6 aca

Succinic acid, histidine, xanthine, CMP, 6-ACA, NCG, and SCMC were reconstituted according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). Succinic acid (1 mM), histidine (100 µM), xanthine (14 µM), CMP (1 mg/mL), 6-ACA (30 µM), NCG (1 mM), and SCMC (10 µM) were added to the hIO culture medium, and the medium was changed every other day. The hIOs were treated with the metabolites for two passages.

Boc-lysine, 6-ACA, acetaldehyde, BSA, KLH, TFA, TMB, and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO). Malondialdehyde bis(dimethyl acetal), the Imject EDC mcKLH Spin Kit, goat anti-rabbit IgG (H+L) antibody with HRP, and goat anti-mouse IgM secondary antibody were obtained from Thermo Scientific (Rockford, IL). EnVision+Single Reagents anti-mouse-HRP and rabbit anti-human IgG F(ab’)2 fragment antibody with HRP were purchased from Dako North America, Inc. (Carpentaria, CA) and Jackson Immuno Research Laboratories (West Grove, PA), respectively.

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.