Anti-Buoyancy Trap for Long-Term Quantitative Density Profiling of Adipocyte Spheroids

Abstract 3T3-L1 adipocyte spheroids are widely used three-dimensional adipose models that undergo pronounced physical property changes during adipogenic maturation, including progressive buoyancy driven by lipid accumulation. We quantitatively profiled the time-resolved evolution of bulk spheroid density over 60 days, revealing a 6.7% reduction from 1.022 to 0.954 g/cm³ associated with intracellular lipid accumulation. This buoyancy-driven displacement hinders standardized interrogation of spheroid physical properties during long-term culture. To enable longitudinal and reproducible analysis of density remodeling, we introduced an anti-buoyancy spheroid trap (AS-Trap) that mechanically stabilizes spheroids during extended culture. Morphometric analysis revealed that lipid droplet areas in mature spheroids (790.68±513.84 μm²) closely approximated in vivo adipocytes (846.34±257.28 μm²) and were substantially larger than those in 2D cultures, providing structural context for the observed density reduction. Notably, the terminal spheroid density converged toward reported values for native white adipose tissue, underscoring the physiological relevance of the material state achieved during maturation. Together, this work establishes density as a quantitative physical phenotype of adipogenic maturation and enables standardized long-term interrogation of adipose spheroids as living materials.