Abstract Acute myeloid leukemia (AML) is an aggressive blood malignancy with a dismal 5-year survival rate below 30%. Dysregulation of mRNA translation is a hallmark and a driver of tumorigenesis, including leukemogenesis. However, the precise contributions of translation factors to AML pathogenesis and their potential as therapeutic targets remain poorly understood. Here, we identify eukaryotic translation initiation factor 4A1 (eIF4A1) as a promising vulnerability of AML; genetic depletion or pharmacological inhibition of eIF4A1 markedly suppresses AML initiation and progression via reprogramming amino acid metabolism. Through unbiased multi-omics analysis, we identified eIF4A1 as the most highly expressed translation factor in AML. Notably, eIF4A1 expression was significantly elevated in AML cells and patient samples compared to healthy controls. eIF4A1 knockout (KO) dramatically inhibited AML cell proliferation, suppressed mitochondrial respiration, and reduced global translation intensity in vitro and substantially delayed AML progression in vivo. While eIF4A1 is traditionally studied within the cap-binding eIF4F complex, our other findings uncover a novel eIF4F complex-independent mechanism. BioID-MS assays, coupled with the validation of PLA assays and co-IP assays, revealed the robust RNA independent proximity of eIF4A1 and mRNA stabilizer Y-box binding protein 1 (YB-1). Integrative RNA-seq and proteomics demonstrated phosphoglycerate dehydrogenase (PHGDH) as a functionally essential target of eIF4A1. Moreover, metabolic profiling combined with isotope tracing (13C) orthogonally confirmed the crucial role of eIF4A1 in rewiring de novo serine metabolism, in which PHGDH serves as the rate-limiting enzyme. Gene specific CLIP-qPCR verified the direct binding of both eIF4A1 and YB-1 to PHGDH mRNA. Furthermore, the KO of either eIF4A1 or YB-1 accelerated PHGDH mRNA decay. Collectively, these findings suggest an eIF4F-independent mechanism of eIF4A1: eIF4A1 cooperates with YB-1, stabilized PHGDH mRNA and reprogrammed amino acid metabolism in AML. Zotatifin, an FDA-approved eIF4A1 inhibitor, administered intraperitoneally (0.5 mg/kg; twice weekly for five weeks), dramatically reduced the leukemia burden and significantly prolonged survival of AML mouse models in vivo (immunodeficient xenograft model: 63 vs. 150 days median survival for PBS vs. Zotatifin, P = 0.0006; immunocompetent bone marrow transplantation model: 40 vs. 75 days, P = 0.001). Moreover, Zotatifin demonstrated strong synergistic activity with the YB-1 inhibitor SU056 in eradicating AML both in vitro and in vivo (P < 0.0001), primarily by disrupting amino acid biosynthesis. Overall, our findings identify eIF4A1 as a key regulator of AML pathogenesis and metabolic homeostasis. Targeting eIF4A1, particularly with Zotatifin, represents a promising therapeutic strategy for AML. Citation Format: Xiaoxu Zhang, Honghai Zhang, Lei Dong, Alexandra Huang, Xueer Wang, Lili Ren, Hongjie Bi, Seán O’Leary, Rui Su. Pharmacological inhibition of eIF4A1 suppresses leukemogenesis via specifically rewiring amino acid biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4037.

Timely and spatially sufficient oxygen delivery remains a significant challenge in large-volume soft tissue repair. Here, we present a novel plug-and-supply flap (PASF) system that integrates hierarchical vasculature and in situ endothelialization to achieve instantaneous perfusion within seconds and provide uniform metabolic support. We fabricated hierarchical vascular networks with high structural precision using a hydrogen-bond reorientation technology, while biofunctionalizing the conduit surface to enable rapid endothelial adhesion via a unique galectin-mediated mechanism, without needing prolonged pre-culture. Computational modeling guided vascular geometry optimization by simulating oxygen diffusion and hemodynamic dynamics, thereby improving both metabolic support and perfusion stability. In a rabbit carotid artery anastomosis model, PASF restored immediate perfusion and enhanced oxidative metabolism, thereby promoting angiogenesis and establishing a pro-regenerative niche. Together, these findings demonstrate a generalizable strategy for engineering perfusable scaffolds that actively modulate the post-implantation niche in soft-tissue reconstruction, where early metabolic support is vital.

