Comparing spontaneous C2C12 organization on isotropic gelatin hydrogels and TCPS

Tissue-level self-organization is driven by intercellular feedback as well as cues from the external environment. Given that C2C12 myoblasts locally self-organize even on conventional isotropic TCPS21, we hypothesized that on highly deformable substrates, more extensive self-organization, may occur. To investigate this hypothesis, we compared C2C12 culture on TCPS to cultures on gelatin hydrogels cross-linked with transglutaminase (TG), which can be formulated with Young’s Modulus as low as single kPa23. In order to quantify the degree of cellular alignment across samples, we utilized the ImageJ Plug-In, OrientationJ24 to evaluate the distribution of cellular alignments and generate an alignment score (0–1) equivalent to 0–100% of the signal falling within the same 20°. In all samples, fluorescent images of cellular F-actin were applied as input. To compare local vs global scale organization, we analyzed incrementally larger field of views (FOV) spanning ~ 350 µm × 350 µm (0.125 mm2) to 4 mm × 4 mm (16 mm2), see Fig. 1a. Using this approach, we observed a rapid decline in the degree of alignment for C2C12 myotubes on TCPS as FOV increases, in agreement with previous work21, see Fig. 1b. Subsequently, we investigated spontaneous myotube alignment on 2.5, 5 and 10% w/v gelatin hydrogels cross-linked with 10 U/mL TG. Interestingly, despite being several orders of magnitude softer than TCPS, hydrogels based on 10 and 5% w/v gelatin, gave rise to cellular self-organization properties that were close to identical to those observed for TCPS, see Fig. 1c,d. For the largest FOVs, the degree of alignment was even lower than that observed on TCPS. However, when gelatin content was lowered further to 2.5% w/v we observed a striking change. Even in the largest FOV of 16mm2, more than 50% of the culture was oriented within 20°. This is comparable to the degree of alignment observed only for the smallest, most local FOVs for the other conditions. Further, the local organization was also increased for the 2.5% w/v gelatin, as more than 75% of the culture fell within 20° for the smallest FOV in the analysis.

Figure 1

Local and global spontaneous myotube alignment. C2C12 myoblasts were cultured on TC polystyrene and stained for F-actin after 9 days of differentiation. (a) Sample images (of n = 4) show that in conventional cell culture the degree of cellular alignment decreases with increasing field of view (FOV). Scale-bars: (left to right) 100 µm, 250 µm, 1 mm (b) Myotube alignment was quantified from F-actin images using the ImageJ Plug-in, OrientationJ, and given as an alignment score (0–1) for multiple FOVs ranging from 0.125 mm2 to 16 mm2. Areas shaded in dark grey indicate values below theoretical point of random alignment (0.11) for the analysis method (c) Large FOV images (16 mm2, scale bars = 1 mm) of C2C12 myotubes on various substrates at day 9 of differentiation. Left to right: TC polystyrene, 10% w/v gelatin—10 U/mL Transglutaminase (TG), 5% w/v gelatin—10 U/mL TG and 2.5% w/v gelatin—10 U/mL TG. (d) Alignment score as a function of FOV for C2C12 myotubes developed on various substrates. Left to right: TC polystyrene, 10% w/v gelatin—10 U/mL TG, 5% w/v gelatin—10 U/mL TG and 2.5% w/v gelatin—10 U/mL TG. Points indicate mean ± s.e.m. (n = 4). Evaluation performed on day 9 after initiation of differentiation.

The mechanical properties of soft gelatin hydrogels regulate C2C12 self-organization

The mechanical properties of enzymatically cross-linked gelatin hydrogels is influenced by both the solid protein content, gelatin bloom, and extent of covalent cross-linking induced by TG23. To investigate the influence of enzymatically introduced covalent cross-linking, we varied the TG concentrations between 10, 2.5 and 0.6 U/ mL, for hydrogels with 10, 5 and 2.5% w/v gelatin content, see Fig. 2. 2.5% w/v gelatin cross-linked with 0.6 U/mL TG failed to form a stable hydrogel. The remaining eight hydrogel compositions were stable and were used as substrate for C2C12 culture. Following proliferation and differentiation into myotubes, we evaluated local and global alignment, for each of the gel compositions, see Fig. 2a,b. We observed a clear global-scale myotube self-organization only for selected compositions: 5% w/v gelatin—0.6 U/ml TG, 2.5% w/v gelatin—10 U/ml TG and 2.5% w/v gelatin—2.5 U/ml TG. For all other compositions, the self-organization was highly similar to that observed for conventional TCPS. These distinct types of organization—only local vs. global—appeared at the onset of myotube formation, see supplementary Fig. 1.

