Dmem f12 ham 1 1

The confluent perichondrial cells were dissociated into single cells with 0.25%
trypsin. Next, 50,000 cells were seeded in a 100 mm dish containing Dulbecco’s
Modified Eagle’s Medium (DMEM)/F12 Ham (1:1) (Sigma-Aldrich), stabilized
antibiotic–antimycotic solution (100×) (Sigma-Aldrich), 0.2 mM ascorbic acid
2-phosphate (AA2P) (Sigma-Aldrich), 10⁻⁷ M dexamethasone
(Sigma-Aldrich), 1× ITS-X (Gibco), 5 ng/mL insulin-like growth factor (IGF)
(Sigma-Aldrich), 10 ng/mL basic fibroblast growth factor (FGF) (Wako), 10 ng/mL
platelet-derived growth factor BB (PDGF-BB) (Peprotech), and 2% FBS. The medium
was changed every other day until the cells became confluent, which occurred
between 7 and 10 days of culture (passage 1 cells).

Approximately 3000 spheroids from day 3 micro 3D culture were collected from the
micropatterned plate by gentle pipetting and transferred to a rotating wall
vessel containing 50 mL of chondrogenic induction medium. The spheroids were
kept still overnight to allow fusion, followed by rotating culture at 15 rpm.
The following week, the medium was replaced with chondrogenic induction medium
containing DMEM/F12 Ham (1:1) (Sigma-Aldrich), stabilized antibiotic–antimycotic
solution (100×) (Sigma-Aldrich), 0.2 mM AA2P (Sigma-Aldrich), 10⁻⁷ M
dexamethasone (Sigma-Aldrich), 1× ITS-X (Gibco), 5 ng/mL IGF (Sigma-Aldrich), 10
ng/mL basic FGF (Wako), 10 ng/mL PDGF-BB (Peprotech), 10 ng/nL TGFβ1
(Peprotech), 20 ng/mL bone morphogenetic protein 4 (R&D), 1% Glutamax
(Gibco), and 1% FBS. The medium was changed weekly for a total of 4 weeks.