Detailed Survey of an in-vitro Intestinal Epithelium Model by Single-Cell Transcriptomics

The gut plays a critical role in maintaining human health by facilitating the absorption of nutrients, regulating metabolism, and interacting with the immune system and gut microbiota. The co-culture of two human colorectal cancer cell lines, Caco-2 and HT29, on Transwell is commonly used as an in vitro gut mimic in studies of intestine absorption pharmacokinetic, gut mechanics, and gut-microbes interplay given the similar morphology, expression of transporters and enzymes, and barrier function. However, to sufficiently evaluate the translatability of insights from such a system to human physiological contexts, a detailed survey of cell type heterogeneity in the system and a holistic comparison with human physiology needs to be conducted rather than by the presence of a few well-studied proteins. Single-cell RNA sequencing provides high-resolution expression profiles of cells in the co-culture, enabling the heterogeneity to be characterized and the similarity to human epithelial cells to be evaluated. 16019 genes transcriptional profile in 13784 cells were acquired and compared to human epithelial cells (GSE185224). We identified the intestinal stem cell, transit amplifying, enterocyte, goblet cell, and enteroendocrine-like cells together with differentiating HT29 cells in the system based on the expression of canonical markers in healthy adult human epithelial cells. The epithelium-like co-culture was fetal intestine-like, with less variety of gene expression compared to the human gut. Transporters for major types of substance (lipid, amino acid, ion, water, etc.) were found transcribed in the majority of the enterocytes-like cells in the system. However, some of the well-studied transporters were absent. Toll-like receptors were not highly expressed in the sample, yet the treatment of LPS still caused a mild change in TEER and gene expression, possibly by the interaction with CD14. Overall, the Caco-2/HT29 co-culture is a cost-effective epithelium model for drug permeability testing or mechanical simulation, but its discrepancy with the real epithelium phenotype-wise is not negligible. As a result, its response to biological factors might not provide transferrable knowledge to the study of human gut physiology, especially the innate immune aspect. Overall design: We cultured Caco-2 (ATCC, passage no. 8) and HT29 (ATCC, passage no.13) separately at 1x105 cells/ cm2 and seeded them together into the apical chamber of three Transwells (Corning Inc, Costar, USA) with a ratio of 9:1 on day 0. Two Transwells were used as controls, where cells were co-cultured and harvested on day 23. The other Transwell was treated with 1 ng/ml Escherichia coli O111:B4 lipopolysaccharides on day 22 and harvested on day 23. Then all samples were prepared for single-cell RNA sequencing per the manufacturer's instruction (10X genomics). Cell morphology agreed with the previous description of the Caco-2/HT29 monolayers. The TEER values reported from control treatments were within range values reported by Hoffmann et aL, indicating the established Caco-2/HT29 co-culture systems possesses comparable properties as other reported cases.