Figure 2

Substrate mechanical properties determine spontaneous myotube alignment. (a) Alignment score as a function of FOV for C2C12 cultures developed on a range of gelatin hydrogels varying in both gelatin concentration (color): 2.5–10% w/v , and transglutaminase concentration (TG) (pattern): 0.6–10 U/ml. Error bars indicate mean ± s.e.m. (n = 4). Evaluation performed on day 9 after initiation of differentiation. (b) Comparison of the alignment score for the largest field of view (16 mm2) for each of the analyzed conditions. Error bars indicate mean ± s.e.m. (n = 4). Statistical significance: ***P < 0.001. (c) Young’s modulus for each hydrogel compositions analyzed in (a-b), error bars indicate mean ± s.e.m., for all conditions n ≥ 3 (d) Stress vs. strain equivalent for 5% w/v gelatin hydrogels cross-linked with 0.6, 2.5 or 10 U/ml TG, under compression. Less strain stiffening is observed for lower degrees of enzymatic cross-linking. Vertical lines indicate 0.1, 0.2 and 0.3 engineering strain (ε) (e) Approximate effective modulus for 5% w/v gelatin hydrogels (0.6–10 U/ml TG) in strain regimes 0.01–0.1, 0.1–0.2 and 0.2–0.3 engineering strain (ε).

For each of the hydrogel compositions, we next estimated their Young’s modulus at ambient conditions, summarized in Fig. 2c. As expected, the stiffness generally increased with increasing gelatin and TG concentrations, with the gelatin solid content having the largest impact on the nominal Young’s modulus. Notably, the three samples that gave rise to global self-organization were the three softest compositions, which all displayed Young’s modulus ≤ 6 kPa. While the three compositions based on 5% w/v gelatin displayed similar Young’s modulus of ~ 7.1, ~ 7 and ~ 6 kPa, we only observed large-scale alignment for the formulation with the lowest amount of TG. Interestingly, this formulation was notably less strain-stiffening, see Fig. 2d,e, which can have significant influence on cellular responses25,26. Based on our observations, we performed a secondary screening of hydrogels in the range of 3–5% w/v gelatin with 0.6 or 0.3 U/mL TG, see supplementary Fig. 2. This confirmed that large-scale self-organization occurs in this range, given the appropriate amount of covalent cross-linking. We thus conclude that several gel formulations can be made to yield isotropic substrates where large-scale self-organization occur, by balancing gelatin concentrations below 5% w/v with an appropriate cross-linking degree. A Young’s modulus of 6 kPa appears to be the upper threshold. To ensure that our observations were not tied to the particular strain of the C2C12 cell line, we performed a control experiment culturing a separate C2C12 line on 2.5% w/v gelatin with 10U/mL TG. Indeed, this also displayed large-scale spontaneous alignment, see supplementary Fig. 3. Lastly, it is worth noting that for very low TG concentrations of 0.3 U/ml, gel stability was diminished, and several layers of cells were observed. A similar behavior was observed for 0.6 U/ml TG, in particular for gelatin concentrations ≤ 3% w/v.

Figure 3

Spontaneous global organization increases myotube lengths. (a) Representative composite image (n = 4) of F-actin in C2C12 myotubes, spanning a full well in a standard 12-well plate. Composite images were composed of 252 frames, and the OrientationJ ImageJ plug-in was used to false-color the image according to the local angle of orientation of F-actin filaments. Top: C2C12 culture on 10% w/v gelatin—10 U/mL TG hydrogels. Bottom: C2C12 culture on 2.5% w/v gelatin—10 U/mL TG hydrogels. Close-up areas: 1cm2, scale bars 5 mm (b) Length of myotubes developed on a range of hydrogel substrates: gelatin: 2.5–10% w/v, TG: 0.6–10 U/ml. Middle bar represents median length, while the upper and lower bound of the box respectively represents the upper and lower quartile. Lines indicate largest and lowest value within 1.5 times the interquartile range. For each gel composition, four separate cultures were used to evaluate at least 100 random myotubes. Statistical significance: *P < 0.05, ***P < 0.001 (c) Distributions of myotube lengths for 2.5, 5 and 10% (w/v) gelatin hydrogels, cross-linked using 10 U/mL TG. Evaluation performed at day 9 after initiation of differentiation.

Large-scale self-organization increases C2C12 myotube length

Native adult myotubes span from several mm to cm in length. In contrast, engineered C2C12 myotubes developed in conventional culture plates rarely exceed 0.5 mm due to their lack of organization21. Therefore, for the soft gelatin substrates that gave rise to spontaneous self-organization, we hypothesized that longer myotubes would simultaneously be obtained. To evaluate the degree of the self-organization, we imaged several full wells in a 12-well plate comparing a high 10% w/v and low 2.5% w/v gelatin hydrogel each cross-linked using 10 U/ml TG,see Fig. 3a,b, and supplementary Fig. 4. We applied the ImageJ OrientationJ plugin to identify and false-color images according to the local angular orientation of the F-actin filaments. As anticipated, we observed large-scale organization for all 2.5% w/v gelatin formulations, while for all 10% w/v gelatin we found distinct ~ 500 µm-sized locally organized domains. Interestingly, for the soft formulation, the large-scale alignment appeared to be influenced significantly by the edges of the well, while one or a few orientations would dominate towards the center. This indicates that the degree of organization could be limited by the presence of external walls. Next, we evaluated the myotubes length on each of the previously investigated gel formulations at day 9 of differentiation. Here, we observed a drastic increase in median myotube length from 0.3 mm to 0.8 mm from the stiffest to softest gel formulation, with myotube lengths frequently reaching 2 mm see Fig. 3b,c. Notably, the measured lengths were restricted by the boundaries of the 3.4 mm × 2.7 mm FOV used in the analysis. Indeed, when extending differentiation time to 21 days and FOV to 15.8 mm × 11.9 mm, we observed several myotubes spanning > 5 mm, thus approaching their natural lengths27, see supplementary Fig. 5.

