Endocrine Abstracts

Background: In obesity the relationship between white adipose tissue expansion and neovascularisation becomes uncoupled leading to inadequate perfusion of adipose tissue. Under these circumstances the secretory profile of adipocytes becomes metabolically unfavourable and pro-atherosclerotic. Therefore, targeting white adipose tissue may provide a potential therapeutic strategy to combat the negative metabolic and cardiovascular consequences of obesity. Here, we validated a murine adipose organoid model, which mimics adipose function including vascularisation.

Methods: To culture adipose spheroids, stromal vascular fraction cells were isolated from epididymal adipose tissues from C57BL/6 mice and seeded in a low adhesion 96-well plate. Adipose differentiation was initiated with differentiation media. Following initiation of adipose differentiation, adipose organoids were maintained in growth media for 8 days. To confirm different cell types were present in cultured organoids, gene expression of various cell-specific markers was quantified using real-time PCR. 3T3-L1 fibroblasts and whole adipose tissue from wild-type mice served as negative and positive controls respectively. To assess the vascular phenotype of the adipose organoids, organoids were fixed for immunofluorescence.

Results: Data was presented in fold changes relative to 3T3-L1 fibroblasts. Gene expression studies revealed cultured adipose spheroids expressed adipocyte markers Adipoq (3600-fold increase), Fabp4 (1200-fold increase) and Hoxc8 (16000-fold increase); endothelial cell markers Cdh5 (100-fold increase), Kdr (200-fold increase) and Pecam1 (20-fold increase); immune cell markers Cd14 (6-fold increase), Cd68 (100-fold increase), Csf2ra (1000-fold increase), Fcgr2b (barely detected in 3T3-L1 fibroblasts but expressed at comparable level to adipose tissue) and Icam1 (1500-fold increase). In addition, using immunofluorescence, cultured adipose spheroids present vessel-like structures stained with isolectin B4.

Conclusion: Here, we demonstrate that this adipose organoid model displays markers of diverse cell types. Immunofluorescence analysis reveals the presence of vessel-like structures within these cultured organoids. These findings demonstrate the suitability of our model for advancing studies on adipose health and function.