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Establishment and validation of a human in vitro model to investigate uptake transport at the Blood-Brain Barrier

Project Objectives

  • Characterise endothelial cell lines and select the most suitable blood-brain barrier (BBB) model.
  • Validate adenoviral overexpression of the uptake transporters OATP1A2 and OATP2B1.
  • Determine transporter localisation via biotinylation assays.
  • Confirm transporter abundance using targeted proteomics.
  • Assess neurosteroid transport across the BBB model.
  • Confirm active drug substrates.
  • Produce SOPs for a validated human-based BBB model.

3Rs Impact

  • Replaces animal BBB studies with a human-based in vitro model.
  • Reduces reliance on rodent and large-animal uptake studies.
  • Improves scientific relevance, as this overcomes species differences.
  • Enables cost-effective, high-throughput experimentation.
  • Potential to spare thousands of animals, given typical BBB sample sizes.

Background

The blood‑brain barrier (BBB) regulates molecular exchange between the bloodstream and the brain, helping to protect the central nervous system from toxic compounds, while allowing the passage of nutrients. While efflux transporters are well studied, uptake transporters remain poorly characterised, despite their significance for neuroactive drugs and neurosteroids. In addition, much of our current knowledge depends on rodent models, which weakens translational power due to significant species differences, such as those related to transporter isoforms and expression levels.

In order to bridge this gap, the research team will establish and validate a human-based in vitro BBB model for studying uptake transporters, such as the Organic Anion Transporting Polypeptide (OATP)1A2 and OATP2B1. After establishment and validation, the model will then be used to assess the effect of these uptake transporters on the transcellular transport of drugs and endogenous molecules, such as neurosteroids. It will also contribute to the replacement of animal models, and will help to reduce the use of animals.

Published : 08.07.25

PROJECT DETAILS 

  

Grant scheme: Doctorate Programme 

Grant number: DP-2022-001 

Status: Complete

Funding amount: CHF 169’428 

Animal use: No license required

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Start date: 02.01.23 

End date: 01.01.26 

Mr Valerio Taggi

Universität Basel

Supervisor:

Prof. Henriette Meyer zu Schwabedissen | Universität Basel

OUTPUT

Taggi, V., Schäfer, A.M., Kinzi, J. et al. Targeted and Untargeted Proteomics-based Comparison of Adenoviral Infected hCMEC/D3 and hBMEC as a Human Brain Endothelial Cells to Study the OATP2B1 Transporter. Mol Neurobiol, 62, 9074–9088 (2025). https://doi.org/10.1007/s12035-025-04807-7

Taggi, V., Schäfer, A. M., Dolce, A., & Meyer zu Schwabedissen, H. E. (2024). A face-to-face comparison of the BBB cell models hCMEC/D3 and hBMEC for their applicability to adenoviral expression of transporters. Journal of Neurochemistry, 168, 2611–2620. https://doi.org/10.1111/jnc.16125

The model was optimized for transporter function, with a strong focus on the OATP2B1 uptake transporter, and without the need for in vivo animal experiments. In replicating the structure and characteristics of the BBB, the validated model enables accurate and reproducible research on drug transport mechanisms.

Comprehensive Standard Operating Procedures (SOPs) and videos have been developed for the model. The SOPs provide detailed guidance on cell culture, adenoviral transduction, barrier characterization, and functional assays, while the videos demonstrate practical lab techniques, such as cell handling, culture maintenance, and model characterization. This multimedia approach lowers the barrier for adoption for other research groups.