Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death

Apical‐out intestinal organoids are a relatively simple method of gaining access to the apical cell surface and have faced increasing scientific interest over the last few years. Apical‐out organoids can thus be used for disease modelling to compare differing effects on the basolateral versus the apical cell surface. However, these ‘inside‐out’ organoids die relatively quickly and cannot be propagated as long as their basal‐out counterparts. Here, we show that apical‐out organoids have drastically reduced proliferative potential, as evidenced by immunohistochemical staining and the incorporation of the thymidine analogue EdU. At the same time, cell death levels are increased. Nevertheless, these phenomena cannot be explained by an induction of differentiation, as the gene expression of key marker genes for various cell types does not change over time. Basal out organoid polarity can easily be reversed by extracellular matrix removal to generate fully apical out intestinal organoids. However, this polarity reversal is accompanied by decreased proliferation coupled with increased cell death, ultimately leading to the death of apical out organoids, questioning their applicability and usefulness for disease modelling. Despite these differences, we demonstrate that transcript expression of key intestinal marker genes is unaffected by polarity reversal.