Live cell imaging of mouse intestinal organoids reveals heterogeneity in their oxygenation

Intestinal organoids are widely applied in stem cell research, regenerative medicine, toxicology, pharmacology, and host-microbe interactions research. The variability of oxidative metabolism for stem and differentiated cell types constituting organoid is known to be important but so far it has not been studied in details. Here, we report the use of live cell microscopy of oxygen via the phosphorescence lifetime imaging microscopy (PLIM) method to address the oxygenation and variability of aerobic metabolism of individual organoids in the culture. Using the cell-penetrating phosphorescent O₂-sensitive probe, we found inhomogeneous O₂ distribution in live organoids, with areas of relatively high oxygenation (up to 73 μM in organoid compared to an average 40 μM O₂) and trans-epithelial O₂ microgradients (up to 0.6-0.83 μM/μm). We also demonstrated that intestinal organoid culture consists of units with different respiration activity and oxygenation (from 27 to 92 μM, equal to 2.8-9.7% O₂), depending on age of the culture and drug treatment. Collectively, our results indicate that ignoring the metabolic heterogeneity of organoid culture can be critical for proper data interpretation. The live cell imaging PLIM method demonstrates a clear advantage of using individual organoids as separate experimental units rather than 'bulk' organoid cultures.