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A deeper understanding of intestinal organoid metabolism revealed by combining fluorescence lifetime imaging microscopy (FLIM) and extracellular flux analyses.

Authors
  • Okkelman, Irina A1
  • Neto, Nuno2
  • Papkovsky, Dmitri B1
  • Monaghan, Michael G3
  • Dmitriev, Ruslan I4
  • 1 Laboratory of Biophysics and Bioanalysis, ABCRF, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, T12 K8AF, Ireland. , (Ireland)
  • 2 Department of Mechanical and Manufacturing Engineering, Trinity College Dublin, Ireland; Trinity Centre for Biomedical Engineering, Trinity College Dublin, Ireland. , (Ireland)
  • 3 Department of Mechanical and Manufacturing Engineering, Trinity College Dublin, Ireland; Trinity Centre for Biomedical Engineering, Trinity College Dublin, Ireland; Advanced Materials and BioEngineering Research (AMBER) Centre at Trinity College Dublin and Royal College of Surgeons in Ireland, Dublin, Ireland. Electronic address: [email protected] , (Ireland)
  • 4 Laboratory of Biophysics and Bioanalysis, ABCRF, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, T12 K8AF, Ireland; Institute for Regenerative Medicine, I.M. Sechenov First Moscow State University, Moscow, Russian Federation. Electronic address: [email protected] , (Ireland)
Type
Published Article
Journal
Redox Biology
Publisher
Elsevier
Publication Date
Feb 01, 2020
Volume
30
Pages
101420–101420
Identifiers
DOI: 10.1016/j.redox.2019.101420
PMID: 31935648
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Stem cells and the niche in which they reside feature a complex microenvironment with tightly regulated homeostasis, cell-cell interactions and dynamic regulation of metabolism. A significant number of organoid models has been described over the last decade, yet few methodologies can enable single cell level resolution analysis of the stem cell niche metabolic demands, in real-time and without perturbing integrity. Here, we studied the redox metabolism of Lgr5-GFP intestinal organoids by two emerging microscopy approaches based on luminescence lifetime measurement - fluorescence-based FLIM for NAD(P)H, and phosphorescence-based PLIM for real-time oxygenation. We found that exposure of stem (Lgr5-GFP) and differentiated (no GFP) cells to high and low glucose concentrations resulted in measurable shifts in oxygenation and redox status. NAD(P)H-FLIM and O2-PLIM both indicated that at high 'basal' glucose conditions, Lgr5-GFP cells had lower activity of oxidative phosphorylation when compared with cells lacking Lgr5. However, when exposed to low (0.5 mM) glucose, stem cells utilized oxidative metabolism more dynamically than non-stem cells. The high heterogeneity of complex 3D architecture and energy production pathways of Lgr5-GFP organoids were also confirmed by the extracellular flux (XF) analysis. Our data reveals that combined analysis of NAD(P)H-FLIM and organoid oxygenation by PLIM represents promising approach for studying stem cell niche metabolism in a live readout. Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.

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