School of Biochemistry and Cell Biology, University College Cork, Ireland.
Department of Anatomy and Neuroscience, University College Cork, Ireland.
Biomaterials. 2017 Nov;146:86-96. doi: 10.1016/j.biomaterials.2017.08.043. Epub 2017 Sep 3.
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 O-sensitive probe, we found inhomogeneous O distribution in live organoids, with areas of relatively high oxygenation (up to 73 μM in organoid compared to an average 40 μM O) and trans-epithelial O 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% O), 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.
肠类器官广泛应用于干细胞研究、再生医学、毒理学、药理学和宿主-微生物相互作用研究。构成类器官的干细胞和分化细胞类型的氧化代谢变异性是很重要的，但迄今为止，这方面的研究还不够详细。在这里，我们报告了使用通过磷光寿命成像显微镜 (PLIM) 方法检测氧的活细胞显微镜来解决单个类器官在培养中的氧合和有氧代谢变异性的问题。使用穿透细胞的磷光 O 敏感探针，我们发现活类器官中存在不均匀的 O 分布，具有相对高氧合的区域（类器官中高达 73 μM，而平均 O 为 40 μM）和跨上皮 O 微梯度（高达 0.6-0.83 μM/μm）。我们还证明，肠类器官培养物由具有不同呼吸活性和氧合的单位组成（从 27 到 92 μM，相当于 2.8-9.7%的 O），这取决于培养物的年龄和药物处理。总的来说，我们的结果表明，忽略类器官培养物的代谢异质性可能会对正确的数据解释产生重大影响。活细胞成像 PLIM 方法表明，将单个类器官用作单独的实验单位而不是“批量”类器官培养具有明显的优势。
