Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels

Abstract

In vitro models of skeletal muscle are critically needed to elucidate disease mechanisms, identify therapeutic targets, and test drugs pre-clinically. However, culturing skeletal muscle has been challenging due to myotube delamination from synthetic culture substrates approximately one week after initiating differentiation from myoblasts. In this study, we successfully maintained aligned skeletal myotubes differentiated from C2C12 mouse skeletal myoblasts for three weeks by utilizing micromolded (μmolded) gelatin hydrogels as culture substrates, which we thoroughly characterized using atomic force microscopy (AFM). Compared to polydimethylsiloxane (PDMS) microcontact printed (μprinted) with fibronectin (FN), cell adhesion on gelatin hydrogel constructs was significantly higher one week and three weeks after initiating differentiation. Delamination from FN-μprinted PDMS precluded robust detection of myotubes. Compared to a softer blend of PDMS μprinted with FN, myogenic index, myotube width, and myotube length on μmolded gelatin hydrogels was similar one week after initiating differentiation. However, three weeks after initiating differentiation, these parameters were significantly higher on μmolded gelatin hydrogels compared to FN-μprinted soft PDMS constructs. Similar results were observed on isotropic versions of each substrate, suggesting that these findings are independent of substrate patterning. Our platform enables novel studies into skeletal muscle development and disease and chronic drug testing in vitro.

Figure 1. Fabrication and characterization of μmolded gelatin hydrogels.

(a) Glass coverslips (blue) were masked with low-adhesive tape (orange), laser-engraved to selectively expose the center region, and chemically activated to facilitate gelatin adhesion. Gelatin solution (yellow) was dropped on the coverslip and molded with a PDMS stamp (gray). After overnight incubation, the stamp and tape were removed. The construct was then incubated in MTG solution (green) for four hours, rinsed, sterilized, and seeded with cells. (b) 10% w/v gelatin solutions were cast in Petri dishes and solidified into hydrogels after overnight incubation at room temperature. The bulk compressive elastic modulus of hydrogel cylinders was measured immediately (no MTG) or after incubation in the 10% w/v MTG solution for four or 24 hours at room temperature (mean ± s.e.m., n = 4 for each sample, *indicates statistically significant difference, ANOVA followed by Tukey’s test for multiple comparisons, p < 0.05). Details of statistical analysis are located in Supplementary Table S1. (c) AFM scan of the surface of a μmolded gelatin hydrogel construct. Color bar indicates height. (d) Values for the height of the cross-section of a μmolded gelatin hydrogel construct, illustrating the dimensions of the μmolded features. (e) Cross-section of a μmolded gelatin hydrogel construct doped with fluorescent beads, illustrating the overall height of the hydrogel construct.

Figure 2. Degradation of μmolded gelatin hydrogels in culture-like conditions.

Measurements of the relative dry mass (a) and relative wet mass (b) of μmolded gelatin hydrogels after incubation for the indicated time in PBS at 37 °C (n = 7 for zero days and one day, n = 6 for seven days, *indicates statistically significant difference compared to zero days, Kruskal-Wallis test followed by Tukey’s test for multiple comparisons, p < 0.05). Details of statistical analysis are located in Supplementary Tables S2 and S3. Features μmolded onto gelatin hydrogels were present both before (c) and after (d) overnight incubation in PBS at 37 °C. Histograms of elastic moduli values collected from μmolded gelatin hydrogels incubated for zero days (e) or one day (f) in PBS at 37 °C. Each color represents values recorded from an independent construct (n = 3 for 0 days and 1 day). Average elastic modulus values ± s.e.m. are indicated on the plots.

Figure 3. Cell adhesion on engineered constructs.

(a) Representative images of C2C12 skeletal myoblasts seeded and differentiated into myotubes on FN-μprinted PDMS (first column), FN-μprinted soft PDMS (second column), and μmolded gelatin hydrogels (third column). Images were collected one week (first row) and three weeks (second row) after initiating differentiation of myoblasts into myotubes. Blue: nuclei, red: sarcomeric α-actinin. Total number of nuclei present on μpatterned (b) and isotropic (c) constructs (n is indicated below each bar, *indicates statistically significant difference compared to PDMS construct at same time point, Kruskal-Wallis test followed by Tukey’s test for multiple comparisons, p < 0.05). Details of statistical analysis are located in Supplementary Tables S4 and S5.

Figure 4. Quantification of myogenic index, myotube width, and myotube length on engineered constructs.

Myogenic index (i), myotube width (ii), and myotube length (iii) for μpatterned (a) and isotropic (b) constructs one week and three weeks after initiating differentiation of myoblasts into myotubes (n is indicated below each bar, *indicates statistically significant difference compared to soft PDMS constructs at Week 1, ^†indicates statistically significant difference compared to soft PDMS constructs at Week 3, Kruskal-Wallis test followed by Tukey’s test for multiple comparisons, p < 0.05). Detailed statistical analyses are located in Supplementary Tables S6–S11.

Figure 5. Myotube length on engineered constructs.

(a) Representative images of C2C12 skeletal myoblasts cultured on FN-μprinted soft PDMS and μmolded gelatin hydrogels, stained one week (Week 1) and three weeks (Week 3) after initiating differentiation into myotubes. (b) Histograms of myotube lengths in FN-μprinted soft PDMS and μmolded gelatin hydrogels.

Figure 6. Tissue thickness and sarcomere formation in myotubes.

(a) 3-D volume of skeletal myotubes cultured on μmolded gelatin hydrogels, illustrating that the tissue is a relatively flat monolayer. Myotubes with visible sarcomeres on μmolded gelatin hydrogels one week (a) and three weeks (b) after differentiation. Blue: nuclei, red: sarcomeric α-actinin.

Figure 7. Global myotube alignment on isotropic and μmolded gelatin hydrogels.

(a,b) For each coverslip, five images of sarcomeric α-actinin (white) were captured at multiple locations and assembled into a five-panel montage. Global myotube alignment was calculated as the orientational order parameter of vectors assigned to sarcomeric α-actinin intensities within each montage. Representative isotropic (a) and μmolded (b) montages with low and high global myotube alignment, respectively. Dotted yellow lines were added to illustrate boundaries between individual images. (c) Global myotube alignment for isotropic and μmolded gelatin hydrogels at each time point (mean ± s.e.m, n is indicated below each bar, *indicates statistically significant difference compared to isotropic gelatin constructs at same time point, ANOVA followed by Tukey’s test for multiple comparisons, p < 0.05). Detailed statistical analyses are located in Supplementary Table S12.