Linoleic acid solution

Equal numbers of iPSCs were mixed with iChons to give a final concentration of 20 million cells/ml in the bioink, and the printed constructs were maintained at 37 °C and 90% humidity in 5% CO₂. After 7 days of culture in the DEF CS™ (TaKaRa ClonTech, Sweden) pluripotent medium mixed with an equal volume of conditioned DEF-CS medium (conditioned DEF medium was taken from 80% confluent pluripotent iPSCs after 24 hours of culture, sterile filtered, and fresh growth factors (GF1, GF2 and GF3; TaKaRa ClonTech, Sweden) were added), the medium was changed every day. Cells were then initiated to differentiate by changing the medium to a defined chondrogenic medium (high-glucose Dulbecco’s modified Eagle’s medium; PAA Laboratories) supplemented with 5.0 μg/ml linoleic acid solution (Sigma-Aldrich), 1x ITS-G premix (10 mg/l insulin, 5.5 mg/l transferrin, 6.7 μg/l selenious acid; Life Technologies), 0.11g/l sodium pyruvate, 1.0 mg/ml human serum albumin (Equitech-Bio, TX, USA), 10 ng/ml TGFβ1 (R&D Systems, Abingdon, UK), 10 ng/ml GDF5, 10 ng/ml BMP2, 100 nM dexamethasone (Sigma-Aldrich), 80 μM L-ascorbic acid (Sigma-Aldrich), and 1x penicillin/streptomycin (PEST; PAA Laboratories). The medium was changed three times a week. Relevant control cultures with only printed irradiated chondrocytes were kept throughout the differentiation protocol.

Control chondrocytes (200,000/well) were centrifuged to form pellets in a 96-well
round-bottom low-attachment plate. The plate was centrifuged for 5 minutes at
500 × g to form pellets, with one in each 96 well to form a
micro tissue following differentiation for at least 2 weeks. Then, the 3D
bioprint with and without chondrocytes and the control chondrocyte micro tissue
pellets were induced to differentiate by replacing the defined medium (DEF) with
chondrogenic differentiation medium, consisting of DMEM-high-glucose
(high-glucose DMEM; PAA Laboratories) supplemented with 5.0 mg/mL linoleic acid
solution (Sigma-Aldrich), 1× ITS-G premix (6.25 mg/mL insulin, 6.25 mg/mL
transferrin, 6.25 ng/mL selenous acid; Life Technologies), 1.0 mg/mL human serum
albumin (Equitech-Bio, Kerrville, TX, USA), 10 ng/mL TGFβ1, 10 ng/mL TGFβ3, 100
nM dexamethasone (Sigma-Aldrich), 80 nM ascorbic acid 2 phosphate
(Sigma-Aldrich), and 1× penicillin/streptomycin (PEST; PAA Laboratories). The
medium was changed 3 times a week. The 3D bioprints and control chondrogenic
pellets were harvested after 14 days for histological and reverse
transcription–polymerase chain reaction (RT-PCR) analysis. The cell number and
viability were counted before and after 3D bioprinting using nucleocounter
NC-200TM in Via1-CasettesTM (ChemoMetec, Denmark).