease in storage modulus G' could indicate network damage \ninduced by the larger shear strains. As in measurements of elastic PAH frequency sweeps, the \nG\n0/i1/i1  values measured were finite but low. \nFigure 1e shows the log-log plots of G´ and G´´ of viscoelastic soft, intermediate, and stiff model \nECMs as a function of shear strain γ . We estimated the zero-shear strain G 0/i1  values to be \nG0/i1 = 3.03 ± 0.67 kPa, G0/i1 = 8.4 ± 0.9 kPa, G0/i1 = 12.1 ± 1.9 kPa for soft, intermediate, and stiff \nviscoelastic model ECMs, respectively. We also estimated zero-shear strain G 0/i1/i1  values to be \nG0/i1/i1 = 0.292 ± 0.04 kPa, G0/i1/i1 = 0.458 ± 0.03 kPa, and G 0/i1/i1 = 0.690 ± 0.05 kPa for soft, \nintermediate, and stiff viscoelastic model ECMs, respectively. Results revealed that within the \ninvestigated shear strain range of 0.01- 100%, the viscoelastic model ECMs' G´ responded \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nlinearly. The G 0/i1/i1  values measured were significantly higher than those of the linear-elastic \ncounterparts. Additionally, the G/i1/i1  remained relatively constant over the shear strain range \ninvestigated. \nFigure 1. (a) Schematic illustrating elastic and viscoelastic PAH model ECM networks. Viscoelastic model ECMs were fabricated by \nadding linear polyacrylamide chains to the elastic network. (b) Rheological properties of the model ECMs as a function of angul ar \nfrequency at a shear strain of 1%. We show the storage modulus ( G/i1, filled symbols) and loss modulus ( G/i1/i1, open symbols) of \nmodel (c) elastic and (d) viscoelastic ECMs, respectively. Rheological properties as a function of shear strain at constant ang ular \nfrequency of 6.28 rad/s (or 1 Hz). We show the storage modulus ( G/i1, filled symbols) and loss modulus ( G/i1/i1, open symbols) for \nmodel (e) elastic and (f) viscoelastic ECMs, respectively. Three independent and freshly prepared samples reported. Symbols \ndenote the mean; vertical bars indicate the standard error of the mean of 3 independent measurements for each test.  \nFigures 2a and 2b show the average zero-shear strain storage modulus G0´ and zero-frequency \nstorage modulus values of elastic and viscoelastic model ECMs, where the exact values have \nbeen described previously in this section. Likewise, figures 2c and 2d show the average loss \nmodulus G0/i1/i1  of elastic and viscoelastic model ECMs. We found that the zero-shear loss \nmoduli values (evaluated at angular frequency of ω  = 6.28 rad/s, or 1 Hz) of elastic model \nECMs were within 2% or less from values estimated for the corresponding viscoelastic model \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nECMs. We propose that the loss moduli of the elastic model ECMs did not significantly \ncontribute to the mechanical responses and were therefore treated as ideal elastic. Taken \ntogether, our results suggest that the differences in the zero-shear strain and zero-frequency \nstorage moduli between the elastic and viscoelastic model ECMs are not statistically significant, \nwhereas differences in the loss moduli are statistically significant. Values are also summarized \nin Table 3 and Table 4.  \nIn addition to frequency- and strain-sweep experiments to estimate the loss and storage moduli \nfrom the shear response, we obtained a complementary metric of the time-dependent \nmechanical response of the ECMs. Specifically, we characterized the relaxation behavior of \nsoft, intermediate, and stiff elastic and viscoelastic model ECMs, as shown in Figure S3 and \nsummarized in Tables S1 and S2. Relaxation data were fitted to expressions with a single \nrelaxation timescale to approximate the dominant dynamical response. \nTable 3.  Limiting values of the shear modulus and loss modulus, G/i1 and G/i1/i1  obtained from shear-sweep and \nfrequency-sweep tests on elastic PAH model ECMs.  \n \nElastic G0/i1, shear strain \n(kPa) \nG0/i1/i1, shear \nstrain (kPa) \nG0/i1, Frequency \nsweep (kPa) \nG0/i1/i1, Frequency \nsweep (kPa) \nSoft E 2.09 ± 0.18 0.001 ± 0.001 2.67 ± 0.05 \n \n0.01 ± 0.00 \nIntermediate E 7.2 ± 0.450 0.01 ± 0.002  6.59 ± 0.357 0.00 ± 0.00 \nStiff E 10.24 ± 1.05 0.005 ± 0.003 10.65 ± 0.578 0.00 ± 0.00 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nFigure 2. (a) Average zero-strain storage modulus, G 0´, from shear strain sweep test for soft, intermediate, and stiff \nelastic and viscoelastic model ECMs, respectively. Experiments were conducted at an angular frequency of 6.28 rad/s (or \n1 Hz) (b) Average zero-frequency storage modulus, G 0´, from angular frequency sweep tests for soft, intermediate, \nand stiff elastic and viscoelastic model ECMs, respectively. Experiments were conducted at a low strain  of 1%.  (c) \nAverage estimated zero-strain values of the loss modulus, G 0´´, from shear strain sweep tests for soft, intermediate, \nand stiff elastic and viscoelastic model ECMs, respectively. (d) Average low-frequency loss modulus values (for 0.1 \nrad/s) estimated from angular frequency sweep tests, G 0´´, for soft, intermediate, and stiff elastic and viscoelastic \nmodel ECMs, respectively. An independent t-test was used to assess whether differences between elastic and \nviscoelastic model ECMs were statistically significant. NS - not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. N = 3 \ngels per condition. \nTable 4. Limiting values of shear modulus and loss modulus, G/i1  and G/i1/i1, obtained from shear-sweep and \nfrequency-sweep tests on viscoelastic PAH model ECMs.  \nViscoelastic G0/i1, shear \nstrain (kPa) \nG0/i1/i1, shear \nstrain (kPa) \nG0/i1, Frequency \nsweep (kPa) \nG0/i1/i1, Frequency \nsweep (kPa) \nSoft VE 3.03 ± 0.39  0.293 ± 0.023 1.96 ± 0.44 0.06 ± 0.004 \nIntermediate VE  8.44 ± 0.546   0.458 ± 0.0184 7.46 ± 2.22 0.20 ± 0.023 \nStiff VE 12.12 ± 1.13 0.689 ± 0.027  8.23 ± 0.212 0.246 ± 0.006 \n \n3.2 Elasticity and viscoelasticity of PAH model ECMs of similar rigidity alter epithelial \nmigratory behavior \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nAs described in the methods section, we conducted 24-hour time-lapse microscopy studies to \nmonitor and quantify the migratory behavior of A549 cells on elastic and viscoelastic model \nECMs. We measured the mean square displacement ( MSD), the cell motility (migration) \nexponent ( α ), and the instantaneous cell speed ( V). For clarity, Figures 3a and 3b show \nrepresentative cell MSD curves for one A549 cell migrating on collagen type-I-coated soft, \nintermediate, and stiff elastic and viscoelastic ECMs over a total period of 24 hours. Figure S4 \nshows MSD curves of all cells recorded and for all investigated model ECMs. The slope of the \nlog-log MSD versus lag time τ was used to extract the cell motility exponent, α , which quantifies \nmigration behavior. When α  = 1, the cell migration mimics pure diffusive behavior. When α  is < \n1, cell migration is sub-diffusive. Finally, when α  > 1, cell migration behavior is considered \nsuper-diffusive; this typically happens due to periods where cells exhibit persistent or directional \ncell motion. We extracted \nα  values by fitting data to Eqn. (6) for specified periods as described \nnext (see SI for details).  The MSD data were separated and analyzed into two time periods, 0-\n10 hours and 10-24 hours, based on an apparent migratory change occurring approximately at \n10 hours.  \nFigure 3c summarizes our estimated α  values for all model elastic and viscoelastic ECMs \ncalculated for cells between 0 and 10 hrs. Averages were computed by taking the average of \nthe single alpha values obtained from each individual cell for each (time) period: 0-10 hours, \nand 10-24 hours. Specifically, we found that \nα  = 1.09 /g3399  0.16, α  = 1.19 /g3399 0.15, and α  = 0.89 /g3399  \n0.16 for elastic soft, intermediate, and stiff model ECMs, respectively. Correspondingly, we \nfound that α  = 0.87 /g3399  0.12, α  = 1.33  /g3399  0.15, and α  = 1.08 /g3399 0.18 for viscoelastic soft, \nintermediate, and stiff model ECMs, respectively. Within each substrate type (within each \nstiffness), we found that the differences in α  values between elastic and viscoelastic ECMs were \nnot statistically significant using Student's t-tests. \nData for the 10-24 hour period complements the 0-10 hour period data. Figure 3d summarizes \nour estimates of the \nα  values for all model elastic and viscoelastic ECMs calculated for cells \nbetween 10 and 24 hrs. We found α  = 1.09 /g3399  0.25, α  = 0.89 /g3399 0.24, and α  = 1.11 /g3399  0.21 for \nelastic soft, intermediate, and stiff model ECMs, respectively. The α  values were α  = 1.26 /g3399  \n0.26, α  = 1.18  /g3399  0.33, and α  = 1.14 /g3399 0.23 for viscoelastic soft, intermediate, and stiff model \nECMs, respectively. Within each substrate type (stiffness), we found that the differences in α  \nvalues between elastic and viscoelastic ECMs were not statistically significant using Student's t-\ntests. \nFigure 3e shows average speeds, v, of A549 cells migrating on collagen type-I coated elastic \nand viscoelastic model ECMs over the 10-hour period. Overall, cells on elastic model ECMs \nexhibited different migration patterns than those on viscoelastic counterparts. Cells on