Via1 casettes

We previously generated iPSC lines from surplus chondrocytes using mRNA-based reprogramming^{6 (link)}. The A2B iPSC line was maintained under feeder-free conditions in Cellartis DEF-CS™ (TaKaRa ClonTech, Sweden). This iPSC line was karyotype-tested, was normal even at late passages, was pluripotent with regards to the expression of pluripotency markers and was able to differentiate into all germ layers^{6 (link)} (Supplementary Fig. 8). This line was also shown to be superior in the differentiation protocol to generate articular cartilage matrix in 3D pellets and was used for 3D printing in subsequent experiments. In addition, iPSC-conditioned DEF medium from confluent clone A2B iPSCs was used after printing since increased survival has been noticed for single cells in a conditioned medium. For co-culture conditions, human surplus chondrocytes were irradiated (iChons) before being mixed with iPSCs to prevent the proliferation of the chondrocytes. The cell number was counted in a nucleocounter NC-200^TM using Via1-Casettes^TM (ChemoMetec, Denmark).

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).