6 aminocaproic acid 6 aca

Muscle bundles were generated as previously described by Bakooshli et al. with minor adjustments^{22 (link)}. Before generation of muscle bundles, bone-shaped PDMS chambers were sterilized and pre-treated with 1% Pluronic F-127 (Sigma-Aldrich) for 1 h at room temperature. Confluent myoblast cultures were dissociated with TrypLE Express Enzyme (Gibco) reagent and 3 × 10^5 cells were used per muscle bundle. Cells were incubated on ice and combined with an ice-cold hydrogel mixture consisting of 4 mg/ml bovine fibrinogen (Sigma-Aldrich) and 20% Growth Factor Reduced Matrigel (Corning). Before loading the cell/hydrogel mixture into a bone-shaped PDMS chamber, 0.8 units of Bovine Thrombin (Sigma-Aldrich) were added to initiate fibrinogen polymerization. Loaded PDMS chambers were incubated for 30 min at 37 °C and after polymerization muscle bundles were kept in normal proliferation medium containing 1.5 mg/ml 6-aminocaproic acid (6-ACA) (Sigma-Aldrich). After 2 days, differentiation was induced by changing medium to differentiation medium (DMEM high glucose (Gibco), 2% horse serum (Gibco), 1% Penicillin G (Sigma-Aldrich) and 2 mg/ml 6-Aca). Seven days after differentiation muscle bundles were fixed overnight at 4 °C with 2% PFA. Muscle bundles were stored in PBS at 4 °C.

3D-TESMs were generated in an Ecoflex Replica mold using a 15-µl hydrogel-cell mixture containing 2 mg/ml fibrinogen (Sigma-Aldrich), 20% Matrigel growth factor reduced (±10 mg/ml, Corning), 240,000 MPCs, and 0.8 units/ml bovine thrombin (Sigma-Aldrich) [15 ]. For the delivery of lentiviral particles, viral concentrates were transferred to the hydrogel-cell mixture before pipetting the mixture into the PDMS chamber or to the culture medium. The 3D-TESMs were incubated for 20 min at 37 °C before the addition of proliferation medium supplemented with 1.5 mg/ml 6-aminocaproic acid (6-ACA) (Sigma-Aldrich) and switched to differentiation medium supplemented with 2 mg/ml 6-ACA after 48 h. 3D-TESMs were cultured on a 65-rpm shaking platform at 37 °C/5% CO2, and half of the differentiation medium was refreshed every 48 h.