Live Cell Microscopy of Intestinal Organoid Oxygenation

Oxygenation of stem cell niche is important metabolic activity indicator for the intestine in normal and diseased states. In vivo , it can be affected by such factors as diet, drugs, metabolites produced by microbiota, and inflammation. We hypothesized that O 2 must be also very important for adequate development of organoids and organs‐on‐chip, growing as 3D cultures. We took advantage of cell‐penetrating O 2 ‐sensitive probes and phosphorescence lifetime imaging microscopy (PLIM), which, in contrast to the end‐point hypoxia stains and electrode‐based measurements, provides high‐resolution real‐time visualization of O 2 . Due to the specialised cultivation conditions for different types of organoids and varied ability for staining with live cell dyes and probes, we optimized the staining parameters with O 2 ‐sensitive probes. Using staining with small molecule Pt‐Glc probe [1] and PLIM technique we analyzed O 2 distribution in organoids from small intestine of mouse. We were able to directly measure oxygenation of organoids without their recovery from Matrigel matrix and multiplexed it with number of live fluorescent probes (Hoechst 33342, Cholera Toxin‐Alexa 488, TMRM, CellTox Green, Calcein Green) and immunofluorescent staining of fixed samples. We next analysed the oxygenation of live organoids treated either with mitochondrial uncoupler FCCP or anti‐diabetic drug metformin, inhibiting activity of mitochondrial complex I [2]. We found that even when produced from the same animal, resting organoids displayed highly heterogeneous oxygenation, from 27 to 88 μM (approx. 2.7~8.8% of air saturation). This can be explained by the growth at different depths in Matrigel matrix and limited diffusion of O 2 and/or highly variable respiration between organoids. Treatment with FCCP, increasing oxygen consumption, resulted in decrease of organoid oxygenation, confirming that the intestinal epithelia tissue has active mitochondria. In contrast, metformin treatment significantly increased the overall oxygenation of organoids in comparison to non‐treated culture. However, the magnitude of response to metformin varied for samples taken from different animals. This data confirms that when accumulating metformin, intestinal epithelia undergoes inhibition of mitochondrial respiration, which can change its oxygenation and lead to glycolytic switch of metabolism. Altogether, our results point at the importance of analysis of organoid culture oxygenation in tissue engineering, and demonstrate how this information can be translated in studies of molecular mechanisms of diabetes. Support or Funding Information Supported by Science Foundation Ireland (SFI) grant 13/SIRG/2144.