@inproceedings{01HK50WPGR5G11V5TJHJEKV76P,
  abstract     = {{We live in a world of ever increasing micro- and nanoplastic pollution. These nanoplastics display a complex chemical and physical composition, broad dispersity and poorly understood effects on the function of the mammalian gastrointestinal tract. To address these knowledge gaps, tractable models together with a selection of the most appropriate (bio)analytical methods, enabling for live multi-parameter analysis with single cell resolution, are much needed. 
Here we report a new method to study interactions of nanoplastics with the gut epithelium, based on combining adult stem cell-derived small intestinal organoid cultures with multi-parameter live Fluorescence Lifetime Imaging Microscopy (FLIM). Normally, gut organoids display a ‘basal-out’ topology with an apical membrane that is difficult to access. To address this, we optimized the conversion of porcine and mouse small intestinal organoid cultures to an ‘apical-out’ topology (confirmed by a phalloidin staining), which improves apical-membrane availability without disturbing a 3D integrity of organoids and facilitates studying epithelial interactions with drugs, microbiota and nanoplastics. Live ‘apical-out’ organoids were exposed to a set of nanoparticles exhibiting different surface charges, sizes of <200 nm and doped with red fluorescent dyes. In contrast to previous work with basal-out gut organoids, we observed an efficient accumulation of the nanoparticles from the apical side over the course of 24 h under regular growth conditions. We also compared staining of basal- and apical-out gut organoids with several fluorescent FLIM probes, enabling visualization of cell redox, mitochondrial membrane potential and lipid droplets. Interestingly, a more efficient uptake was observed for many dye structures including the lipid-sensing Nile Red and the membrane tension-sensing Flipper-TR probes. 
Together with segmentation of stem cells with tracers such as Lgr5-GFP, labeling cell redox and ‘apical-to-basal’ membrane transport, the presented methodology enables to address the effects of nanoplastics on stem cell and enterocyte function, their metabolism and efficiency of nutrient absorption, in a live and multi-parameter setting. 

Supported by Special Research Fund (BOF) by Ghent University and Research Foundation Flanders (FWO) grants.}},
  articleno    = {{P1769}},
  author       = {{Okkelman, Irina and Devriendt, Bert and Dmitriev, Ruslan}},
  booktitle    = {{MOLECULAR BIOLOGY OF THE CELL}},
  issn         = {{1059-1524}},
  language     = {{eng}},
  location     = {{Boston, MA, USA}},
  number       = {{1}},
  pages        = {{P1769:480--P1769:480}},
  title        = {{Navigating the fate of intestinal epithelial cells in the world of nanoplastic pollution via Fluorescence Lifetime Imaging Microscopy (FLIM)}},
  url          = {{https://www.molbiolcell.org/doi/10.1091/mbc.E23-10-0402}},
  volume       = {{35}},
  year         = {{2024}},
}