Engineering a skeletal muscle model to study extracellular vesicle dynamics

Figures

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  Figure 1.. Diagram illustrating the fabrication of tissue engineered skeletal muscle C2C12 constructs. After passaging the cells, they were resuspended in the collagen/Matrigel® mix. Constructs were incubated for 4 days in GM. From day 4 onwards, GM was replaced with myogenic DM. For EV collection, CM was recovered every 48 h intervals and SM-EVs isolated.

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  Figure 2.. SM model composition. The impact of Matrigel® concentration (a–f) and cell density (g–i) on SM parameters: (a) longitudinal images evidence the presence of mature SM structures in constructs composed of (i) 20%, (ii) 40% and (iii) 60 Matrigel® (red: F-actin, blue: DAPI, scale bars = 100 µm), (b) cross sections displaying MyHC (green) and DAPI (blue) at all Matrigel® concentrations (scale bars = 50 µm), (c) normalised MyHC positive area (%) from quantification of fluorescence staining, (d) myotube CSA (µm), (e) quantitative force (µN) measurements for tetanus and twitch, (f) graphical representation of individual tetanus and twitch measurements, (g) quantification of total nuclei per CSA for all cell concentrations, (h) quantification of myotube CSA (µm), and (i) representation of normalised MyHC positive area (%) after fluorescence quantification. (N = 3).

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  Figure 3.. Profiling EVs dynamics in an SM model: (a) cross sectional and longitudinal immunofluorescence images of 3D SM model at days 14 and 16 of differentiation (green: MyHC, red: Rhodamine and blue: DAPI). Scale bar: 20 µm, (b) particle distribution for samples collected at day 14, day 16, day 18, day 20 and combined samples, (c) boxplots displaying (i) average particle concentration measurements and (ii) average particle sizes, (d) western blots displaying Alix, Annexin A2, CD63, CD9 and calnexin at days 14, 16, 18, 20 and combined (comb), (e) table presenting particle concentration, average size, protein concentration, particle-to-protein (PTP) ratio and concentration of particles isolated per individual construct, (f) representative TEM image displaying EV material isolated using the UF + SEC protocol, (g) percentage MyHC coverage, (h) zeta potential following supplementation with leucine, (i) western blot for Alix, α-SCGA, β-SCGA and APOA1, and (j) semi-quantitation of Alix expression in leucine supplemented SM model (N = 3). *p < 0.05. **p < 0.01. ***p > 0.001.