A deeper understanding of intestinal organoid metabolism revealed by combining fluorescence lifetime imaging microscopy (FLIM) and extracellular flux analyses
Preprint posted on September 16, 2019 https://www.biorxiv.org/content/10.1101/771188v1
Article now published in Redox Biology at http://dx.doi.org/10.1016/j.redox.2019.101420
To say that organoid culture technologies have revolutionized human biology and medicine in recent years is nothing short of an understatement. The discovery and development of methods to generate 3D organoids for virtually all tissues have enabled the study of self-organized complex systems at a depth previously painfully limited by accessibility of human samples. Consequently, organoids have seen use in a wide range of applications—from developmental biology to disease modeling, drug screening, personalized medicine, and even regenerative medicine.
Several challenges remain, however. For all their advantages, the very heterogeneity that distinguishes and defines organoids also renders it difficult to perform assays previously developed for homogeneous 2D cultures. Intestinal organoids, this study’s chosen model system, are especially notable among the many organoid models for their dramatic 3D spatial heterogeneity—far more than being cellular aggregations, they display clear apicobasal polarity with crypt domains containing LGR5+ stem cells, and villus domains containing differentiated enterocytes and enteroendocrine cells.
Current methods to study metabolism generally fall into either of two categories: 1) bulk methods that take extracellular measurements (most commonly of oxygen and pH) of media containing a whole organoid, or 2) disruptive methods following flow sorting of dissociated organoids. Here, the authors pioneer a third approach: microscopy-based techniques to measure oxygen (O2) and NADH/NADPH in intact intestinal organoids while preserving spatial information.
To measure oxygen levels, they use a small molecule—Pt-Glc—whose phosphorescence is quenched by oxygen. After mouse intestinal organoids are stained with the probe, oxygen levels can be determined by phosphorescence lifetime imaging microscopy (PLIM). The authors also measure fluorescence along with phosphorescence in LGR5-GFP organoids, where GFP marks the stem cell niche and non-fluorescent regions contain more differentiated cells. Using this setup, they find higher oxygenation in GFP+ stem cell-containing regions compared to GFP– differentiated regions across various culture conditions of metabolic stress (namely low glucose and pyruvate withdrawal).
Next, the authors use two-photon excitation microscopy to measure NAD(P)H autofluorescence in stem cell-containing and differentiated regions of LGR5-GFP organoids. Analysis of the fluorescence decay curves provides information on properties of NADH/NADPH: the proportion of enzyme-bound (vs free) NAD(P)H, and mean fluorescence lifetime of bound NAD(P)H. Here, the authors report a decrease in NAD(P)H lifetime in GFP+ regions compared to GFP– regions, as well as some differences in the proportion of bound NAD(P)H in GFP+ and GFP– regions across various media conditions.
As with every nascent technology, however, there were several limitations and complications: firstly, NADPH and NADH must often—as in this case—be measured together due to their very similar fluorescence properties. Secondly, only protein-bound NAD(P)H could be accurately measured, as the picosecond-range fluorescence lifetime of free NAD(P)H was too short. Additionally, autofluorescence was seen in the intestinal organoid lumen. Finally, the biological significance of changes in these properties was not entirely straightforward to determine.
Nonetheless, the authors provide evidence to indicate distinct metabolic dynamics in stem and differentiated cells within intestinal organoids, demonstrating the utility of these techniques for studying metabolism within specific regions of a complex organoid system.
Questions for the authors
- What other complementary techniques can be used to cross-validate oxygen and NAD(P)H measurements?
- Which other pathways or molecules might be most interesting to look at, alongside NAD(P)H & O2?
- You saw clear differences in O2 levels in GFP+ vs GFP– regions, but nothing as obvious for NAD(P)H—I’m curious if you would attribute that to limited sensitivity of the technique, or do you think that biological differences aren’t as large?
- Similarly, in Figure 5 you show that organoids with more distinct LGR5-GFP domains have larger differences in O2 level than more ‘mosaic’ organoids. Do you think this is due to imaging resolution/sensitivity, or biological differences (perhaps organoids with more distinct GFP domains had higher quality differentiation than organoids that became more mixed)?
Posted on: 30th October 2019Read preprint
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