Development of novel human in vitro vascularized adipose tissue model with functional macrophages

Figures

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  Fig. 1. Identity and functionality of macrophages. Macrophages engulfed dead cell parts (shown in green), imaged with Cell-IQ (Chipman technologies). In image a in the first time point, the well of cell culture plate is still full of small particles seen as fuzzy green background. b At 5 h time point, the background is clean, because macrophages have engulfed the dead cell debris. c Double positive macrophage (CD68-FITC (green) and CD11b-V450 (blue)). d Hematoxylin–eosin stained macrophages isolated from adipose tissue. Nuclei of the macrophages marked with arrowhead. c and d obtained with Nikon Eclipse TS100 inverted fluorescence microscope and digital sight DS-U2 –camera. Scale bar 50 µm

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  Fig. 2. Adipocyte–Macrophage model (AMM). a Phase contrast image overlaid with fluorescence image of the model with and without macrophages. Triglycerides stained with Adipored, seen in green. The presence of macrophages seems to lead to larger lipid accumulation in adipocytes. Scale bar in both columns 100 µm. b Triglyceride accumulation per cell calculated from AdipoRed (total amount of triglycerides) and WST-1 (relative cell number) results. Images obtained with Nikon Eclipse TS100 inverted fluorescence microscope and digital sight DS-U2 –camera

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  Fig. 3. Adipocyte–Macrophage model with IFNγ (AMMI). Image a Phase contrast image of adipocytes and macrophages. Image b Close-up of macrophage in the culture: phase contrast image combined with AdipoRed (green) stained adipocytes and macrophage stained with CD11b (red). Macrophages marked with arrow heads and lipids stained with adipored marked with arrow. Macrophage nucleus can be seen in the close-up (asterisk). Obtained with Nikon Eclipse TS100 inverted fluorescence microscope and digital sight DS-U2 –camera. Scale bar 50 µm. c Triglyceride accumulation significantly differs between cultures with and without macrophages. Results depicted as mean ± SD, **p < 0.01

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  Fig. 4. Cytokine expression in Adipocyte–Macrophage model with IFNγ (AMMI). Amounts of TNFα, IGF, Resistin, IL-6, PAI, TGFβ, Adiponectin and leptin were determined. Adiponectin shows significant increase in the presence of macrophages. Results depicted as mean ± SD, *p < 0.05

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  Fig. 5. Characteristics of Vascularized Adipose Tissue model with Macrophages with IFNγ (VATMI). a Vasculature in VATMI stained with DAB. b Adipocytes and vessel structures in the phase contrast image of VATMI. Both obtained with Nikon Eclipse TS100 inverted fluorescence microscope and digital sight DS-U2 –camera. Vasculature marked with arrows and adipocytes with asterisks. Scale bar in both 100 µm. c Triglyceride accumulation differs between cultures with and without macrophages. Results depicted as mean ± SD

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  Fig. 6. Cytokine expression in Vascularized Adipose Tissue model with Macrophages with IFNγ (VATMI). Increase is seen in IL-8 and IP-10 although none of the differences were significant. Results depicted as mean ± SD