S33 - Establishing a Blood Brain Barrier Spheroid Model for Studying Nanoplastic Trafficking

Introduction: : The blood-brain barrier (BBB) is a dynamic and selectively permeable interface that protects the brain from harmful substances while regulating nutrient exchange. Comprised of endothelial cells, pericytes, and astrocytes, the BBB is critical for maintaining central nervous system (CNS) health. Emerging studies suggest that nanoplastics, ubiquitous byproducts of plastic degradation, may cross biological barriers, including the BBB, raising concerns about their potential neurotoxicity. However, the mechanisms governing nanoplastic trafficking across the BBB and their neurological implications remain poorly defined. Animal models offer some insight but are limited by cost, throughput, and ethical constraints. This study aims to establish a physiologically relevant, 3D in vitro spheroid model of the human BBB. This model will serve as a foundational tool to study nanoplastic permeability and inform future assessments of environmental pollutant exposure on brain health, contributing to both scientific understanding and responsible model development.

Materials and

Methods: : We first developed a 3D co-culture spheroid model using primary Human Vascular Pericytes (HVPs) and Human Cerebral Microvascular Endothelial Cells (HCMECs). Before the spheroid formation process, cells were cultured separately in T-75 flasks for 5 days. On Day 1 of the spheroid process, 2,000 HVPs were seeded into each well of an ultra-low attachment 96-well spheroid microplate. After 24 hours of aggregation, 1,000 HCMECs were added to each spheroid to enable self-organization into BBB-like architecture. By Day 4, the spheroids were mature and ready for permeability assays.

To assess barrier function, we introduced FITC-Dextran (3–7 nm) and imaged the spheroids after a 16 hour incubation. A subsequent gentle wash step was applied to reduce background signal from non-internalized tracers. To evaluate pollutant permeability, 100 nm fluorescent polystyrene nanoplastics were introduced on Day 4 and allowed to incubate for 16 hours. On Day 5, spheroids were imaged using a Cytation 10 confocal imaging system. Z-stack projections were used to evaluate both structural organization and nanoparticle distribution. Control conditions included spheroids without nanoplastic or FITC-Dextran treatment, and imaging parameters were held constant across all replicates.

Results, Conclusions, and Discussions:: The co-culture BBB spheroid model consistently formed compact, reproducible 3D structures. Confocal imaging demonstrated the presence of concentric cellular organization, with endothelial cells enveloping the pericyte core. FITC-Dextran assays revealed a marked decrease in internal fluorescence intensity, indicating that the spheroids possess selective permeability consistent with intact barrier function. In contrast, 100 nm fluorescent nanoplastics were clearly detected within the spheroid cores, suggesting there was successful permeation through or around the modeled barrier. The data demonstrates that, unlike FITC-Dextran, nanoplastics of this size can traverse the in vitro BBB model.

These results raise urgent questions about the CNS exposure risks posed by airborne or waterborne nanoplastics, especially in urban environments. Future work will enhance physiological relevance by integrating astrocytes and introducing variable particle properties (size, surface chemistry, charge) to simulate environmental exposures more comprehensively.

This research provides an experimentally tractable system for evaluating nanoparticle trafficking across the BBB. Our long-term goal is to use this platform to elucidate mechanisms of nanoplastic neuroinvasion and inform the development of public health guidelines that protect vulnerable populations from pollutant-associated neurological damage.

Acknowledgements and/or References (Optional)::