Fig. 1: Establishment and characterization of macrophage-augmented intestinal organoids.

a Schematic overview of constructing three different types of macrophage-augmented intestinal organoids (MaugOs). Created in BioRender.com. https://BioRender.com/cegs5ln. b Representative images of time-lapse confocal microscopy during MaugOs formation. Unstained intestinal organoid fragments and CFSE pre-labelled macrophages differentiated from THP-1 monocytes were integrated for 18 hours. Images were taken every 30 minutes (also see Supplementary Movie 1). c Immunofluorescence staining images of MaugOs incorporating organoids from three donors. Epithelial membrane of organoids was stained by EpCAM (red); THP-1 monocytes-derived macrophages stained by CFSE (green). Scale bar, 150 μm. d Bright field and immunofluorescence staining images depicting organoids, THP-1 macrophages and MaugOs. e Venn diagram of overlapped differentially expressed genes in macrophages (Mφ), intestinal organoids and MaugOs. f Significantly enriched pathways by KEGG analysis of MaugOs. (p < 0.05). g The expression of inflammatory genes quantified by qRT-PCR upon stimulation of 1 μg/mL LPS for 1 hour and 12 hours. Each group was compared to its respective control without LPS treatment (n = 3). h Quantification of IL-1β protein production by ELISA in MaugOs integrated THP-1 monocytes-derived macrophages (n = 4). i Immunofluorescence staining images of MaugOs integrated PBMC monocytes-derived macrophages and iPSCs-derived macrophages. Epithelial membrane of organoids was stained by EpCAM (red); PBMC monocytes-derived macrophages were stained by CFSE, and iPSCs-derived macrophages expressed H2B-mEGFP protein (green). Scale bar, 50 μm. j The expression of inflammatory genes quantified by qRT-PCR upon stimulation of LPS in MaugOs integrated iPSCs-derived or PBMC monocytes-derived macrophages (n = 2). k Quantification of IL-1β protein production by ELISA in MaugOs integrated PBMC monocytes-derived macrophages and iPSCs-derived macrophages (n = 4). Data were presented as means of biological replicates ± SEM. Statistical analysis was performed using the two-tailed Mann–Whitney test. *p < 0.05.