soft, \nelastic ECMs migrated faster, with higher instantaneous speeds, than those on stiff, elastic \nECMs. Interestingly, cells on intermediate elastic ECMs migrated faster than cells on both soft \nand stiff ECMs. The average speeds were v = 0.5 ± 0.1 \n/i1 m/min, v = 0.6 ± 0.1 /i1 m/min, and v = \n0.4 ± 0.1 /i1 m/min for cells migrating on soft, intermediate, and stiff elastic model ECMs, \nrespectively. Cells on soft viscoelastic model ECMs migrated at similar speeds as cells on \nintermediate viscoelastic model ECMs. However, cells on stiff viscoelastic model ECMs \nmigrated faster than on both soft and intermediate model ECMs. We found v = 0.4 ± 0.1 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\n/i1 m/min, v = 0.3 ± 0.1 /i1 m/min, and v  = 0.6 ± 0.1 /i1 m/min for soft, intermediate, and stiff \nviscoelastic, respectively. In summary, cells on intermediate viscoelastic model ECMs migrated \n54% slower than on their elastic counterparts, and cells on stiff elastic model ECMs migrated \n29% slower than in their viscoelastic counterparts. \n \nFigure 3. (a) Mean square displacement (MSD) curves versus lag time, /g2028, for A549 cells on soft, intermediate, and \nstiff elastic model ECMs over 24 hours, respectively. (b ) MSD curves versus lag time for A549 cells on soft, \nintermediate, and stiff viscoelastic model ECMs over 24 hours, respectively. (c) Average A549 cell motility exponent, \nα, on soft, intermediate, and stiff elastic and viscoelastic model ECMs over 0 to 10 hours. (d) A549 cell migration \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nspeed on soft, intermediate, and stiff elastic and viscoelastic model ECMs from 0 to 10 hours, respectively. (e) \nAverage A549 cell motility exponent, α, on soft, intermediate, and stiff elastic and viscoelastic model ECMs over 10 to \n24 hours. (f) A549 cell migration speed on soft, intermediate, and stiff elastic and viscoelastic model ECMs from 10 to \n24 hours, respectively.  NS - not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. N = 10 cells per condition. \nFigure 3f shows the average speed of A549 cells migrating on collagen type-I coated elastic and \nviscoelastic model ECMs for the period from 10-24 hours. Overall, cells on elastic model ECMs \nexhibited different migration patterns than those on their viscoelastic counterparts. In particular, \ncells on soft elastic ECMs migrated faster compared to those on stiff elastic ECMs. Interestingly, \ncells on intermediate elastic ECMs migrated faster in comparison to both soft and stiff ECMs. \nWe estimated v = 0.5 ± 0.1 \nµ m/min, v = 0.6 ± 0.1 µ m/min, and v = 0.4 ± 0.0 µ m/min for cells \nmigrating on soft, intermediate, and stiff elastic model ECMs, respectively. Cells on soft \nviscoelastic model ECMs migrated at similar speeds to cells on intermediate viscoelastic model \nECMs. However, cells on stiff viscoelastic model ECMs migrated faster than cells on both soft \nand intermediate model ECMs. We estimated v  = 0.4 ± 0.1 \nµ m/min, v = 0.3 ± 0.1 µ m/min, and v \n= 0.6 ± 0.1 µ m/min for cells migrating on soft, intermediate, and stiff viscoelastic, respectively. In \nsummary, our experiments indicate that cells on intermediate viscoelastic model ECMs migrated \n41% more slowly than on their elastic counterparts, and cells on stiff elastic model ECMs \nmigrated 32% more slowly than on their viscoelastic counterparts.   \nTo assess whether cell speeds remained similar or changed dramatically over the 24-hour \nobservation period, average cell speeds were calculated for 4 intervals: 0-6 hours, 6-12 hours, \n12-18 hours, and 18-24 hours. Figures S5 and S6 show the speeds of cells recorded on all \ninvestigated model ECMs. Overall, A549 cell speed on soft, intermediate, and stiff elastic and \nviscoelastic model ECMs varied with time, as evidenced by differences across the 4 time \nperiods. Cells migrating on elastic and viscoelastic soft model ECMs migrated at similar speeds. \nSimilar behaviors were observed in cells migrating on elastic and viscoelastic stiff model ECMs, \nthat is, migrated at similar speeds. However, this was not the case for cells migrating on elastic \nand viscoelastic intermediate-model ECMs. Cells on the elastic intermediate model ECMs \nmigrated faster. Overall, these findings indicate that while ECM stiffness generally drives \ncomparable migration speeds on soft and stiff elastic and viscoelastic substrates, intermediate \nstiffness uniquely reveals a pronounced dependence on ECM mechanics. \n3.3 Projected cell areas of migratory cells are similar on elastic and viscoelastic PAH \nmodel ECMs \nTo study the impact of the loss modulus on cell motility and cell-substrate mechanics, we \ninvestigated time-dependent projected cell areas as cells moved on the model ECMs. For each \nframe imaged, an instantaneous cell area was determined as described in the methods section. \nFigures 4a and 4b show the average projected cell area, A, for cells on soft, intermediate, and \nstiff elastic and viscoelastic model ECMs. We observe that cells moving on elastic model ECMs \nreached homeostatic behavior after ~10 hours and followed the expected trend of increasing \narea with increasing stiffness. Cells on viscoelastic model ECMs reached homeostatic behavior \nfaster, after approximately 7 hours.  \nInterestingly, cells on intermediate viscoelastic model ECMs had smaller projected cell areas \nthan on soft and stiff viscoelastic model ECMs, whereas they exhibited similar projected cell \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nareas on soft and stiff viscoelastic ECMs. In contrast, cells exhibited a larger projected cell area \non intermediate elastic ECMs than on viscoelastic ECMs. Finally, cells had a larger cell area on \nstiff elastic ECMs than on stiff viscoelastic ECMs. \nFigure 4.  (a) A549 cells projected cell area on soft, interm ediate, and stiff elastic model ECMs over 24 hours, \nrespectively. (b) A549 cells projected cell area on soft, intermediate, and stiff viscoelastic model ECMs over 24 hours, \nrespectively. (c) Cell area on soft, intermediate, and stiff elastic and viscoelastic model ECMs from 10 – 24 hours. (d) \nCell area on soft, intermediate, and stiff elastic and viscoelastic model ECMs after 24 hours. An independent t-test \nwas used to assess whether differences in cell behavior between elastic and viscoelastic model ECMs were \nstatistically significant. NS - not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. N = 10 cells per condition.  \nFigure 4c and 4d show the average cell area between 10 hours and 24 hours ( i.e., \nhomeostasis/steady state) and the average cell area after 24 hours for soft, intermediate, and \nstiff elastic and viscoelastic model ECMs. Averages were calculated from 10 cells per condition. \nFigure 4c shows the average cell area from 10 hours to 24 hours for cells on elastic and \nviscoelastic model ECMs. The average measured cell areas were 1298.3 ± 210.3 \nµ m2, 1371.4 ± \n167.0 µ m2, and 2072.4 ± 387.0 µ m2 for elastic soft, intermediate, and stiff model ECMs, \nrespectively. Similarly, the average measured cell areas were 1370.0 ± 126.5 µ m2, 537.9 ± \n135.0 µ m2, 1522.2 ± 208.1 µ m2 for viscoelastic soft, intermediate, and stiff model ECMs, \nrespectively. Figure 4d shows the final cell area at t = 24 hours for cells on elastic and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nviscoelastic model ECMs. The average measured cell areas were 1527.5 ± 390.1 µ m2, 1467.4 ± \n179.7 µ m2, and 2089.96 ± 469.5 µ m2 for elastic soft, intermediate, and stiff model ECMs, \nrespectively. Similarly, the average measured cell areas were 1364.6 ± 192.3 µ m2, 557.5 ± \n154.9 µ m2, 1524.8 ± 279.7 µ m2 for viscoelastic soft, intermediate, and stiff model ECMs, \nrespectively. Interestingly, average cell areas at 24 hours on viscoelastic intermediate model \nECMs were 62% smaller in comparison to the average for cells on elastic intermediate model \nECMs. Lastly, average cell areas on elastic stiff model ECMs were similar to those on \nviscoelastic stiff model ECMs after 24 hrs. \n3.4 Focal adhesion size depends on loss modulus for soft and stiff PAH model ECMs, but \nnot for intermediate  \n \nFigure 5. (a) Immunofluorescence image of paxillin in A549 cells on soft elastic and viscoelastic model ECMs. (b) \nImmunofluorescence image of paxillin in A549 cells on intermediate elastic and viscoelastic model ECMs. (c) \nImmunofluorescence image of paxillin in A549 cells on stiff elastic and viscoelastic model ECMs. (d) Focal adhesion \narea of A549 cells on soft, intermediate, and stiff elastic and viscoelastic model ECMs. An independent t-test was \nused to determine if the differences in cell behavior betw een elastic and viscoelastic model ECMs were statistically \nsignificant. NS - not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. \nTo gain insight into the interplay between elasticity and viscoelasticity and focal adhesion \ncomplexes, which are involved in the mechanosensory machinery of migratory cells, we \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nquantified paxillin at focal adhesions to estimate focal adhesion sizes in A549 cells on elastic \nand viscoelastic model ECMs after a 24-hour time-lapse. Figures 5a, 5b, and 5c show \nrepresentative images of paxillin stained on elastic and viscoelastic soft, intermediate, and stiff \nmodel ECMs. Figure 5d summarizes the focal adhesion area, A\nFA, for soft, intermediate, and \nstiff elastic and viscoelastic model ECMs. Cells on soft elastic model ECMs exhibited a smaller \nfocal adhesion area compared to cells on intermediate elastic model ECMs. However, cells on \nstiff elastic model ECMs exhibited larger focal adhesion areas than those on soft and \nintermediate elastic model ECMs. This trend is expected and has been observed for multiple \nadherent cell types.