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Caption : supplementary figure 1. scheme of spheroid fabrication. (a) schematic representation of spheroid fabrication using human fibroblasts (hfbs) and human macrophages (hmps) in non-adhesive agarose molds. 


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Caption : supplementary figure 2. characterization of differentiated human macrophages. (a) fluorescence activated cell sorting (facs) for pbmcs labeled with macrophage marker cd68. (b-c) bright-field images of macrophages at day 1 and day 14 after facs. (d) confocal image of macrophages stained dapi, cd86 and cd206. scale bars: (b-d) = 20 μm.


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Caption : supplementary figure 3. single channel images from immunofluorescent staining of hybrid spheroids to identify macrophages and fibroblasts. confocal images of frozen sections of hybrid spheroids (fibroblasts to macrophages from 2:1, 4:1, 8:1, 16:1, 32:1, 64:1 and 100:0) stained with hsp47 and cd68. 16:1 showed the highest expression level of fibroblast activity (hsp47). dapi is nuclear marker, hsp47 is fibroblast marker, cd68 is pan macrophage marker. scale bars: 50 μm.


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Caption : supplementary figure 4. immunofluorescent staining of hybrid macrophage-fibroblast spheroids. (a) confocal images of frozen sections of hybrid spheroids (fibroblasts to macrophages from 2:1, 4:1, 8:1, 16:1, 32:1, 64:1 and 100:0) stained with hsp47 and cd68. immunofluorescence markers dapi (nuclei), hsp47 (fibroblast/collagen maturation), and cd68 (pan-macrophage marker). (b) relative expression levels of cd64 and cd68 as pan-macrophage genes by qpcr (n=3). scale bars: (a) = 50 μm. 


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Caption : supplementary figure 5. immunofluorescent staining of hybrid spheroids for proliferation activity. confocal images of hybrid spheroids frozen sections (fibroblasts to macrophages from 2:1, 4:1, 8:1, 16:1, 32:1, 64:1 and 100:0) stained with ki67 (proliferation marker) and dapi (nuclear marker). scale bars: 50 μm.


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Caption : supplementary figure 6. f16 ratio hybrid spheroids demonstrate increased macrophage pro-fibrotic activity on immunofluorescent staining. higher magnification confocal images of hybrid spheroid frozen sections (fibroblast to macrophage ratios 16:1) stained with ccr7 (a) and pstat1 (b) as m1 macrophage markers respectively with pan-macrophage marker cd68 and dapi as nuclear marker. white arrows indicate cd86-positive macrophages in f16 spheroid that have polarized toward an m1 phenotype. scale bars: 50 μm.


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Caption : supplementary figure 7. immunofluorescent staining of positive controls for asma antibody. confocal images of human trachea scar tissue and isolated fibroblasts from it stained with asma as myofibroblasts and dapi as nuclear marker. scale bars: 100 μm.