15 ml conical tube

For cryopreservation experiments, Bioblock-derived hPS cells generated by above described dissociation procedure were used. E8 + RI-suspended cells were counted and the respective amount of cells was transferred into a 15 mL conical tube (Greiner AG) for subsequent centrifugation at 300×g and 4 °C for 3 min. Collected cells were re-suspended in a respective amount of cryopreservation medium consisting of 90% E8, 10% dimethyl sulfoxide (DMSO; Merck), 0.1% Pluronic F-68 (Thermo Fisher Scientific) and 10 µM RI (Tocris Bioscience) to achieve the desired cell concentration followed by aliquoting of 1 mL into CRYO.S freezing tubes (Greiner AG). Cryopreservation was performed with a Planer Kryo 10 Series III controlled-rate freezer (Planer Limited) with a defined protocol (Additional file 1; Table S4) followed by long-term storage at −150 °C. Cells have been cryopreserved for at least 3 weeks before further cultivation. For direct suspension culture inoculation in bioreactors, cells were thawed in a water bath at 37 °C for ≈3 min, diluted in cooled 10 mL of E8 + RI and collected by centrifugation at 300 × g and 4 °C for 3 min for re-suspension in 20 mL E8 + RI followed by cell counting. Bioreactors were inoculated as described above at the established density of 5 × 10⁵ cells/mL.

A total of 150,000 cells were cultured in the tip of a 15 mL conical tube (Greiner, Kremsmünster, Austria) to enable cell culture in micromass with chondrogenic medium (Stem MACS Chondro Diff Media, Miltenyi Biotec). Cells were resuspended carefully and cultured at 37 °C in a 5% CO₂ humidified atmosphere with the cap slightly screwed. Half of the chondrogenic medium was replaced weekly. On Day 21, aggregates were stained with Alcian blue (Sigma) to highlight cartilage proteoglycans. In some cases, cryosectioned pellets were stained with Alcian blue to confirm chondrogenic differentiation.

A total of 150,000 cells were cultured in the tip of a 15 mL conical tube (Greiner, Vilvoorde, Belgium) to enable cell culture in micromass with chondrogenic medium (Stem MACS Chondro Diff Media, Miltenyi Biotec). Cells were resuspended carefully and cultured at 37 °C and 5% CO₂ in a humidified atmosphere with a slightly screwed cap. Half of the chondrogenic medium was replaced weekly. On day 21, aggregates were stained with Alcian blue (Sigma–Aldrich, Diegem, Belgium) to highlight cartilage proteoglycans. In some cases, cryosectioned pellets were stained with Alcian blue to confirm chondrogenic differentiation.