\n21,30,40 The average measured focal adhesion areas were 0.3 ± 0.1 µ m2, 0.6 \n± 0.1 µ m2, 0.8 ± 0.1 µ m2 for soft, intermediate, and stiff elastic, respectively.  Interestingly, cells \non stiff viscoelastic ECMs formed smaller focal adhesion areas than on both soft and \nintermediate viscoelastic ECMs. The average measured focal adhesions were 0.8 ± 0.1 \nµ m2, \n0.7 ± 0.1 µ m2, and 0.5 ± 0.1 µ m2 for soft, intermediate, and stiff viscoelastic, respectively. \nFinally, we compared the focal adhesion area of cells on elastic model ECMs with those on \nviscoelastic model ECMs. Cells on soft elastic model ECMs assembled focal adhesion areas \nthat were 65% smaller compared to those on soft viscoelastic model ECMs. However, cells on \nintermediate elastic model ECMs showed focal adhesion areas comparable to those on \nintermediate viscoelastic ECMs. Interestingly, cells on stiff elastic model ECMs exhibited a 65% \nlarger focal adhesion area than those on stiff viscoelastic model ECMs. \n4. Discussion \nWhile PAH based viscoelastic model ECMs have been reported in the literature, currently used \nsubstrates span a limited range of stiffness values. Furthermore, the effects of ECM \nviscoelasticity on migratory cells remain incompletely understood. Therefore, our first objective \nwas to expand the range of mechanical properties (that is increase attainable values in the \nstiffness-viscoelastic phase space) and thereby increase the library of tunable PAH for \nmechanobiology studies. The motivation for focusing on PAH hydrogels is, in part, due to the \nrelative ease in tuning PAH’s mechanical and chemical properties,\n47 and the mechanobiology \nfield's familiarity with PAHs.48–54 The protocols we describe and use enable independent control \nof the storage and loss moduli over a wider range of stiffness than previously reported.21,25,31,32,37 \nSpecifically, consistent with previous studies, we tuned the loss modulus by incorporating linear \nacrylamide polymer chains into elastic PAH networks crosslinked with acrylamide and \nbisacrylamide. Our fabrication protocols enabled us to cast viscoelastic PAHs with shear moduli \nup to 12 kPa (equivalent to Young's modulus of ~32 kPa), exceeding values typically reported in \nthe literature. While we did not explore higher values of stiffness, the strategies described here \ncan be expanded easily to fabricate very stiff viscoelastic substrates.  For the library of PAHs we \nstudy, the loss modulus is within ~10% of the storage modulus, similar to ratios reported for \nstromal or connective tissues, \n55–58 making these substrates biomimetically relevant.  \nWe performed several rheological tests to validate the independent tunability of the storage and \nloss moduli of our model PAH ECMs: shear-strain sweeps, angular-frequency sweeps, and \nrelaxation modulus measurements. Frequency sweep and strain sweep data were used to \nestimate the loss and storage modulus in the linear viscoelastic limit, valid for low strain and at \nlow frequencies (~ 0.1-1 rad/s relevant to mechanobiology studies). We observed similar \nstorage moduli between elastic and viscoelastic model ECMs in both shear tests, indicating that \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\ndifferences were not statistically significant. However, the differences in the loss modulus \nbetween the elastic and viscoelastic ECMs were statistically significant. The responses were \nvalid for small strains and low frequencies, relevant to timescales of relevant cellular processes, \nsuch as focal adhesion turnover,\n59–61 conformational changes of mechanotransducers, 62–64 or \nlamellipodial and filopodia formation. 