Stem cell-derived organoids cultured two-dimensionally: The benefits of organoids with the dosing convenience of epithelial cell lines

Abstract: Despite numerous advantages of stem cell derived intestinal organoids (SIOs) over epithelial cell lines, the originally described three-dimensional (3D) culture in Matrigel (1) has two issues with respect to testing compounds. First, they consist mainly of crypt structures with few differentiated cells, whereas our intestine consists for the majority of differentiated enterocytes. Secondly, the apical or gut lumen side is on the inside of 3D SIOs, making administration and dosing of compounds for experiments very challenging.
To circumvent these issues, we developed two-dimensional (2D) culture models for mouse, porcine and human SIOs in which both the crypt structures as well as the differentiated villus cells (enterocytes, goblet cells and enteroendocrine cells) are represented. We show that these 2D organoid cultures maintain location-specific gene expression and responses, e.g. that the artificial sweetener rebaudioside A (derived from Stevia) stimulates production of GLP1 specifically in ileal enteroendocrine cells. Furthermore, we are able to grow SIOs in transwell formats to stable, confluent layers with a build-up of electrical resistance, low FD4 leakage (<1% leakage/h) and expected absorptions of model compounds for transcellular transport (antipyrine) and paracellular transport (atenolol). Additionally, stimulation of the monolayer with known immunogenic stimulants Flagellin and IL-1B showed an increase in IL-8 secretion compared to the blank. Taken together, our results indicate that the 2D SIO model allows studying the effects of compounds beyond the existing possibilities with standard epithelial cell lines. We are currently exploring the potential of this culture method in organ-on-a-chip applications and in co-culture methods with immune cells and bacteria.