Figure 1.
Schematic of polyacrylamide gel incorporation into a multiwell plate.
PA gels are cast using an array of coverglass to sandwich polymerization solutions within a multiwell plate, followed by ligand conjugation and sterilization.
Figure 2.
Measurement of substrate elastic modulus and thickness.
(A) Acrylamide: bisacrylamide content was extrapolated from Yeung et al. [10] to target a broad physiologically relevant stiffness range. Young's modulus was determined by AFM microindentation of gels cast within three separate 96 well plates. Data are mean ± SD (n = 3). (B) Final gel thicknesses are within 11% CV for each stiffness condition, but variable between stiffness due to differences in gel swelling with bisacrylamide crosslink content. Data are mean ± SD (n = 5) from one 96 well plate.
Figure 3.
Surface density of collagen across matrix stiffness.
(A) Chemiluminescence-based detection of gel-bound collagen. The no sulfo-SANPAH controls assess non-specific adsorption of collagen to the gels. (B) Average pixel intensities of the chemiluminescent signals imaged in (A). (C) Relationship between applied collagen concentration and signal intensity measured across all stiffness conditions in (B).
Figure 4.
Automated imaging of cell morphology in a 384 well plate.
Seven cell types (Table 1) cultured across increasing substrate stiffness, stained for F-actin (red) and nuclei (blue). Images were obtained at 200X magnification.
Figure 5.
Stiffness-dependent growth profiles.
12 cell types (Table 1) cultured across 5 stiffness conditions in 96 well plates. Fold change represents number of attached cells at 72 h versus the 4 h attachment density. Cells are classified as normal (green), SV40- or adenovirus-transformed (orange), or spontaneously immortalized (blue). Error bars indicate mean ± SD (n = 3). P values from one-way ANOVA are indicated in each plot. *p<0.05 vs. growth on the softest substrate (0.3 kPa) by Tukey's test.
Table 1.
Cell types.
Figure 6.
Substrate stiffness interactions with soluble and matrix factors.
(A) Effect of varying collagen ligand density on cell accumulation. Applied collagen concentrations are indicated. Fold change represents number of attached cells at 72 h versus the 4 h attachment density. (B) NHLFs accumulate more rapidly on stiffer substrates in high serum concentrations. No significant differences in accumulation are detected in low serum concentrations, but both 0% and 1% conditions exhibit statistically positive trends with increasing stiffness (p = 0.0032, p = 0.013, respectively, by two-tailed t-test). (C) Increasing matrix stiffness promotes BrdU incorporation while suppressing caspase 3/7 activity in cells cultured in 10% serum. Values are normalized per cell number and expressed relative to the maximal value. Error bars indicate mean ± SD (n = 3). *p<0.05 vs. effect on the softest substrate (0.3 kPa) by Tukey's test. #p<0.05 vs. effect at the lowest collagen density by t-test.
Figure 7.
Modification of drug responses on 1 kPa vs. glass.
(A) Compounds that inhibit cell growth with similar potency on 1 kPa and collagen-coated glass (rigid). For each stiffness condition, fold change represents the number of cells at the indicated dose (µM) vs. a zero dose control after 72 hours in culture. (B) Compounds that affect relative cell growth differentially (p<0.05) between the two stiffness contexts. (C) Stiffness-independent effects of a Rac1 inhibitor. (D) Reduced potency of an FGF1 receptor inhibitor on 1kPa vs. glass. (E) Increased potency of a PP1 inhibitor on 1 kPa vs. glass. (F) Stimulation of cell growth on 1 kPa by a myosin II inhibitor. (G) Absolute cell numbers following blebbistatin treatment in F. Error bars indicate mean ± SD (n = 3) in A–G. (H) Blebbistatin (10 µM) stimulates cell growth on 1 kPa substrates, with no effect on 20 kPa substrates. Fold change indicates cell number over no drug control. Error bars indicate mean ± SEM (n = 5 experiments). #p<0.05 vs. the rigid condition by t-test for all panels.
Table 2.
Molecular actions of tested compounds.