Single-cell transcriptomics of Caco-2 cells cultured in a human gut-on-a-chip under a mechanodynamic culture condition

The microphysiological human gut-on-a-chip has demonstrated in vivo-relevant cellular fidelity of intestinal epithelium compared to its cultures in a static condition1, 2. Microfluidic control of morphogen gradients and mechanical cues robustly induced morphological histogenesis with villi-like three-dimensional (3D) microarchitecture, lineage-associated cytodifferentiation, and physiological functions of a human intestinal Caco-2 epithelium3, 4. However, transcriptomic dynamics that orchestrates morphological and functional reprogramming of the epithelium in a microphysiological culture remains elusive. Single-cell transcriptomic analysis revealed that a gut-on-a-chip culture that offers physiological motions and flow drives three distinctive subclusters that offer distinct gene expression and unique spatial representation in 3D epithelial layers. The pseudotemporal trajectory of individual cells visualized the evolutionary transition from ancestral genotypes in static cultures into more heterogeneous phenotypes in physiodynamic cultures on cell cycles, differentiation, and intestinal functions including digestion, absorption, drug transport, and metabolism of xenobiotics. Furthermore, the inversed transcriptomic signature of oncogenes and tumor-suppressor genes of Caco-2 cells verified that a gut-on-a-chip culture drives a postmitotic reprogramming of cancer-associated phenotypes. Thus, we discovered that a physiodynamic on-chip culture is necessary and sufficient for a cancer cell line to be reprogrammed to elicit in vivo-relevant heterogeneous cell populations with restored normal physiological signatures. Caco-2 cells cultured in a gut-on-a-chip were harvested and run for single-cell RNA sequencing. Cells cultured in Transwell inserts were used as a control.