Effects of embedding muscle fibers in a 3D fibrin/basement membrane hydrogel during culture. (A) Graphical illustration of the 3D culture experiment (image made in BioRender). (B) Viability classification of muscle fibers cultured in 2D and 3D at days 1 and 7. (C) Dedifferentiation classification of muscle fibers cultured in 2D and 3D at days 1 and 7. (D) Representative confocal maximum intensity projection images stained for α-actinin (green), myosin (red), and nuclei (blue) in muscle fibers cultured for 7 days in 2D and 3D. Note the lack of sprouting of the muscle fiber end in the 3D cultured muscle fibers. (E) Quantification of muscle fiber length cultured in 3D at days 1 and 7 of ex vivo culture. Dashed lines represent the mean length results of the 2D experiment in Fig. 1. (F) Quantification of muscle fiber width cultured in 3D at days 1 and 7 of ex vivo culture. Dashed lines represent the mean length results of the 2D experiment in Fig. 1. (G–I) Contractile measurements of muscle fibers kept in culture for 1 and 7 days in 2D and 3D culture systems. (G) Quantification of resting sarcomere length at days 1 and 7 cultured in 2D and 3D. (H) Quantification of percentage of sarcomere shortening at days 1 and 7 cultured in 2D and 3D. (I) Quantification of contractile velocity at days 1 and 7 cultured in 2D and 3D. Data are means ± SEM; large dots represent mean values per mouse, and small dots represent single muscle fibers. Significance was determined using χ-square test in panel B, Student’s t test in panels E and F, and two-way ANOVA for panels G–I with P < 0.05 considered as significant with ** = P < 0.01 and **** = P < 0.001.
Figure 2.

Effects of embedding muscle fibers in a 3D fibrin/basement membrane hydrogel during culture. (A) Graphical illustration of the 3D culture experiment (image made in BioRender). (B) Viability classification of muscle fibers cultured in 2D and 3D at days 1 and 7. (C) Dedifferentiation classification of muscle fibers cultured in 2D and 3D at days 1 and 7. (D) Representative confocal maximum intensity projection images stained for α-actinin (green), myosin (red), and nuclei (blue) in muscle fibers cultured for 7 days in 2D and 3D. Note the lack of sprouting of the muscle fiber end in the 3D cultured muscle fibers. (E) Quantification of muscle fiber length cultured in 3D at days 1 and 7 of ex vivo culture. Dashed lines represent the mean length results of the 2D experiment in Fig. 1. (F) Quantification of muscle fiber width cultured in 3D at days 1 and 7 of ex vivo culture. Dashed lines represent the mean length results of the 2D experiment in Fig. 1. (G–I) Contractile measurements of muscle fibers kept in culture for 1 and 7 days in 2D and 3D culture systems. (G) Quantification of resting sarcomere length at days 1 and 7 cultured in 2D and 3D. (H) Quantification of percentage of sarcomere shortening at days 1 and 7 cultured in 2D and 3D. (I) Quantification of contractile velocity at days 1 and 7 cultured in 2D and 3D. Data are means ± SEM; large dots represent mean values per mouse, and small dots represent single muscle fibers. Significance was determined using χ-square test in panel B, Student’s t test in panels E and F, and two-way ANOVA for panels G–I with P < 0.05 considered as significant with ** = P < 0.01 and **** = P < 0.001.

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