65,66 The elastic and viscoelastic responses were further \nconfirmed by compression-relaxation tests; as expected, elastic model ECMs exhibited an \ninstantaneous response, whereas viscoelastic model ECMs exhibited a time-dependent \nresponse. Notably, the relaxation time for the intermediate viscoelastic model ECMs was \nsignificantly longer. \nOne of the reported advantages of PAH ECMs is their intrinsic optical transparency, which \nfacilitates imaging of cellular processes using inverted optical and epifluorescence microscopes. \nAll our elastic PAH model ECMs have retained this property. This suggests that the linear \npolyacrylamide chains were evenly dispersed in the elastic network. However, among the \nviscoelastic PAH model ECMs, only the intermediate-rigidity ECM retained optical transparency. \nFor the soft and stiff viscoelastic PAH model ECMs, nevertheless, the resulting substrates were \ntranslucent. UV-Vis absorbance measurements validated this observation, as shown in Figure \nS7. This is most likely due to a combination of  immiscibility at the working concentrations, and \nmicrophase separation of the linear polyacrylamide chains during the curing process of the \nelastic network. Imaging cells through these ~150 µm-thick translucent substrates (as estimated \nfrom Z-stacks) posed challenges for accurately tracing cell boundaries with our computational \ntools (the Marker tracker tool described in Materials and Methods) and thus required manual \ntracing. To overcome this optical limitation, upright microscopy can be used, as was the case to \nimage focal adhesions. In summary, these PAH-based model ECMs expand the tunability of the \nmechanical niche microenvironment for mechanobiology studies by combining complementary \nimaging modalities.  \nOur next objective was to evaluate how the loss modulus affected epithelial cell \nmechanobiology, specifically focusing on cell mean-squared displacement, migration, cell area, \nand focal adhesion area. Previous studies have shown that cells can differentiate between \nelastic and viscoelastic model ECMs, and their responses vary depending on the specific cell \ntype.\n30,37 In these studies, cellular responses varied depending on the ECM ligands presented to \ncells (e.g., collagen, fibronectin, or laminin), the model ECM crosslinking parameters, and the \nlocation of immobilized ECM ligands, either within the elastic network, within embedded linear \npolymer chains, or both. 21 A previous study showed that when only linear polymer chains were \nfunctionalized with collagen, cells did not adhere; however, when fibronectin was used, cells \nadhered.\n33 Here, we functionalized the entire surface using the UV-activated crosslinker Sulfo-\nSANPAH and collagen type I at 100 µ g/mL, functionalizing both model ECM components, the \nelastic network and surface-exposed linear polyacrylamide chains.  \nIt is well known that increased substrate stiffness promotes cell migration, area, and \nproliferation, including A549s. For example, collective cell migration of A549s was higher on \npolydimethylsiloxane (PDMS) on substrates with a stiffness of 18.3 MPa than on 1.4 or 3.4 MPa \nPDMS.\n67 Tissues, however, are viscoelastic, rather than purely elastic, as is the case with \nPDMS and other model mechanobiology substrates, such as PAHs. Understanding how energy \ndissipation in soft materials, quantified by the loss modulus or relaxation constants of model \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nECMs, regulates cellular mechanisms has attracted considerable attention in recent years. Our \nstudy focused on single-cell migration of A549s on PAH-based model ECMs with tunable loss \nmodulus. The combination of cell-type and model ECMs suggests that our study can be \nconsidered as a relevant model system that can be further extended to investigate ECM \nmechanics in cancer metastasis. For instance, ECM mechanics influences how \nadenocarcinoma cells that have undergone an epithelial-to-mesenchymal transition (EMT) \nmigrate and become invasive, a process required for metastasis. Our results show that an \nincrease in the loss modulus affected cell velocity on intermediate and stiff substrates, but not \non soft substrates. Furthermore, the responses between intermediate and stiff substrates were \nof opposite trend, suggesting, combined with previous literature, that there is no universal trend \nand that potentially different mechanosensory signaling pathways were activated. Our stiff \nviscoelastic findings are analogous to those observed for non-tumorigenic human MCF10A cells \nseeded on alginate-based model ECMs, in which cell migration increased on viscoelastic \nsubstrates compared with elastic counterparts.\n68 \nAnother study using MCF10A cells as well demonstrated that on extremely soft viscoelastic ( E \n~0.3 kPa) PAH-based model ECMs, cells migrated faster than on their elastic counterpart, while \nmigration speed decreased on stiff viscoelastic compared to stiff elastic. 