Transcriptional analysis of the intestinal cells
To gain a comprehensive overview of the transcriptional responses to basal fluid, we performed RNA-seq analysis of intestinal epithelial cells in the chip. Differentially expressed genes were identified to elucidate the transcriptomic differences between the presence and absence of fluid. Based on the biological repeated correlation test of the samples, a clear distinction could be observed between the perfused and static groups, while within each group, independent reproducibility was confirmed (Fig. 4B) . By applying the criteria of an absolute value of log fold change > 1 and a p-value < 0.05, we identified 151 up-regulated genes and 304 down-regulated genes in the perfused group compared to the static condition (Fig. 4A) .
The biological processes associated with these genes were further characterized using GO and KEGG pathway enrichment analysis. Using the GO database, we classified the differentially expressed genes into biological process, molecular function, and cell component categories. Among all the enriched terms, the biological process classification was the most prominent and of primary interest. Fig. 4C presents the top 20 terms in descending order of p-value. Notably, in the context of our system, several up-regulated genes of cell under fluid flow were associated with cellular activities, including “regulation of epithelial cell proliferation”, “negative regulation of endothelial cell apoptotic process”, and “cellular response to dexamethasone stimulus”. Additionally, terms related to material metabolism, such as ”negative regulation of cholesterol storage”, ”linoleic acid metabolic process”, and ”glutathione derivative biosynthetic process”, indicated that fluid stimulation impacts the metabolic function of cells. Interestingly, in the enrichment analysis of down-regulated genes, we identified the terms “cellular response to fluid shear stress” and “cellular response to mechanical stimulus”. Furthermore, terms related to cell adhesion, such as “cell surface” and ”anchored component of membrane”, were also present. Whether this is a negative feedback regulation of cells in the gut-on-a-chip system we constructed, it still needs a further exploration.
Furthermore, the KEGG enrichment analysis (Fig. 4D) revealed that the presence of fluid in the culture enhances several pathways associated with drug metabolism compared to static culture. These pathways include “Metabolism of xenobiotics by cytochrome P450”, “Drug metabolism - other enzymes”, and “Drug metabolism - cytochrome P450”. Moreover, the enrichment of up-regulated genes in pathways such as “Fat digestion and absorption” and “Metabolic pathways” indicates an improvement in the metabolic function of cells in the presence of fluid. These findings underscore the ability of our platform to achieve better physiological function simply by providing fluid with a rocker, as compared to regular static cultivation.
Assessment of drug bioavailability after exposure to