Part 1: A Novel Model for Three-Dimensional Culture of 3T3-L1 Preadipocytes Stimulates Spontaneous Cell Differentiation Independent of Chemical Induction Typically Required in Monolayer

Differences in monolayer and three-dimensional (3D) culture systems have been recognized for several years. Despite the recognized importance of 3D systems, low cost and convenience of monolayer culture are still readily used for metabolic and nutritional studies. Here, we present part 1 of a 2-part series that will highlight (1) a novel and cost-effective model for culturing 3T3-L1 preadipocytes in 3D agarose as well as (2) an initial study showing the successful use of this 3D model for experimental analysis of these cells treated with cinnamon extract while suspended in agarose. In part 1, we provide a full characterization of the model system for the 3T3-L1 cells that demonstrate the functionality and convenience of this system. Importantly, we note spontaneous differentiation to adipocytes while cultured under these methods, independent of chemical induction. We present a 2.5-week time course with rounded cells forming vacuoles as early as 24 hours and accumulation of lipid detectable by Oil Red O stain at 0.5 weeks. Serum selection, lipid volume determination, and cell size are characterized. We conclusively demonstrate adipogenesis based on a peroxisome proliferator-activated receptor γ (PPARγ) detection using immunohistochemistry (IHC) of sections from these 3D cultures. Methods, materials and recommendations are described as well as proposed benefits to the use of this culture system for 3T3-L1 cells.