30 In our case, cells \nmigrated at higher speeds on stiff viscoelastic than on stiff elastic, while cells migrated at slower \nspeeds on intermediate viscoelastic than on intermediate elastic. Interestingly, on soft PAH \nmodel ECMs, speeds were similar on viscoelastic and elastic substrates. Yet the migration or \nmotility exponent, which quantifies the form of the mean square displacement, changed \nsignificantly from soft elastic (\nα  = 1.09) to soft viscoelastic (α  = 0.87) ECMs. Cells on soft, elastic \nsurfaces displayed hindered migratory behavior rather than purely random (Brownian-like) \ndiffusive motion. Indeed, cell migration was significantly hindered on intermediate viscoelastic \nmodel ECMs (G\n/i1  ~ 8 kPa, with relaxation times of ~3 seconds), as seen in Figure S3. However, \nthe MSD curves remained relatively similar for cells moving on intermediate elastic ( α  = 1.19) \nand intermediate viscoelastic (α  = 1.13) model ECMs. In contrast, cells on stiff model ECMs (G/i1  \n~ 12 kPa, with shorter relaxation times of approximately 0.5 seconds) promoted cell migration. \nAs expected, MSD curves differ qualitatively for cells on stiff elastic ( α  = 0.89) and on stiff \nviscoelastic (α  = 1.06) model ECMs. A possible explanation for this, motivated by motor clutch \nmodels and theories for cells migrating on viscoelastic model ECMs, 25,37 is that cells on \nintermediate viscoelastic model ECMs may undergo motor clutch dynamics following \"load and \nfail\" regimes. This may lead to weaker focal adhesion forces (indicating immaturity), increased \nretrograde flow, reduced spreading, and subsequently slower migration. In contrast, cells on stiff \nviscoelastic model ECMs may experience \"frictional slippage,\" resulting in smaller focal \nadhesion sizes and shorter adhesion lifetimes, whereas larger focal adhesions form on stiff \nelastic ECMs, as shown in Figure 5d. Previous studies using fibroblasts (HMF3) have shown \nsimilar findings: viscoelastic model ECMs with higher storage modulus and enhanced cell \nmigration.\n40  \nMatrix elasticity significantly influences cell spreading area; however, results in the literature \nregarding cell area for cells interacting with viscoelastic model ECMs have been mixed. \nPrevious studies have shown that fibroblasts exhibit a larger cell area on elastic model ECMs \n(2428.93 ± 864.71 μ m²) compared to cells seeded on viscoelastic ECMs (1296.73 ± 311.62 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nμ m²) and cells seeded on glass (1792.61 ± 487.09 μ m²).40 This decrease in cell area may be \ndue to the cells' inability to form large and stable focal adhesions. However, some studies have \nreported a higher spreading area for cells on viscoelastic ECMs than on their elastic \ncounterparts.37 To further characterize the behavior of migratory cells, we analyzed their \ninstantaneous projected cell area over 24 hours. We then compared the cell area at the \nbeginning and end of the time-lapse study, as defined in Section 2.10 . Our findings showed that \nthe initial and final cell areas of migratory cells were not statistically different on soft or stiff \nviscoelastic vs elastic model ECMs. However, cells on intermediate model ECMs and on \nviscoelastic ECMs showed decreased cell spreading (projected cell area) in both the first half \n(parsed from t = 0 to 10 hours) and the second half (parsed from t = 10 to 24 hours) of the time-\nlapse studies. This decrease in cell area may be due to cells' inability to form strong or mature \nfocal adhesions, which prevented them from spreading or slipping during migration, as observed \nin the cell migration data. Similar trends in cell area have been reported for human airway \nsmooth muscle (HASM) and human prostate carcinoma epithelial (22Rv1) cells on soft tunable \nelastic and viscoelastic model ECMs. \nLastly, we analyzed the focal adhesion area in relation to the elastic and viscoelastic model \nECMs to better understand cell migration. Focal adhesion size may predict or correlate with cell \nmigration on elastic substrates.\n69 We observed that cells formed larger focal adhesions as \nsubstrate stiffness increased; this trend was not seen for cells on viscoelastic substrates. Some \nhave reported no significant variation of epithelial cell focal adhesion area on what others have \nreferred to as soft (~ 0.3 kPa) and stiff (~ 3 kPa) elastic and viscoelastic model ECMs. 30 While \nfibroblasts have displayed significant differences in stiff (~14 kPa) model elastic and viscoelastic \nECMs.