Speaker
Description
INTRODUCTION: The increased prevalence of obesity over the last few years has intensified the need for physiologically relevant models for the characterization of the mechanisms regulating obesity-linked adipogenic and inflammatory responses. In obesity, there is an unhealthy expansion of adipose tissue, at the expense of hypertrophic processes responsible for the enlargement of adipocytes. This leads to the generation of a chronic low-grade inflammation, altered adipokine secretion and increased lipolysis. Current in vitro obesity research focuses mainly on adipose tissue models on two-dimensional cell culture that fall short of mimicking the three-dimensional (3D) in vivo environment. To surpass that, this work aims at developing 3D adipose-like tissue analogues bearing hypertrophied adipocytes within an inflammatory milieu.
METHODS: Human adipose-derived stem cells were seeded at sub-confluency and cultured for 3 days with ascorbic acid supplementation and then for 6 days in adipogenic differentiation medium plus 9 days in adipogenic maintenance medium1. The generated cell sheets were then submitted to different time combinations of TNF-α (100 ng/mL), an inflammatory cytokine, and palmitic acid (PA) (0.5 mM), a saturated fatty acid that promotes adipocyte enlargement, for further 13 days. Adipocyte size (image analysis), lipid accumulation (LipidTOX) and glycerol release (Glycerol Assay Kit) were assessed as adipogenic indicators, and the secretion of TNF-α and IL-6 (ELISA) was used as an indicator of the inflammatory status. The expression of adipogenic (PPARγ and FABP4) and lipolytic (pHSL, ATGL and PLIN) markers was determined at the mRNA (RT-PCR) and protein (western blot) levels. Additionally, to further increase the complexity of our system, differentiated cell sheets were stacked together to produce 2- and 3-layered constructs, which were then submitted to the same treatments as the single cell sheets and assessed regarding the same parameters.
RESULTS & DISCUSSION: Altogether, our analysis showed that the combination of hypertrophic and inflammatory stimuli (PA+TNF-α) for 13 days, and the treatment with PA alone for 10 days followed by a combination of PA+TNF-α for 3 days resulted in adipogenic cell sheets with higher TNF-α and IL-6 secretion, larger adipocytes with increased accumulation of neutral lipids and increased glycerol release. These features were found in both single cell sheets and 3D constructs.
CONCLUSION(S): These results showed that by tuning the culture conditions of cell sheets with inflammatory and hypertrophic stimuli we are able to modulate their adipogenic phenotype. Overall this allowed obtaining 3D adipogenic analogues presenting enlarged adipocytes with a pro-inflammatory profile resembling the characteristics of the obese adipose tissue.
REFERENCES:
1. Lago MEL, et al. Modulation of the secretory potential of in vitro adipose tissue microenvironments. Eur Cells Mater 2017;33:729
83767227924
