Navigating the fate of intestinal epithelial cells in the world of nanoplastic pollution via Fluorescence Lifetime Imaging Microscopy (FLIM)
Year: 2024
Type: conference-paper
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.
Institutions: Ghent University, American Society for Cell Biology
Language: English
Subfield: Pollution
Field: Environmental Science
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7068081947