Preparation of MSC-laden 3D bioprinting scaffold and SG induction

Type B gelatin (Sigma-Aldrich, USA) and sodium alginate (Sigma-Aldrich) were dissolved in 0.5 × PBS at 3% and 1% (w/v), respectively, and the final ratio between alginate and gelatin was 1:3. Modified pasteurization was used to sterilize the alginate–gelatin solution. Briefly, the solution was sterilized at 70°C for 30 minutes three times with an interval of 4°C for 10 minutes. After that, the solution was stored at 4°C and incubated at 37°C for 30 minutes before use. The formation of alginate–gelatin gel (AG) was described previously [13]. Briefly, a mixture of 10 mL sterilized alginate–gelatin solution, 200 μL 5 × 10⁶ single-cell suspension of MSCs and 1 mL PD were prepared, followed by gelation at 4°C for 30 minutes. An extrusion-based 3D bioprinter (Bio-Architect PRO, Regenovo, China) was used to bioprint the mixture within a temperature-controlled chamber in which the temperature was settled within the gelation region of gelatin. Cylindrical scaffold was bioprinted layer by layer using nozzles with a 260 μm inner diameter, flow rate of 8–13 mm/s and pressure of 0.05–0.3 MPa. The diameter of the scaffold was 20 mm, with a gap between each printed line of 1.5 mm and totaling 6 layers in height. After the bioprinting process, the printed AG scaffolds were immersed in pre-cooling 2.5% (w/v) calcium chloride for 5 minutes for crosslinking, then washed with Dulbecco’s modified Eagle medium/Ham’s F12 nutrient medium (DMEM/F12, Hyclone, USA) three times. The crosslinked AG scaffold was cultured in an atmosphere of 5% CO₂ at 37°C with SG induction culture medium (SGCM; Table S1). The medium was replaced with fresh SGCM every three days. Cell morphology was examined and recorded using an automated inverted research microscope (DMI4000 B, Leica, Germany). SG scaffolds would be fully induced in SGCM after 7-days after which the scaffolds would be seeded with HF spheroids.