Adipocyte Differentiation and Exosome Stimulation

Mesenteric fat preadipocytes from non-IBD, CD, and UC patients were collected from a previous study and stored in liquid nitrogen [28 (link)]. The human preadipocytes were thawed and cultured in DMEM/F12 media containing 10% calf serum and 1% P/S (Invitrogen) until >60% confluence was achieved. The preadipocytes were dissociated by trypsin/EDTA solution (Invitrogen) and seeded to 6-well plates (400,000 cells per plate) in DMEM/F12 media containing 10% calf serum and 1% P/S. Two days later, the preadipocytes underwent differentiation process by incubating with induction media (DMEM with FBS, P/S/G, bovine insulin (Sigma I-5500; 1μg/mL), dexamethasone (Sigma D-4902; 1μM) and isobutylmethylxanthine (IBMX; Sigma I-5500; 115μg/mL) for two days, insulin media (DMEM with FBS, P/S/G and insulin (1 μg/mL)) for two days, and DMEM + FBS + P/S for two days [29 (link)]. The adipocytes were regarded as differentiated by the observation of lipid droplet deposition under microscope. The differentiated adipocytes were serum-starved for 8 hours, followed by incubation with human serum exosomes (100μg/mL) for 16 hours. The conditioned cells were then switched to serum-free DMEM media for 6 hours to let the cells secrete elafin. The conditioned media were collected for elafin ELISA.