Pancreatic ductal adenocarcinoma (PDAC), a refractory cold tumor, is characterized by a dense extracellular matrix (ECM) and stress-triggered neuroimmune crosstalk, which cause T cell exhaustion and poor efficacy of anti-T cell immunoglobulin and mucin domain-3 antibody (aTIM-3) monotherapy. Here, we report a sucrose-hybridized, Pan02 pancreatic tumor cell membrane-coated ultrasound (US)-responsive biomimetic nanocarrier (CT@SM) co-loading carvedilol (β-blocker) and aTIM-3. The Pan02 tumor cell membrane shell enables homologous tumor targeting, prolonged circulation, and reduced off-target distribution, while intercalated sucrose modulates vesicle acoustic impedance to generate US-triggered inertial cavitation. US activates cavitation to disrupt the ECM and co-releases cargos deep in tumors: carvedilol relieves norepinephrine-mediated T cell exhaustion, and aTIM-3 suppresses M2-type tumor-associated macrophages. Consequently, compared to free aTIM-3, up to an 11.23-fold increase of aTIM-3 in the tumor region was obtained in the CT@SM + US group. Immunotherapy efficacy was significantly enhanced by reactivating T cells, leading to suppressed tumor growth and prolonged survival in the PDAC mice stress model. Overall, this study offers a promising strategy based on a dual-modified biomimetic acoustic delivery platform for immunotherapy in PDAC and other cold tumors.

Segmental airway reconstruction requires tracheal grafts that emulate the hierarchical structure and biochemical functions of native trachea. However, achieving both biomimetic fidelity and efficient vascularization remains challenging. Here, we present a digitally light-processed tissue-engineered trachea featuring anatomically biomimetic C-shaped cartilage rings, alternating fibrous rings, and dorsal fibrous bands, precisely replicating the structural hierarchy of native trachea. To promote rapid neovascularization, we develop a stress-relaxing and degradable alginate-based hydrogel capable of dynamic vascular endothelial growth factor loading and sustained release, thereby facilitating endothelial cell migration and angiogenesis. Within the fibrous regions, pre-engineered vascular channels are incorporated to guide host vascular ingrowth, resulting in a 2.6-fold increase in neovascular density compared to no-channel scaffolds. This platform integrates spatial control through precise structural design with temporal bioactive signal modulation, enabling synchronized vascularization. When transplanted via end-to-end anastomosis with native tracheae, the vascularized grafts exhibit enhanced survival and functional integration, offering a robust strategy for tracheal tissue engineering and segmental airway reconstruction.

Regeneration of hyaline cartilage remains a major clinical challenge. Current first-line treatments for focal articular cartilage defects, typically induce fibrocartilage instead of hyaline cartilage, resulting in inferior mechanical and biological outcomes. Here we report a biomimetic strategy inspired by the biosilicification process of diatoms, for the de novo synthesis of biosilica nanocages exhibiting effective binding to the hyaline chondrogenic cytokine transforming growth factor β1 (TGF-β1). To enhance clinical applicability, an injectable biosilica nanocage-based hydrogel (Si-aGel hydrogel) was further engineered to conformally fill cartilage defects of diverse geometries while exhibiting excellent mechanical performance. Si-aGel hydrogel preserves long-term hyaline chondrogenic commitment of rat primary bone marrow-derived mesenchymal stem/stromal cells (BMSCs) in vitro. Moreover, when injected into the full-thickness cartilage defects, Si-aGel hydrogel effectively sustained stable TGF-β1-activated chondral differentiation of BMSCs and promoted complete hyaline cartilage repair with a type II collagen- and aggrecan-rich hyaline matrix deposition without any exogenous supplements. In contrast, those without intervention or treated with hydrogel lacking biosilica nanocages developed fibrocartilaginous and hypertrophic repair tissue, marked by a disorganized matrix with abundant type I and type X collagen. Together, our study suggested the biosilica nanocage-based therapy has the great potential for hyaline cartilage regeneration.