\n40 In this study, we examined the focal adhesion area 24 hours after cell seeding on \nelastic and viscoelastic model ECMs. Interestingly, we observed a larger focal adhesion area on \nsoft viscoelastic substrates compared to their elastic counterparts; however, cell migration did \nnot show significant differences. While the focal adhesion area on intermediate model ECMs \nremained similar, their migratory behavior differed. Finally, cells on stiff viscoelastic model \nECMs exhibit smaller focal adhesions, while migration increases compared to their elastic \ncounterparts. \nCell signaling via the underlying mechanical substrate has been demonstrated for cells on \nsubstrates that are neither too stiff nor too compliant.\n9,14,15 Recent theoretical studies by us and \ncollaborators show that substrate stiffness, cell migration rates, and cell-substrate stresses \naffect the frequency of contacts between neighboring cells, and the ability of migratory cells to \nmove persistently . 70,71 Our experimental findings highlighting the complex interplay between \nsubstrate (ECM) elastic and viscoelastic properties in regulating epithelial cell responses \nstrongly suggest that the role of substrate viscoelasticity must also be considered to understand \ncell-cell interactions, and emergent long-ranged behavior such as durotaxis and adurotaxis.  \n5. Conclusion \nWe created a tunable PAH-based viscoelastic platform with storage moduli comparable to those \nof their elastic counterpart to investigate the response of cell mechanobiology to loss moduli. \nUsing Adenocarcinoma lung epithelial cells (A549s) as a model cell line, we evaluate mean-\nsquared displacement, cell migration, cell area, and focal adhesions on both elastic and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 17, 2026. ; https://doi.org/10.64898/2026.02.04.703912doi: bioRxiv preprint \n\nviscoelastic model ECMs. Our analysis shows that A549 cell migration is enhanced or hindered \non model ECMs with storage moduli above ~3 kPa, depending on the substrate relaxation time. \nWe also observed a significant decrease in focal adhesion size on stiff viscoelastic model \nECMs, correlating with an increase in cell migration speed. Our results suggest that \nviscoelasticity influences cell migration above a certain stiffness value, depending on the \nrelaxation time of the substrate. We also observe that there is no true correlation between cell \nmigration and focal adhesion on a viscoelastic substrate, as traditionally observed on elastic \nsubstrates with increasing storage modulus.  \n6. Conflicts of interest \nNo conflicts of interest to declare \n7. Acknowledgements \nA.M.S., A.G., and R.C.A.E. acknowledge funding from the NSF- CREST: Center for Cellular and \nBiomolecular Machines through the support of the National Science Foundation (NSF) Grant \nNo. NSF-HRD-1547848. A.M.S and R.C.A.E. acknowledge funding from the Tobacco-Related \nDisease Research Program through the support of the University of California Office of the \nPresident Grant No. T31KT1583 awarded to R.C.A.E. A.M.S., and A.G. acknowledge funding \nfrom the CAREER NSF Grant No. CBET 2047210 awarded to A.G., A.M.S. acknowledges \nfunding from the UC Merced Graduate Dean's Dissertation fellowship. \n8. Supplementary Information \nFigure S1. Stability of the viscosity of linear polymer chains \nFigure S2. Cell centroid for speed calculations  \nFigure S3. Relaxation of soft, intermediate, and stiff elastic and viscoelastic model ECMs \nTable S5. Relaxation values of elastic model ECMs \nTable S6. Relaxation values of viscoelastic model ECMs \nFigure S4. Total mean square displacement of soft, intermediate, and stiff elastic and \nviscoelastic model ECMs over 24 hours \nFigure S5. A549 cell migration speed on soft, intermediate, and stiff elastic and viscoelastic \nmodel ECMs \nFigure S6. Average cell speed across different time increments on soft, intermediate, and stiff \nelastic and viscoelastic model ECMs. \nFigure S7. UV-Vis spectra comparison of Polyacrylamide soft, intermediate, and stiff \nviscoelastic model ECMs \n9. References \n1. Karamanos, N. K. et al. A guide to the composition and functions of the extracellular matrix. \nFEBS J. 288, 6850–6912 (2021). \n2. Pally, D. & Naba, A. Extracellular matrix dynamics: A key regulator of cell migration across \nlength-scales and systems. Curr. Opin. Cell Biol. 86, 102309 (2024). \n3. Naba, A. Mechanisms of assembly and remodelling of the extracellular matrix. Nat. Rev. \nMol. Cell Biol. 25, 865–885 (2024). \n4. Seo, B. R. et al.  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