Dmem f12

We obtained leukemia macrophages (raw264.7) from Cyagen Biosciences (Wuhan, China). Rat nucleus pulposus cells were extracted from the caudal intervertebral disc of 4-week-old SD rats, as previously reported [54 (link)]. The cells were then grown in DMEM high glucose or DMEM/F12 (Cyagen, Wuhan, China) with 1% penicillin–streptomycin (P/S, Cyagen, Wuhan, China) and 10% fetal bovine serum. The culture media was then replaced every two days, while the cells were maintained at 37 °C and 5% carbon dioxide. Extracts were prepared from each group according to the following steps: 400 µL of hydrogel (height: 5 mm, diameter: 10 mm) was soaked in 3 mL of medium and cultured at 37 °C for 24 h. Extraction solution was filtered via a 0.22 µm filter membrane. The culture medium was then gathered, and fetal bovine serum and P/S were added as necessary. Prior to use, the obtained media were kept at 4 °C [55 (link)].

iMPMECs were generated in our laboratory [34 (link)]. The cells were used at passages 3–10, and maintained in DMEM-F12 (Gibco, USA) supplemented with 5% foetal bovine serum (FBS) (ExCell, China), 1% penicillin and streptomycin (Gibco, USA), 1% endothelial cell growth supplement (ECGS) (ScienCell, USA), 100 IU/ml heparin (Solarbio, China), and 92 mg/L D-valine (Sigma, USA) and incubated at 37 °C in 5% CO₂. The cells were grown to 70–80% confluence, washed 3 times with PBS, and treated with or without 1 µg/ml LPS (Sigma, USA) for 24 h in conditioned culture medium (CCM) containing exosome-depleted FBS. CCM was collected after 24 h. Exosome-depleted FBS was prepared by centrifuging FBS for at least 18 h overnight at 100,000 × g at 4 °C, after which the centrifuged FBS were passed through a 0.22 μm filter (Millipore, USA). MSCs derived from the bone marrow of C57BL/6 mice (Cyagen Biosciences, China) were used at passages 3–8 and cultured in DMEM-F12 supplemented with 10% FBS and 1% penicillin and streptomycin, and these cells were tested for their osteogenic, adipogenic, and chondrogenic differentiation potentials to ensure they met the characteristic of stem cells (Supplementary Fig. S1).