The harsh microenvironment characterized by avascularity and hypoxia presents a significant challenge for bone regeneration following refractory bone defects. Tissue engineering combined with electrotherapy has emerged as a promising alternative for repairing bone defects, offering advantages such as accelerated healing and the restoration of physiological functions in regenerated bone. In this study, we propose a strategy for constructing tissue-engineered cartilage derived from bone marrow stem cells (BMSCs) for bone regeneration, utilizing 3D-printed triboelectric scaffolds (TES). The TES scaffold is fabricated from biodegradable bioelastomer and conductive biomaterial, featuring excellent biomimetic elasticity and hydrophobicity. The TES contains numerous hydrophobic microporous units, enabling in situ self-powered stimulation in vivo. The conductivity of the TES has been shown to enhance the chondrogenic differentiation potential of BMSCs during in vitro induction into tissue-engineered cartilage. Notably, the TES scaffold was more effective in promoting endochondral ossification of tissue-engineered cartilage in vivo. The in vivo osteogenesis mechanism of the TES group was further analyzed through proteomics, revealing that TES facilitated actin cytoskeleton remodeling, activated the PI3K-Akt pathway, provided metabolic support, and enhanced intercellular communication to drive the endochondral ossification process. Finally, in situ skull defect repair in rabbits successfully demonstrated the efficacy of TES electrical stimulation in promoting tissue-engineered endochondral ossification, thereby achieving bone defect regeneration and providing an effective biological strategy for the repair of refractory bone defects.

Achieving spatiotemporal control over cellular self-organization when fabricating functional tissues remains a key challenge. Here, we develop bioFLOAT (bioprinted free-form living architectures via osmotic adaptive templates) that harnesses the phase behavior of liquid-liquid phase separation (LLPS) to mediate morphological multicellular assembly. This approach confines cells within a floating phase that is wettable to cells but dewets the container surface, allowing its shape to be defined by geometric confinement. This system programs cellular self-assembly through boundary restriction and osmotic gradients, generating lumen-like or solid tissues without scaffolds or cellular spheroids. By precisely controlling LLPS-driven crowding, bioFLOAT achieves predictable cellular compaction and rapid (<24 h) fabrication of multiscale tissue architectures with high fidelity. Engineered myocardial tissues from human embryonic stem cell-derived cardiomyocytes exhibit appropriate sarcomeric organization and spontaneous contractility. Overall, bioFLOAT represents a scalable platform for manufacturing functional tissues by harnessing the fluid-physical behavior of phase separation and wetting.

Silicon (Si) participates in diverse biological processes, yet its specific roles remain elusive because of the lack of tools for visualizing silicon in living organisms. In biological contexts, silicon predominantly occurs as silicates with varying polymerization states and structural heterogeneity, posing fundamental challenges for recognition and imaging using existing chemical or analytical methods. Here, we introduce a molecular recognition strategy that leverages single-stranded DNA aptamers selected through a three-dimensional silicified hydrogel screening system, yielding high-affinity binders with nanomolar dissociation constants. We validated these aptamers using electrophoretic mobility shift assays (EMSA) and microscale thermophoresis (MST), and molecular docking revealed the sequence-structure motifs governing silicate recognition. Using these aptamers, we engineered two fluorescent sensors that map intracellular silicate distributions with high specificity. To our knowledge, these are the first aptamers that selectively recognize silicates with distinct polymerization states, providing a powerful tool for real-time visualization of silicon in living cells. This approach opens new avenues for biological imaging and for elucidating the functional roles of biogenic silicon in living organisms.

