DOI: 10.5061/dryad.rxwdbrvq2
title: Data for: Rigidity sensing of inclusions directs differentiated cell elongation and force generation across phenotypes

description: Fibrosis is driven in part by the transition of healthy fibroblasts to a
 contractile phenotype, called myofibroblasts. The mechanics of the
 extracellular matrix play a crucial role in regulating the cell fates and
 behaviors during this transition. However, most studies to date focus on
 cells grown on 2D surfaces and matrices with homogeneous properties. This
 leaves open how local rigidity differentially regulates the behaviors of
 both phenotypes in 3D environments, including polarization, contraction,
 and maintenance of phenotypes, during remodeling. Here, we engineer 3D
 microgel-in-collagen composites by embedding low-volume fractions of
 cell-scale microgels with two levels of rigidity, mimicking healthy and
 pathological tissues, that are stiffer than the surrounding collagen but
 do not significantly change the bulk modulus. We find that microgels serve
 as mechanical centers: both phenotypes polarize toward microgel
 inclusions. The polarization response decays as a power-law with distance
 ∼ r−n, decreasing more slowly for myofibroblasts (n ≈ 0.35) than
 fibroblasts (n ≈ 0.81), indicating that myofibroblasts are more sensitive
 to small mechanical variations. In-situ measurement finds that
 forces are highest for myofibroblasts near stiff microgels and lowest for
 fibroblasts near soft microgels. Local rigidity also stabilizes the
 myofibroblast phenotype: Both the ordering of the proinflammatory marker
 α-SMA and nuclear YAP localization persist for cells cultured with stiff
 microgels over several days, but diminish quickly for those cultured with
 soft microgels and in pure collagen. Together, these results reveal a
 rigidity- and phenotype-dependent feedback loop: stiff inclusions induce
 cell polarization and collagen remodeling via contractile force, which in
 turn, maintain the myofibroblast phenotype. Our study positions mechanical
 heterogeneity as a useful and sensitive handle to probe and potentially
 modulate early fibrotic progressions.
description: # General README for Dataset --- ## Overview This repository (Dryad.zip)
 contains all data, scripts, and analyses supporting the results presented
 in the associated publication: 'Rigidity sensing of inclusions
 directs differentiated cell elongation and force generation across
 phenotypes' by Yuxin Luo and Yimin Luo. Each figure has a
 corresponding folder, which includes raw data, processed data, and
 analysis scripts used to generate the figure panels. --- ## General Notes
 ### Dataset Organization The dataset is organized by figure, with each
 folder named according to its corresponding figure (e.g., Figure_3,
 Figure_4, etc.). Each figure folder has contents as follows: ### Data
 Contents * **MATLAB Scripts**: Primarily used for figure generation,
 statistical analysis, and visualization. These scripts are self-contained
 and reproduce the corresponding figure panels when run. They are prefixed
 with the figure number (e.g., `Figure_7b.m`, `Figure_7d.m`). * **Processed
 Data (CSV files)**: Cleaned and prepared datasets for analysis and
 visualization are included directly alongside corresponding scripts. These
 files contain quantitative measurements such as rheology values, collagen
 contraction areas, anisotropy indices (DOA), and YAP ratios. * **Raw
 Data**: Included where applicable, such as cell tracking trajectories from
 TrackMate, or Trios files from the rheometer. ### Experimental Conditions
 Descriptions of setups of experiemts such as bulk rheometry are briefly
 described in figure-specific README files. --- ## Overview of Figures ###
 **Figure 3** * **Content**: Frequency sweep rheology of storage modulus
 (G′) for bulk PEGDA gels and collagen composites. ### **Figure 4** *
 **Content**: Polarization of fibroblasts and myofibroblasts relative to
 microgels, quantified by angle distribution and distance-dependent
 correlation. ### **Figure 5** * **Content**: Collagen contraction assays
 in 96-well plates, quantified by normalized pellet area over 72 hours. ###
 **Figure 6** * **Content**: Tracer bead displacement analysis for
 quantifying forces exerted by fibroblasts and myofibroblasts near stiff or
 soft microgels. ### **Figure 7** * **Content**: Examining the extent of
 myofibroblast activation through analyzing degree of anisotropy (DOA) of
 alpha-SMA staining and nuclear-to-cytoplasm YAP staining. ### **Figure
 S2** * **Content**: Joint analysis of cell alignment and spatial proximity
 to microgels. Includes polarization angle–distance datasets, power-law
 decay of ⟨cos(2θ)⟩, boxplots of alignment factor distributions, cell-count
 histograms per radial bin, and polar heatmaps visualizing the spatial
 distribution of alignment around microgels. ### **Figure S3** * Content:
 Local collagen fiber alignment relative to the cell–microgel axis.
 OrientationJ hue maps are combined with cell outlines and microgel
 centroids to compute fiber deviation Δθ, rectangular masked regions along
 the cell–microgel direction, and corresponding histograms and cos(2Δθ)
 maps quantifying fiber reorientation influenced by cell–microgel
 interactions. ### **Figure S4** * Content: Frequency sweep rheometry of
 the storage (G’) moduli measured for bulk pure collagen and composite with
 microgels of different stiffness after decellularizing the matrix. ###
 **Figure S5** * **Content**: Representative polar plots for trajectories
 of fibroblasts and myofibroblasts near microgels, and mean speed
 comparison across conditions. ### **Figure S6** * **Content**: Histograms
 and volume fraction analysis of PEGDA microgel size distributions. --- ##
 Citation If you use this dataset in your work, we request that you cite
 the corresponding publication: **Title**: Rigidity sensing of inclusions
 directs differentiated cell elongation and force generation across
 phenotypes\ **Journal**: ACS Biomaterials Science & Engineering --- ##
 Contact For questions regarding the data or scripts: * Refer to the
 figure-specific README files for detailed information. * Contact
 information is provided in the associated publication. ---