(a) Differential gene expression analysis was carried out to identify genes that are up- or down-regulated in Duodenum Intestine-Chip compared to organoids (blue circle) (Figure 3—source data 1) and adult duodenum compared to organoids (yellow circle) (Figure 3—source data 2). The gene lists were then compared to determine how many genes overlap between those two comparisons (Figure 3—source data 3), and the results are shown as a Venn diagram. 305 genes were identified as common and responsible for the closer resemblance of Duodenum Intestine-Chip to human adult duodenum than organoids from which chips were derived. Sample sizes were as follows: Duodenum Intestine-Chip, n = 3 (independent donors); Organoids, n = 3 (independent donors); Adult duodenum, n = 2 (independent biological specimens). Intestinal crypts derived from the same three independent donors were used for the establishment of Duodenum Intestine-Chip and organoid cultures. Both chips and organoids were cultured in parallel, in the presence of expansion media for 6 days, followed by 2 days of differentiation media. Experiment was terminated and samples were processed for analyses 8 days post-seeding. (b) The list of overlapping genes was subjected to GO analysis to identify enriched biological processes (GO terms) (Figure 3—source data 4). The results are shown as REVIGO scatterplots in which similar GO terms are grouped in arbitrary two-dimensional space based on semantic similarity. Each circle corresponds to a specific GO term and circle sizes are proportional to the number of genes included in each of the enriched GO terms. Finally, the color of a circle indicates the significance of the specific GO term enrichment. GO terms enriched in the overlapping gene set demonstrate that Duodenum Intestine-Chip is more similar to human duodenum with respect to important biological functions of the intestine, including digestion, transport and metabolism. (c) The results of the KEGG pathway analysis using the 305 differentially expressed genes showed seven significantly enriched (FDR adjusted p-value<0.05) pathways related to absorption, metabolism, digestion and chemical carcinogenesis. The size of the bars indicates the fold-enrichment of the corresponding pathways. (d) Curated heatmaps were generated to examine particular genes that belong to the enriched KEGG pathways and to show the expression levels (loge(FPKM)) of these genes across different samples. Genes belonging to five different pathways, including: ‘mineral absorption’, ‘fat digestion and absorption’, ‘retinol metabolism’, ‘metabolism of xenobiotics by cytochrome P450’ and ‘protein digestion and absorption’, are shown. The expression levels of genes associated with ‘chemical carcinogenesis’ (CYP3A4, GSTA2, UGT2B17, CYP1A1, SULT2A1, ADH4, UGT1A4, ADH6, ADH1A, UGT2A3, UGT2B15, UGT2B7) and ‘drug metabolism – cytochrome 450’ CYP3A4, GSTA2, UGT2B17, ADH4, UGT1A4, ADH6, ADH1A, UGT2A3, UGT2B15, UGT2B7) were included in the heatmap representing ‘metabolism of xenobiotics by cytochrome P450’ as they showed to overlap in between three different pathways. Each heatmap has its own color scale, which corresponds to a different range of loge(FPKM) values, as indicated on the color bars located to the left. The provided results further demonstrate that Duodenum Intestine-Chip (Chip) is more similar to adult duodenum (In vivo) than are the organoids (Organoids). Sample sizes were as follows: Duodenum Intestine-Chip, n = 3 (independent donors); Organoids, n = 3 (independent donors); In vivo, adult duodenum, n = 2 (independent biological specimens). Intestinal crypts derived from the same three independent donors were used for the establishment of Duodenum Intestine-Chip and organoid cultures. Both chips and organoids were cultured in parallel as described in (a). See also Figure 3—figure supplement 1, Figure 3—figure supplement 1—source data 1 and Figure 3—figure supplement 1—source data 2 showing the results of DGE analysis followed by functional enrichment performed between Organoids or Duodenum Intestine-Chip and Adult Duodenum.