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. 2021 Jun 3;11:11695. doi: 10.1038/s41598-021-91203-5

Figure 4.

Figure 4

3D culture of primary human skeletal muscle enhances acetylcholine receptor clustering, produces functional force and upregulates transcription of post-synaptic mRNAs. (a) Confocal tile scans of single z-plane (Tissue engineered 3D) and fluorescence images (Monolayer 2D) of human skeletal muscle labelled for AChR on the post-synaptic membrane, pan-myosin heavy chain (MF-20), laminin (monolayer only) and nuclear DNA (DAPI) across 1, 2 and 3 weeks culture. Morphological analysis of (b) myotube diameter, (c) AChR density and (d) cluster size. Transcriptional analysis of myogenic; (e) MYOD and (f) MYOG, and post-synaptic; (g) MUSK and (h) LRP4 mRNAs. Blue bars = Monolayer (2D), Red bars = Spheroid (3D). (i) Zoom images of AChR in monolayer (2D) and tissue engineered (3D) skeletal muscle. (j) Functional tetanus and twitch force data and (k) representative traces in engineered tissues. Δ change indicative of normalised data to the earliest monolayer time-point. Beginning of representative tetanus and twitch contractions represents initiation of electrical field stimulation, time elapsed before this point is un-stimulated baseline recording. Data presented ± standard deviation (SD). *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. Scale bars = (a) 100 µm, (i) 20 µm.