3 isobutyl 1 methylxanthine ibmx

For the generation of multicellular spheroids with adipocytes, ASCs were cultured in adipogenic differentiation medium consisting of growth medium with insulin (final concentration 1.7 µM; PromoCell, Heidelberg, Germany), dexamethasone (1 µM; Sigma-Aldrich), 3-isobutyl-1-methylxanthine (IBMX, 500 µM; Serva-Electrophoresis, Heidelberg, Germany), and indomethacin (200 µM; Sigma-Aldrich). Cells were differentiated in conventional 2D culture for 14 days prior to detachment and subsequent spheroid formation. Successful differentiation was verified by qRT-PCR of adipogenic marker genes and determination of intracellular triglyceride content. Adipocyte-containing mono- and co-spheroids were generated as described above for ASCs.

After printing or casting, ASC spheroid-laden constructs were cultured in an adipogenic differentiation medium consisting of Preadipocyte Basal Medium-2 (Lonza, Basel, Switzerland) supplemented with 10% FBS, 1% penicillin/streptomycin, and the hormonal inducers insulin (final concentration 1.7 µM; PromoCell, Heidelberg, Germany), dexamethasone (1 µM; Sigma-Aldrich, St. Louis, MO, USA), 3-isobutyl-1-methylxanthine (IBMX, 500 µM; Serva-Electrophoresis, Heidelberg, Germany) and indomethacin (200 µM; Sigma-Aldrich, St. Louis, MO, USA). Constructs were cultured for up to 21 days, with media exchange every other day.