Methacrylated human recombinant collagen peptide as a hydrogel for manipulating and monitoring stiffness-related cardiac cell behavior

Abstract

Environmental stiffness is a crucial determinant of cell function. There is a long-standing quest for reproducible and (human matrix) bio-mimicking biomaterials with controllable mechanical properties to unravel the relationship between stiffness and cell behavior. Here, we evaluate methacrylated human recombinant collagen peptide (RCPhC1-MA) hydrogels as a matrix to control 3D microenvironmental stiffness and monitor cardiac cell response. We show that RCPhC1-MA can form hydrogels with reproducible stiffness in the range of human developmental and adult myocardium. Cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (cFBs) remain viable for up to 14 days inside RCPhC1-MA hydrogels while the effect of hydrogel stiffness on extracellular matrix production and hPSC-CM contractility can be monitored in real-time. Interestingly, whereas the beating behavior of the hPSC-CM monocultures is affected by environmental stiffness, this effect ceases when cFBs are present. Together, we demonstrate RCPhC1-MA to be a promising candidate to mimic and control the 3D biomechanical environment of cardiac cells.

Keywords: Biomaterials; Cell biology; Materials in biotechnology; Stem cells research.

Graphical abstract

Figure 1

Reaction scheme for RCPhC1 modification

Figure 2

RCPhC1-MA material characterization (A and B) Young’s modulus varies with different illumination time, dosage, and the degree of methacrylation (%DS, n = 3). (C) Young’s modulus of RCPhC1-MA hydrogels upon varying %DS (n = 3-9). (D) Mass swelling percentage differs with %DS (n = 3). (E) Degradation of RCPhC1-MA hydrogels with various %DS in the presence of collagenase (n = 3). (F-H) SEM of surface relief of RCPhC1-MA gels of varying %DS. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗∗ = p < 0.0001. Scale bar = 5 μm. All data are presented as mean ± SEM. For multiple group comparison, a one-way analysis of variance (ANOVA) with Tukey’s post-hoc test was used.

Figure 3

Cytocompatibility of RCPhC1-MA (A and B) Quantification of cFB (A) and hPSC-CM (B) viability in 3D culture inside RCPhC1-MA hydrogels with varying %DS over 1, 7, and 14 days (n = 3). (C-E) hPSC-CMs and cFBs (70:30) cultured in low %DS and intermediate %DS exhibited a clustered morphology, as opposed to the more spread-out morphology in the high %DS. Notable organized sarcomeric α-actinin is found in all constructs after 7 days of encapsulation. ∗∗ = p < 0.01. Scale bar = 20 μm. All data are presented as mean ± SEM. For multiple group comparison, a one-way analysis of variance (ANOVA) with Tukey’s post-hoc test was used.

Figure 4

Collagen production and hydrogel stiffness with time over 14-day culture period (A, D, and G) Micro-indentation data depicting hydrogel stiffness over time in cell-free (- cells) and cell-laden (+cells) hydrogel constructs (n = 3 per condition). ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. All data are presented as mean ± SEM. For multiple group comparison, a one-way analysis of variance (ANOVA) with Tukey’s post-hoc test was used. (B, C, E, F, H, I) Representative fluorescent images demonstrating collagen production (green, CNA-35-OG) and F-actin (magenta, phalloidin) in the RCPhC1-MA constructs with time. Scale bar = 100 μm.

Figure 5

CM contractility is affected by both culture time and hydrogel stiffness (A and B) Bar graphs indicating the effect of culture time and %DS on hPSC-CM beating frequency in monocultures (A) and hPSC-CM/cFB co-cultures (B), accompanied by a summary of the two-way Analysis of Variance (ANOVA) results to analyze interaction effects. (C and D) Bar graphs indicating the effect of culture time and %DS on hPSC-CM contraction amplitude (a.u.) in monocultures (C) and hPSC-CM/cFB co-cultures (D), accompanied by a summary of the two-way ANOVA results to analyze interaction effects. a, significance vs. day 1 group; b, significance vs. day 3 group; c, significance vs. day 8 group; ∗, significance vs. low %DS within each time period; #, significance vs. intermediate %DS within each time period. Significant differences between timepoints and between %DS were determined using one-way ANOVA (n = 3-15 cell clusters analyzed per condition). p < 0.05. All data are presented as mean ± SEM. Moreover, two-way ANOVA was performed to assess the interaction effects of culture time and %DS on hPSC-CM contraction frequency and amplitude. See also Video S1: Encapsulated hPSC-CMs in the low %DS hydrogels after eight days of culture. Related to Figure 5, Video S2: Encapsulated hPSC-CMs in the intermediate %DS hydrogels after eight days of culture. Related to Figure 5, Video S3: Encapsulated hPSC-CMs in the high %DS hydrogels after eight days of culture. Related to Figure 5.