370 Generation of homogeneous, scaffold-free adipose spheroids for screening of lipolytic agents

Abstract

Adipose tissue plays a critical role in energy balance and lipid metabolism. Disorders of adipose function can lead, amongst others, to type II diabetes, obesity and unsightly conditions such as cellulite. Monolayer cell culture systems have proven useful to understand many aspects of fat biology, but these 2D in vitro systems do not reflect the complexity of human adipose tissue. To overcome this limitation, we took advantage of the ability of pre-adipocytes to form spheroids when cultured in ultra-low attachment systems. Therefore, the goal of our study was to develop adipose spheroids and validate their lipolysis responsiveness, when stimulated with reference molecules. Human pre-adipocytes were seeded in GravityTRAP ULA™ 96-well plates and cultured for 17 days using proliferation, differentiation and maturation media. We first showed that cells were able to generate spheroids with homogeneous size distribution (about 400 μm of diameter), to produce extracellular matrix and to secrete adiponectin in a culture plate format suitable for screening. Quantitation of spheroid volume demonstrated that spheroids expanded over time, mainly by accumulation of lipid droplets increasing in size within the spheroid. We showed that adipocyte maturation was inhibited (-30% of spheroid volume and strong decrease of lipid droplet size) when TNF-a was added in both differentiation and maturation media. We then characterized the lipolytic effects of caffeine (C) and evodiamine (E) by showing that both molecules decreased spheroid volume (C: -26%, E: -14%) in concomitance with fewer (C: -70%, E: -47%) and smaller-sized (C: -72%, E: -55%) lipid droplets. We finally showed that these lipolytic effects resulted in an increased release of free fatty acids (C: +25%, E: +26%) and a decreased secretion of adiponectin (C: -80%). In summary, we showed that adipose spheroids provide an interesting in vitro model of pre-adipocyte maturation and adipocyte expansion. They represent relevant in vitro assays to study adipogenesis, adipose tissue dysfunction and evaluate pharmacological agents.