H dmem

The mouse neuroblastoma cell line N2a was obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in High glucose Dulbecco’s modified Eagle medium (H-DMEM, Sigma-Aldrich, Saint Louis, MO, USA) containing 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO₂. OGD treatment was used to simulate ischemia and hypoxia conditions in vitro. After replacing the complete medium with glucose-free DMEM, the N2a cells were cultured in a hypoxic chamber containing 95% N2 and 5% CO₂ for 4 h. Then, the cells were cultured in the complete medium under normoxic culture conditions for 24 h. The N2a cells in the control group were not exposed to OGD. 10 μg/ml ADSC-Exo were administrated in the ADSC-Exo group.

The colonies cultured in the presence or absence of succinate were picked and cultured in 1640 medium (Gibco), supplemented with 0.2% FBS and 100 ng/mL activin A (Sigma) for 1–3 days. After the given incubation time the culture medium was replaced with a new medium that consisted of 1640, 500 nM PdBU (Sigma), 100 ng/mL FGF10 (Sigma) and 2% FBS, and the colonies were cultured during days 4–8; The culture medium was then exchanged with a third medium, supplemented with H-DMEM (Sigma), 1% B27, 50 ng/mL Noggin, 2 μM RA and 0.25 μM Sant1, in which colonies were cultured during days 9–14. Next, the culture medium was exchanged with a fourth medium supplemented with H-DMEM, 1% B27, and 1 μM Compund-E, and the colonies were cultured during days 14–18. After the final incubation, the colonies were placed into KRBE, consisting of 10% FBS for 12 h. The final culture medium was 100 µL KRBE, supplemented with 0.2% FBS and 16.7 mM theophylline, and colonies were incubated in this medium for 2 h at 37 °C. The insulin secreted from the colonies was in the medium, followed by a direct ELISA test utilizing an insulin kit (JiangLai), following a standard procedure.

MG-63 human osteosarcoma cells (ATCC, Manassas, VA, USA, CRL-1427) were obtained from Flow Laboratories. The cells were maintained in a standard medium composed of Minimum Essential Medium Eagle-Alpha Modification (α-MEM) (Sigma-Aldrich, St. Louis, MO, USA, M0644), 1% penicillin–streptomycin (Euroclone, Milan, IT, ECB3001D) (penicillin 10.000 Units/mL, and streptomycin 10 mg/mL) and 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA, F7524) in a humidified incubator at 37 °C, 95% air and 5% CO₂. For passaging Trypsin/EDTA (Trypsin 0.05%–EDTA 0.02% in PBS, Euroclone, Milan, IT, ECB3052D) was used.
Saos-2 human osteosarcoma cells (ATCC, Manassas, VA, USA, HTB-8) were purchased from ATCC. Cells were maintained in high/low glucose (1:1) Dulbecco Modified Eagle’s Medium (H-DMEM, Sigma-Aldrich, St. Louis, MO, USA, D6429) (L-DMEM, Euroclone, Milan, IT, ECM0070L), 1% penicillin–streptomycin (Thermo Fisher Scientific, Massachusetts, USA, 15140122) and 10% FBS (Corning Inc., New York, USA, 35-079-CV), without any additional supplement in a humidified incubator at 37 °C, 95% air and 5% CO₂. For passaging Trypsin/EDTA (Trypsin 0.05%– EDTA 0.02% in PBS, Sigma-Aldrich, St. Louis, MO, USAT4174) was used.
The medium was refreshed every 3 days for both cell cultures.

RAW264.7 cells is a macrophage cell line (American Type Culture Collection, ATCC) that was established from a tumor in a male mouse induced with the Abelson murine leukemia virus. And the cell line was used for macrophage membranes collection in the current study. RAW264.7 cells cultured in Dulbecco’s modified Eagle’s medium (H-DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Excell Bio) and 1% penicillin-streptomycin (HyClone, United States) at 37°C in a 5% CO₂ atmosphere. Gram-negative bacteria E. coli (ATCC 43888) was grown in LB medium at 37°C in an aerobic environment, and bacteria used in all experiments were in the mid-exponential growth period. The bacterial suspension was diluted to 1*10⁶ colony forming units (CFU per mL) for experimental use after measuring the optical density at 600 nm using a microplate reader (ELX800, Gene Company Limited, China).