Chondrocyte isolation and culture
Proximal tibias and distal femurs were dissected from 3 to 5 day old
rats (Charles River Laboratory, Wilmington, MA, USA). These were
digested with 0.3% collagenase type IA (Sigma-Aldrich) in DMEM/F12
aseptically, as described.(Spath, Andrade, Chau, Baroncelli, & Nilsson,
2018) Briefly, dissected cartilage pieces were washed twice in PBS with
1% penicillin/streptomycin, 50ng/ml fungizone (both from Thermo Fisher
Scientific), next incubated with 0.1% EDTA (Sigma-Aldrich) in PBS for
15 minutes and 0.125% trypsin (Thermo Fisher Scientific) in PBS for 30
minutes, all steps at 37°C while shaking. Cartilage pieces were washed
twice in PBS with 1% penicillin/streptomycin, 50ng/ml fungizone, and
digested at 37°C while shaking with 0.3% collagenase type IA
(Sigma-Aldrich) in DMEM/F12 by repeating cycles of 30 minutes until
digestion was complete. Cell suspension was filtered through a 70µm-cell
strainer (BD Biosciences, San Jose, CA, USA), spun at 300g for 5
minutes, washed twice in PBS containing 1% penicillin/streptomycin,
50ng/ml fungizone (Thermo Fisher Scientific). The isolated chondrocytes
were resuspended and cultured at 37°C, 5% CO2 in
DMEM/F12 with GlutaMAX (Thermo Fisher Scientific) supplemented with 10%
fetal bovine serum (FBS), 50µg/ml ascorbic acid (Thermo Fisher
Scientific), 1% penicillin/streptomycin and 50ng/ml fungizone.