Okkelman Irina A, Zhou Hang, Borisov Sergey M, Debruyne Angela C, Lefebvre Austin E Y T, Leomil Zoccoler Marcelo, Chen Linglong, Devriendt Bert, Dmitriev Ruslan I
Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, The Core, C. Heymanslaan 10, 9000, Ghent, Belgium.
Ghent Light Microscopy Core, Ghent University, 9000, Ghent, Belgium.
Light Sci Appl. 2025 Aug 12;14(1):272. doi: 10.1038/s41377-025-01949-0.
Increased micro- and nanoplastic (MNP) pollution poses significant health risks, yet the mechanisms of their accumulation and effects on absorptive tissues remain poorly understood. Addressing this knowledge gap requires tractable models coupled to dynamic live cell imaging methods, enabling multi-parameter single cell analysis. We report a new method combining adult stem cell-derived small intestinal organoid cultures with live fluorescence lifetime imaging microscopy (FLIM) to study MNP interactions with gut epithelium. To facilitate this, we optimized live imaging of porcine and mouse small intestinal organoids with an 'apical-out' topology. Subsequently, we produced a set of pristine MNPs based on PMMA and PS (<200 nm, doped with deep-red fluorescent dye) and evaluated their interaction with organoids displaying controlled epithelial polarity. We found that nanoparticles interacted differently with apical and basal membranes of the organoids and showed a species-specific pattern of cellular uptake. Using a phasor analysis approach, we demonstrate improved sensitivity of FLIM over conventional intensity-based microscopy. The resulting 'fluorescence lifetime barcoding' enabled distinguishing of different types of MNP and their interaction sites within organoids. Finally, we studied short (1 day)- and long (3 day)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function, total cell energy budget and epithelial inflammation. We found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells. The presented FLIM approach will advance the study of MNP toxicity, their biological impacts on gastrointestinal tissue and enable the tracing of other fluorescent nanoparticles in live organoid and 3D ex vivo systems.
微塑料和纳米塑料（MNP）污染的增加带来了重大的健康风险，然而它们在吸收性组织中的积累机制及其影响仍知之甚少。解决这一知识空白需要与动态活细胞成像方法相结合的易处理模型，以实现多参数单细胞分析。我们报告了一种将成体干细胞衍生的小肠类器官培养与活细胞荧光寿命成像显微镜（FLIM）相结合的新方法，用于研究MNP与肠道上皮的相互作用。为此，我们优化了具有“顶端向外”拓扑结构的猪和小鼠小肠类器官的活细胞成像。随后，我们制备了一组基于聚甲基丙烯酸甲酯（PMMA）和聚苯乙烯（PS）的原始MNP（<200 nm，掺杂深红色荧光染料），并评估了它们与显示可控上皮极性的类器官的相互作用。我们发现纳米颗粒与类器官的顶端和基底膜相互作用方式不同，并呈现出物种特异性的细胞摄取模式。使用相量分析方法，我们证明了FLIM比传统的基于强度的显微镜具有更高的灵敏度。由此产生的“荧光寿命条形码”能够区分不同类型的MNP及其在类器官内不同的相互作用位点。最后，我们研究了基于PMMA和PS的MNP对线粒体功能、总细胞能量预算和上皮炎症的短期（1天）和长期（3天）暴露效应。我们发现，即使是原始的MNP也会破坏肠道上皮细胞中的趋化因子产生和线粒体膜电位。所提出的FLIM方法将推动对MNP毒性、它们对胃肠道组织的生物学影响的研究，并能够在活类器官和3D体外系统中追踪其他荧光纳米颗粒。
