Recombinant human insulin

Adipogenic differentiation of culture-expanded, and scaffold-seeded non-GFP-Tg ASC or GFP-Tg ASC was performed over a 15 day period as previously described^{4 (link)}. Briefly, cultured ASC were grown in Stromal Medium (Dulbecco’s modified Eagle’s-Ham’s F-12 medium supplemented with 10% fetal bovine serum, and 1% penicillin/streptomycin). ASC were then trypsinized and plated in 24-well plates in ASC culture media at 3×10⁴ cells/cm² for 24 hrs to allow attachment. On day 1 (24 hours after plating), the medium was removed and cells were incubated for three days in adipogenic differentiation medium (Dulbecco’s modified Eagle’s-Ham’s F-12 medium supplemented with 10% fetal bovine serum, 15 mM HEPES (pH 7.4), biotin (33 µM), pantothenate (17 µM, Sigma), human recombinant insulin (100 nM, Boehringer Mannheim), dexamethasone (1 µM), 1-methyl-3-isobutylxanthine (IBMX; 0.25 mM), and rosiglitazone (1 µM). For the remaining 9 days of the adipocyte differentiation maintenance period, the medium was removed every 3 days and replaced with the same medium that did not contain IBMX and rosiglitazone (maintenance medium).

For scaffold seeded GFP-Tg SVF cells and GFP-Tg ASC, one day prior to seeding, scaffolds were autoclaved and pre-soaked in adipogenic medium in 37°C, 5% CO₂, 95% relative humidity. GFP-Tg ASC were then trypsinized and plated loaded onto pre-soaked scaffolds in ASC adipogenic differentiation media by pipet in two aliquots of 50 µL each containing a total of 2.5 × 10⁵ cells to opposite faces of a cylindrical HFIP silk scaffold as per published methods^{33 (link)}. The cells were incubated for three days in adipogenic differentiation medium (Dulbecco’s modified Eagle’s-Ham’s F-12 medium supplemented with 10% fetal bovine serum, 15 mM HEPES (pH 7.4), biotin (33 µM), pantothenate (17 µM, Sigma), human recombinant insulin (100 nM, Boehringer Mannheim), dexamethasone (1 µM), 1-methyl-3-isobutylxanthine (IBMX; 0.25 mM), and rosiglitazone (1 µM). For the remaining 9 days of the adipocyte differentiation maintenance period, the medium was removed every 3 days and replaced with the same medium that did not contain IBMX and rosiglitazone (maintenance medium). Similarly, control cultures were maintained in parallel in the absence of adipogenic stimulants. All seeded and nonseeded (control) scaffolds were cultivated in a humidified incubator at 37°C with 5% CO₂. Following the in vitro cultivation period, seeded scaffolds were evaluated for their extent of adipogenesis.