Figure 4

Long term C2C12 culture on compliant gelatin hydrogels generates myotubes of high contractile maturity. (a) Confocal images of immunostained myotubes at day 21 on 2.5% w/v gelatin—10 U/ml TG substrate. Blue: DAPI, Red: Actin, Green: α-actinin. Scale bars, top: 100 µm, Bottom: 25 µm (b) Bright field microscopy images of spontaneously contracting myotubes generated on 2.5% gelatin—10 U/ml TG hydrogels, at day 12 of differentiation. Grid interval: 0.5 mm, scale-bar 250 µm. Inserted circles mark immediately traceable points, and their displacement upon contraction. Corresponding videos can be found in the supplementary information (c) Western-blot evaluation of selected myotube proteins across various substrates and time-points. For each protein, the expression relative to that obtained for day 21 of differentiation on the 2.5% gel is shown. Error bars indicate mean ± s.e.m. Corresponding representative western blots are depicted below each graph. Full gel images can be found in the supplementary information. Two statistical analysis were performed: i) Two-way ANOVA followed by Sidak’s post hoc test for 2.5% and 10% samples at days 7 and 21, Statistical significance indicator: *P < 0.05 significant difference between day 7 and day 21 differentiation ii) One-way ANOVA followed by pairwise Tukey’s post hoc test for multiple comparison between samples (n = 2 samples excluded).Statistical significance indicators: #P < 0.05, ##P < 0.01, significant difference with day 7 TCPS.

Long-term culture of C2C12 myotubes on compliant gelatin improves their contractile maturity

Having established that C2C12 myoblasts can self-assemble into aligned and extended myotubes on compliant gelatin substrates, we next sought to evaluate the functional maturity of these myotubes. An important benefit of applying compliant substrates is that these allow extended culture duration of myotubes19, while myotubes tend to delaminate after approximately a week of differentiation on conventional TCPS. Thus, we monitored the functional maturity of myotubes developed on the gelatin hydrogel substrates for up to three weeks of differentiation. We compared three gel compositions: ‘soft’ ~ 1 kPa (2.5% w/v gelatin—10 U/ml TG) and ‘medium’ ~ 6 kPa (5% w/v gelatin—0.6 U/ml TG) where large scale self-organization occur, and ‘hard’ ~ 20 kPa (10% w/v gelatin—10 U/ml TG) gels where no large-scale organization is observed. After 21 days of differentiation, we performed immunohistochemistry and confocal imaging. For all three compositions, this revealed highly fused and striated myotubes, see Fig. 4a and supplementary Fig. 6. Interestingly, after approximately 10 days of differentiation, we observed strong spontaneous contractions, see Fig. 4b and supplementary movies 1–4. These contractions would continue for the remaining culturing period. For the observed samples, see supplementary movies, the contractions appear more concerted on the soft and medium substrates. However, we did not perform a stringent analysis and cannot rule out that these qualitative observations were due to chance. We next compared a number of skeletal muscle markers for myotube samples at day 7 and 21 of differentiation on gel substrates, see Fig. 4c. We further compared these to myotubes differentiated for 7 days on conventional TCPS, the longest reliable maturation time on this substrate. For β-actin, serving as an equal protein loading control, we observed no notable changes in relative expression level across any of the tested substrates and time points. For skeletal muscle α-actin isoform, which is associated with myoblast fusion and muscle formation28, we also observed similar expression across all samples, apart from the softest gel sample at day 7, which was notably lower. This could indicate that myoblast fusion was slower on the softest substrates. Similarly, myogenin–a key regulator of myoblast differentiation and fusion28– had lower relative expression on both the soft and hard gel substrates at day 7, as compared to TCPS. A similar trend was observed when analyzing these markers prior to fusion at the onset of differentiation, see supplementary Fig. 7, however, after 21 days of differentiation, these became comparable to TCPS. These observations indicate that some aspects of myotube fusion are slower on the softest hydrogel substrates, but reach a similar level to that observed on TCPS after extended culture. Lastly, to evaluate the functional maturity of the myotubes, we compared the expression level of the late-stage maturation markers contractile myosins MyHC-I (slow) and MyHC-II (fast)29 for all samples. Interestingly, for all day 7 samples we did not detect expression. In contrast, for all three hydrogel-based substrates we detected both MyHC-I and MyHC-II after 21 days of differentiation, with MyHC-I expression being notably higher on the medium gel formulation.

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