ORIGINAL RESEARCH article
Front. Toxicol.
Sec. In Vitro Toxicology
In Vitro BBB Triculture Assay and Preliminary Computational Model Development to Predict Brain Exposure
Provisionally accepted
- 1Purdue University, West Lafayette, United States
- 2Indiana University, Bloomington, United States
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Neurotoxicity is a critical liability for many environmental pollutants. Current in vitro neurotoxicity screens rely on direct exposure of cultured neurons to xenobiotics, often at exceeding physiologically relevant levels due to the restrictive nature of the blood–brain barrier (BBB). To address this limitation, we have developed a novel human in vitro direct-contact triculture BBB model that more closely mimics the in vivo barrier. The triculture is formed by layering primary astrocytes, primary pericytes, and then brain microvessel endothelial cells (BMECs, HBEC-5i) in direct contact, increasing the restrictive nature of tight junctions and allowing cell-cell signaling that mimics the configuration found in the in vivo BBB. Using this model, we quantified the apparent bidirectional permeability (Papp) of more than 50 compounds, including environmental pollutants and central nervous system (CNS) drugs, primarily by paracellular, passive transcellular and transporter-mediated pathways, to help develop a risk of exposure. In parallel with our in vitro BBB model, we are using the high-throughput toxicokinetics (HTTK) R library developed by the U.S. Environmental Protection Agency (EPA) as our model to predict brain exposure. Briefly, using our measured values Papp, calculated efflux ratio values, and EPA physiologically based pharmacokinetic (PBTK) reference data for compound parameters, we are developing predictions of toxicant accumulation in the brain parenchyma after chronic exposure in steady state. Through the development of this model, we postulate that future investigators could simply perform in vitro BBB permeability studies to determine the relative risk of potential brain accumulation and the risk of neurotoxicity.
Keywords: Apparent permeability, Blood-Brain Barrier, CNS Exposure Prediction, Efflux ratio, High Throughput Toxicokinetics in R, HTTK-R, In Vitro—In Vivo Extrapolation, IVIVE
Received: 05 Jan 2026; Accepted: 16 Feb 2026.
Copyright: © 2026 Xu, Sluka, Zhang and Knipp. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
James Patrick Sluka
Gregory Thomas Knipp
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