A Two-Dimensional Organoid Culture on Invasin or Basement Membrane Extract to Study Host Bacteria Interactions

Organoid technology provides an experimental in vitro platform for studying human physiology in health and disease. Compared with other in vitro systems, such as cancer-derived cell lines or explants, tissue stem cell (TSC)-derived organoids offer key advantages: they can be expanded indefinitely while maintaining genomic stability and are amenable to CRISPR modification. Since these organoids are generated from normal TSCs, they contain the cell types of their tissue of origin. This makes organoids particularly valuable for investigating interactions between bacteria and differentiated, non-tumoral human cells. Intestinal organoids are grown as two-dimensional polarized monolayers on cell culture inserts coated with Basement Membrane Extract (BME) or invasin, an animal-free, cost-effective alternative, and can be differentiated into major intestinal epithelial cell types for infection with pathogenic adherent-invasive E. coli. Key infection readouts include monitoring epithelial gene expression by Reverse Transcription quantitative PCR (RT-qPCR), visualizing bacteria-epithelium interactions by confocal microscopy, and quantifying epithelial barrier breaching. The protocol can be readily adapted to study other organs and bacterial species, providing a versatile platform for investigating host-microbe interactions in organoids.