Spinal cord injury (SCI) creates a mechanically discontinuous and biochemically hostile lesion niche marked by oxidative stress, persistent inflammation, vascular dysfunction, and poor graft retention, which together limit cell-based repair. Here, we report a coordination-engineered living therapeutic interface that converts extracellular matrix (ECM)-rich human umbilical cord mesenchymal stem cell sheets (CSs) from passive grafts into microenvironment-regulating implants. A zinc metal–phenolic network (Zn MPN), formed through tannic acid/zinc ion coordination, was assembled across the sheet surface and accessible ECM domains to generate ZM-CS. This lesion-facing interface preserved sheet architecture, increased construct-level Zn incorporation, slowed degradation, and showed a qualitatively more sustained lesion-associated fluorescence signal after implantation. Under hydrogen peroxide-induced stress, ZM-CS reduced intracellular reactive oxygen species (ROS), preserved the viability of PC12 neuronal-like cells and human umbilical vein endothelial cells (HUVECs), and improved endothelial invasion and tube formation more effectively than unmodified CS. It was also associated with a repair-supportive shift in BV2 microglial inflammatory phenotype, with reduced iNOS-associated pro-inflammatory features and enhanced Arg-1-associated reparative characteristics. Transcriptomic profiling associated this cytoprotection with a repair-compatible neuronal-like cell state characterized by coordinated redox adaptation and enrichment of proliferation-, cytoskeleton-, and repair-associated programs. In a rat spinal cord hemisection model, ZM-CS improved long-term locomotor recovery, gait-related parameters, vascular remodeling, neural structural preservation, and lesion-site inflammatory regulation.

The limited regenerative capacity of injured meniscal tissue necessitates the development of advanced tissue-engineered alternatives. However, conventional homogeneous meniscal implants often fail to replicate the inherent area-specific architecture of the native meniscus. Herein, we present a synergistic regeneration strategy to fabricate a Biomimetic Area-specific Meniscus (BAM), integrating biomimetic microenvironmental modulation with spatially organized multicellular patterning. This study engineered a composite scaffold by combining meniscus-derived decellularized extracellular matrix (Me-dECM) with a 3D-printed polycaprolactone (PCL) framework, which demonstrated favorable bioactivity and mechanical robustness. Through a post-occupancy sacrifice (POS) strategy employing thermosensitive Pluronic F-127 hydrogel, this study achieved precise spatial arrangement of fibrochondrocytes (FCs) and fibroblasts (FBs), resulting in a continuously tripartite meniscal construct with heterogeneous spatial organization. In vitro and in vivo assessments confirmed that this tripartite design effectively recapitulates native meniscal regional heterogeneity, exhibiting gradient distributions of collagen types I and II (COL I/II) and sulfated glycosaminoglycans (GAGs), thereby accomplishing structural and functional biomimicry. Collectively, the BAM strategy enables the regeneration of a mechanically competent, gradient-heterogeneous meniscus, offering a promising translational pathway for functional meniscal reconstruction.

Osteonecrosis of the femoral head (ONFH) is a debilitating condition often leading to joint collapse. While corticosteroids use and alcohol consumption are known risk factors, the pathophysiology, especially in idiopathic cases, which account for one-third population, remains unclear. This study aimed to investigate the potential role of human cytomegalovirus (HCMV) reactivation in the pathogenesis of ONFH, focusing on its presence, distribution, and reactivation status. Blood and femoral head samples were obtained from ONFH patients and fracture controls. Human cytomegalovirus exposure was assessed through serology and viral DNA quantification, and reactivation was confirmed by gB immunohistochemistry and IE-1 mRNA RT-qPCR. Tissue samples from different regions of the femoral head (necrotic, transitional, and healthy zones) were analyzed for viral content, reactivation, and localization. Results showed significantly higher HCMV DNA levels in necrotic and transitional zones of ONFH, strongly correlated with lesion volume. Furthermore, gB localization was predominantly found in the microvascular structures, such as small vessels and capillaries, suggesting that HCMV reactivation may contribute to microvascular damage and ischemia. IE-1 transcripts, markers of viral reactivation, further confirmed reactivation. Notably, HCMV reactivation was observed across all ONFH etiologies-corticosteroid-related, alcohol-related, and idiopathic-indicating its broad involvement in ONFH progression. This study provides the first clinical evidence linking HCMV reactivation to ONFH, offering potential therapeutic avenues, including antiviral treatments, to address this condition.

