Myogenic progression of immortalized human myoblasts in different 3D biomaterial scaffolds in vitro.

Immortalized human myoblasts C25Cl48 were embedded in collagen I (500 000 cells / 100 μl: A and D), Fibrin (500 000 cells / 100 μl: B and E) or PEG-Fibrinogen (500 000 cells / 100 μl: C and F), cultured in proliferation medium for 2 days (A-C) and then switched to differentiation medium for 6 days (D-F). After 2 days in proliferation medium, cells were pulsed with the thymidine analogue 5-Ethynyl-2’deoxyuridine (EdU) for 2 hours and then fixed, before EdU incorporation was visualised and myoblasts immunolabelled for β-Tubulin and nuclei counterstained with DAPI. After 6 days in differentiation medium, cells were fixed and immunolabelled for myosin heavy chain (MyHC) to visualise myotubes and counterstained with DAPI. Proliferating EdU positive (arrow-heads) and β-Tubulin myoblasts were aligned in collagen I (A) and Fibrin (B) gels but randomly orientated in PEG-Fibrinogen scaffold (C). After 6 days in differentiation medium many MyHC positive cells were present but only a few small myotubes were detected in collagen I (D) or Fibrin (E) gels. In PEG-Fibrinogen scaffold (C), small myotubes were randomly orientated. Scale bar represents 100 μm. Representative images from 3 or more independent experiments. Expression of the sarcomeric proteins Myosin Heavy chain (MYH2, 3, 8) (G), Actinin α3 (ACTN3) (H) and Tropomyosin 1 (TPM1) (I) and creatine kinase (CKM) (J) after 7 days of differentiation were analysed by RT-qPCR. Expression was normalized to the house keeping gene TBP. Data are mean±SEM from 3 independent gels where an asterisk denotes a significant difference (p<0.05) from the Collagen gel using an unpaired two-tailed Student's t-test.