Detection of Time-Dependent Uptake of Microplastics by Joint Utilization of Hepatic Organoids and a 3D-Printed...

Microplastics (MPs, <5 mm) are pervasive in foods, the environment, and humans, posing emerging health risks. Traditional two-dimensional (2D) cell models inadequately replicate micron-scale MP uptake, whereas three-dimensional (3D) organoids better mimic tissue complexity. Here, we developed a hepatic organoids (HOs)-in-cage system using human pluripotent stem cell-derived HOs and a 3D-printed porous poly(ε-caprolactone) carrier for efficient organoid retrieval. We established a label-free Nile Red (NR)-based spatiotemporal imaging and flow cytometric quantification pipeline to investigate the uptake dynamics and hepatotoxicity of UV-aged, size-mixed polypropylene (PP, 1 to 20 μm), as well as to colocalize the MP and potential biomarkers within HOs. Contrary to 2D models where >5 μm particles show negligible internalization, 3D HOs exhibited significant accumulation of intact micron-scale MPs via tissue-layer penetration and paracellular retention, with PP uptake peaking at ∼40% within 48 h and ∼18% retained long-term. This triggered dose- and time-dependent hepatotoxicity (40-4000 ng/mL), marked by CD36 upregulation from 8 h, mitochondrial impairment, and elevated LDH and AST levels. Notably, NR fluorescence intensity was governed by polymer chemistry rather than surface roughness, enabling material-specific detection. This work provides novel label-free techniques within 3D in vitro models to explore the depot and effective concentration of micron-scale MPs, advancing understanding of MP-induced cellular damage upon uptake and deposition.