Fetal bovine serum f7524

The myoblasts were isolated and cultured according to the protocol described by Fauconneau and Paboeuf (2000) [103 (link)]. The fast-twitch muscles were collected from the epaxial region and mechanically dissociated with scalpels. To release the muscle cells, the fragments were enzymatically digested with 0.2% collagenase type I (C9891) and 0.1% trypsin (T4799) (Sigma-Aldrich, St. Louis, MO, USA). The cell suspension was filtered in cell strainers (Corning, New York, NY, USA), allowing for the removal of debris, centrifuged, and the cell pellet was resuspended in DMEM medium (DMEM (D7777), 9 mM NaHCO₃ (S5761), 20 mM HEPES (H3375), pH 7.4), with 1% antibiotics (A5955) and 10% fetal bovine serum (F7524) (Sigma-Aldrich, St. Louis, MO, USA). The cells were diluted at a concentration of 2 × 10⁶ cells/mL and plated in 6-wells plates, previously treated with poly-L-lysine (P6282) and laminin (L2020) (Sigma-Aldrich, St. Louis, MO, USA), which have high affinity for the myoblasts. The myoblasts were incubated at 28 °C, with the medium changed every day, and the myoblasts morphology was monitored regularly under a microscope (Olympus, Tokyo, Japan). The results were achieved from 3 independent cell cultures.

Sodium alginate (W201502-1KG; Sigma-Aldrich, Milan, Italy), type A gelatin from porcine skin (G2500-100G; Sigma-Aldrich, Milan, Italy), tyramine, 3-aminophenylboronic acid, N-hydroxysuccinimide (NHS), MES buffer 0.1 M, phosphate-buffered saline (PBS), peroxidase from horseradish, lyophilized, powder ~150 U/mg (HRP enzyme; 77332-100MG; Sigma-Aldrich, Milan Italy), hydrogen peroxide solution, fetal bovine serum (F7524; Sigma-Aldrich, Italy), penicillin–streptomycin (P0781-100ML; Milan, Sigma-Aldrich, Italy), and McCoy’s 5A modified medium powder for cell culture were provided by Sigma-Aldrich (Milan, Italy). N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) was purchased from Tokyo Chemical Industry Co. (TCI, Zwijndrecht, Belgium). Live/Dead Cell Viability Assay kit for 3D and 2D cell culture was obtained from EMD Millipore Corp (Milan, Italy) and HT29 cells (human colorectal adenocarcinoma cell line) were purchased from LGC Standards S.r.L (Milan, Italy).