Cartilage injury and osteochondral defects remain major clinical challenges owing to the limited intrinsic regenerative capacity of cartilage and the inability of current treatments to restore durable hyaline tissue. Conventional hydrogels often fail under joint loading because of insufficient mechanical stability, poor interfacial integration, and limited biological responsiveness. In this review, high-viscosity hydrogels (HVHs) are considered within a performance-based framework that integrates post-deployment cohesion and structural retention, viscoelastic energy dissipation, and reversible network reconfiguration to provide both mechanical support and biological regulation. Through multilevel design strategies, including double-network structures, reversible crosslinking, nanocomposite reinforcement, and stimuli-responsive modules, HVHs establish highly cohesive yet reconfigurable matrices capable of stress dissipation, self-adaptation, and sustained bioactive factor delivery. Beyond structural functions, HVHs actively regulate mechanobiological signaling by modulating matrix stiffness, stress relaxation, immune responses, and stem-cell fate, thereby promoting chondrogenic differentiation, suppressing hypertrophy, and facilitating extracellular matrix deposition. Recent advances in gradient and biphasic HVH systems further enable coordinated regeneration of cartilage and subchondral bone, supporting functional osteochondral integration through spatially controlled mechanical and biochemical cues. Emerging technologies such as 3D/4D printing, artificial intelligence-assisted material optimization, and organoid-based validation are accelerating the development of personalized and adaptive regenerative platforms. Although challenges remain regarding long-term mechanical durability, degradation-regeneration matching, and clinical standardization, HVHs represent a promising paradigm that integrates dynamic network engineering, mechanobiological regulation, osteochondral reconstruction, and translational adaptability. This review summarizes recent advances in HVH design and application, highlighting their potential to reshape future strategies for cartilage and osteochondral regeneration through intelligent, biomimetic, and clinically translatable material systems.

BACKGROUND:We report orthotopic combined whole-liver and bilateral-kidney xenotransplantation (xeno-CLKT) from a six-gene-edited pig into a 53-year-old human decedent recipient. METHODS:After simultaneous implantation and reperfusion, graft function was monitored for nearly 5 days using continuous clinical assessments. Host responses were further characterized with single-cell RNA sequencing, proteomics, and metabolomics of blood and graft tissues collected before and after transplantation. FINDINGS:All grafts maintained basic physiological function throughout the observation period, with no evidence of hyperacute rejection. Early post-transplant immune analysis showed expansion of S100A12+ neutrophils, which emerged as central hubs in inferred intercellular communication networks. Systems-level metabolomic profiling indicated that post-transplant metabolic patterns remained positively correlated with the recipient's pre-transplant baseline, with generally higher magnitudes. CONCLUSIONS:This study provides initial evidence for the feasibility of pig-to-human orthotopic whole-liver plus bilateral-kidney transplantation and identifies early immune and metabolic features that may inform perioperative management and future clinical translation. FUNDING:The research was supported by the Guangxi Key Research and Development Program (AB24010059), the Shenzhen Medical Research Fund (SMAF) (B2302008), and the Guangdong Province Guangdong-Shenzhen Joint Key Project (2023B1515120083).

Cells must efficiently locate and engage for tissue formation and immune coordination, yet classical receptor-ligand binding is limited to nanometre distances and is inherently slow. Here, we uncover a previously unrecognised physical principle, liquid-like adhesion by phase separation (LAPS). This process creates dynamic wetting layers on cell surfaces, functioning as 'liquid bridges' that enable robust, long-range cell capture across tens of micrometres. Remarkably, this wetting- mediated attraction remains effective at nanomolar concentrations-conditions where bulk phase separation would not be expected-and facilitates high-fidelity cell sorting through competitive wetting. By integrating aqueous two-phase systems, endogenous proteins (Galectin-3, CCL5), and fluid-particle-dynamics simulations, we demonstrate that extracellular liquid-liquid phase separation not only mediates long-range cell capture but also acts as a physical catalyst for contact-dependent signaling. These findings establish extracellular phase separation as a key physical principle complementing molecular recognition in multicellular systems, offering new opportunities for understanding immune response, tissue morphogenesis, and therapeutic strategies targeting the extracellular environment. ### Competing Interest Statement The authors have declared no competing interest. FDCT, No. 0001/2021/AKP, 0024/2023/AFJ, 0209/2024/AGJ, 0031/2023/ITP1, and 005/2023/SKL The National Natural Science Foundation of China, 32361163656, 32022088, 32230056, 22503076 The Natural Science Foundation of Jiangsu Province, BM2023008 Guangdong Provincial Committee for Basic and Applied Basic Research, EF2023-00209-ICMS University of Macau, MYRG-GRG2023-00136-ICMS-UMDF, MYRG-GRG2024-00189-ICMS-UMDF, and MYRG-CRG2023-00009-IAPME Zhuhai UM Science & Technology Research Institute, CP-102-2024 JSPS KAKENHI Grant, JP20H05619

