Atdc5 cell line

The ATDC5 cell line was purchased from RIKEN Cell Bank (Tsukuba, Japan) and cells were cultured in a DMEM/F-12 growth medium containing 5% FBS, 1% PS, 10 μg/mL human transferrin, and 3 × 10⁻⁸ M sodium selenite. Bovine insulin (10 μg/mL) was added to the medium for chondrogenic differentiation. Three-dimensional cultures of ATDC5 cells were carried out in a spinner flask. OHA/GC/ADH/ALG hydrogels encapsulating ATDC5 cells (1 × 10⁷ cells/mL) were prepared in the shape of disks. Then, the cell-encapsulated gel disks were incubated at 37 °C.

Mouse primary chondrocytes from WT or Gpr4^−/− mice were isolated from articular cartilage postnatal day 4 pups and digested overnight with 0.1% collagenase. Primary chondrocytes were cultured in DMEM/F12 supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. Cells were passaged when confluence reached 80–90%. P1 cells were used for each experiment according to specific experimental requirements. The ATDC5 cell line was purchased from Riken Cell Bank (Ibaraki, Japan) and maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.

A chondrogenic ATDC5 cell line was purchased from Riken Cell Bank (Ibaraki, Japan) and was used as a reference for the analysis. ATDC5 cells were grown at 37 °C in a cell incubator with 5% carbon dioxide in DMEM/F12 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For control experiments, ATDC5 cells were divided into four groups. To observe the GLA in IL-1β stimulation cartilage cells play a role. (1) Sham group, GLA and IL-1β were not administered to the ATDC5 cells; (2) IL-1β group, In the case of ATDC5 cells, only IL-1β (10 ng/ml) was used; (3) GLA group, Only the GLA (0.5 μM) was applied to the ATDC5 cells; (4) GLA+IL-1β group, we treated ATDC5 cells with IL-1β (10 ng/ml) and GLA (0.5 μM). GLA is dissolved in DMSO. In this experiment, the concentration of DMSO in all cell culture media was kept below 0.05%, and there was no significant effect on the growth of cells.

Mouse chondrogenic ATDC5 cell line was obtained from the RIKEN cell Bank (Tsukuba, Japan) and was cultured in DMEM/F-12 (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum and 100 U/mL penicillin. The cells were incubated in a wet incubator with 5% CO₂ and 95% air at 37 °C for normoxic conditions and in a hypoxic incubator (SMA-30D; Astec, Fukuoka, Japan) with 5% CO₂ and 1% O₂ balanced with N₂ at 37 °C for hypoxic conditions.

Mouse chondrocytes ATDC5 cell line was obtained from Riken Cell Bank (Tsukuba, Japan). The cells were cultured in a 1:1 blend of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F-12; GIBCO, USA) supplemented with 10% fetal bovine serum (GIBCO), in a wet incubator containing 5% CO₂ and 95% air at 37 °C. Culture medium was refreshed every 3 days. Then, the cells were exposed to increasing concentrations of LPS (0, 1, 5, and 10 μg/ml) at 37 °C for 24 h.

The chondrogenic ATDC5 cell line was purchased from Riken Cell Bank (Ibaraki, Japan) and maintained in DMEM/F12 containing FBS (10%), and penicillin/streptomycin (1%). Before the following experimental treatment, ATDC5 cells were incubated for 2 weeks in the presence of ITS (10 μg/ml insulin, 5.5 μg/ml transferrin, and 6.7 ng/ml sodium selenite; Invitrogen) to induce chondrocytic differentiation. (Wang et al., 2020 (link)). The RAW264.7 murine macrophage cell line was obtained from American Type Culture Collection (Rockville, MD, United States) and cultured in DMEM containing FBS (10%) and penicillin/streptomycin (1%). Primary bone marrow macrophage cells (BMMs) were collected from the bone marrow of C57BL/6 mice (4–6 weeks old). Briefly, cells were flushed from the femur bone marrow with DMEM containing M-CSF (30 ng/ml), FBS (10%), and penicillin/streptomycin (1%), and cultured in a T75 flask for 24 h. Non-adherent cells were then removed, and the adherent cells were cultured for a further 3–4 days until cells were fully confluent. (Liu et al., 2014 (link)). Before the following experiments, all the cells were maintained under standard adherent conditions at 37°C under 5% CO₂ and humidified atmosphere.