Spinal cord injury (SCI) repair has been a great challenge worldwide because of its complex regeneration mechanisms and limited self-healing. The biomimetic construction of a bioactive scaffold represents a promising direction for SCI repair. Inspired by the efficient self-healing properties of the neonatal spinal cord, this study developed a neonatal spinal-cord-like scaffold (NSLS) aimed at regulating SCI repair at different stages. The NSLS features a neonatal spinal cord matrix, multilevel biomimetic structures, and matching mechanical strength via personalized laser processing and dual-network cross-linking. The microenvironments of the NSLS activate energy metabolism, synaptic formation, and the gliogenesis of neural stem cells (NSCs). Notably, the NSLS could achieve rapid hemostasis and integration with the host spinal cord, facilitating nutrient infiltration and establishing a stable connection in the early stage. Furthermore, NSCs loaded with NSLS (NSLT) promoted nerve repair by promoting microglial M2 polarization to decrease local inflammatory responses in the intermediate stage. Finally, axons grow directionally within the channels and form new connections to enhance neural repair and functional recovery in the late stage. Therefore, NSLT could significantly enhance nerve regeneration and functional recovery after SCI via stage-specific regulation.

Abdominal aortic aneurysm (AAA) is a fatal cardiovascular disease with hidden onset and high risk of rupture death, which is a major public health concern worldwide. Notably, aortic adventitia is the earliest lesions and the last barrier to rupture, and adventitial fibroblasts (AFs) are the key cellular components of aortic adventitia. Current treatments mainly focus on surgical resection with prosthetic graft replacement and endovascular stent grafting, yet failed to reconstruct the adventitia and abdominal aorta's natural structure effectively. In this study, we proposed an extravascular synergistic cocktail (ESC) therapeutic strategy to treat AAA by incorporating mechanical supporting and vascular remodeling inhibition based on a self-healing bioelastomer with antifibrotic drug of lanifibranor. The single-cell RNA-sequencing revealed potential myogenic transformation of AFs in AAA with the release of chemokines like monocyte chemoattractant protein-1 (MCP-1). The underlying mechanism of lanifibranor in maintaining the phenotype of AFs via promoting lipid metabolism was disclosed. The bioelastomer with multiple reversible dynamic bonds exhibits remarkable arterial-like mechanical properties and ultra-fast self-healing ability under blood condition, and enables rapid in situ AAA wrapping treatment. In both the rat and dog AAA models, it has been fully demonstrated that the ESC therapy can provide effective mechanical support to prevent vascular dilation while continuously releasing lanifibranor to regulate myogenic transformation of AFs to inhibit the development of AAA. Additionally, we investigated the feasibility of minimally invasive laparoscopic implantation in bama pigs, emphasizing its potential for clinical translation and application.

Thymic hypofunction due to primary defects, aging, infection, or cytoreductive therapies causes profound restriction of the T-cell receptor (TCR) repertoire and increases rates of morbidity and mortality. Restoring thymic function is therefore critical. Here, the distinctive niche within the spleen has inspired the development of intrasplenic thymus regeneration (ISTR), an approach in which cultured thymic tissue fragments are implanted into the spleens of athymic nude mice, aged mice, thymectomized mice undergoing hematopoietic stem cell transplantation (HSCT), and humanized mice. The spleen's abundant vasculature and supportive stromal milieu enable rapid thymic organogenesis, leading to the reconstitution of phenotypically diverse and functional T-cell compartments, as well as protective responses to viral and tumor challenges. Relative to intramuscular implantation, ISTR drives faster and more complete thymic development, generates greater T-cell output, and is associated with a lower incidence of graft-vs.-host disease. In humanized animals, intrasplenic thymus grafts also promote the rapid emergence of functional human T cells. These results identify the spleen as an optimal ectopic niche for thymus regeneration and provide a promising strategy for clinical immune reconstitution and regenerative immunology.

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.