Development of In Vitro Blood-Brain Barrier Models using hCMEC/D3 and HBEC-5i Cell Lines for Drug Permeability Assessment

Abstract

Calmodulin-dependent phosphorylation of the water channel, aquaporin-4, promotes its accumulation in astrocytic membranes during ischemic stroke and traumatic brain injury, contributing to cytotoxic edema and blood-brain barrier (BBB) disruption. Inhibiting calmodulin therefore represents a potential therapeutic approach. However, existing inhibitors such as trifluoperazine (TFP) exhibit high toxicity and adverse side effects due to their anti-dopaminergic activity. In-house analogues of TFP have been synthesised to retain calmodulin-inhibitory properties while reducing dopaminergic receptor antagonism. In silico tools such as the SwissADME BOILED-Egg model predict their solubility and permeability, but experimental validation using an in vitro BBB model is necessary to characterise their relative permeability and cytotoxicity to TFP. In this thesis, two immortalised human brain microvascular endothelial cell lines, hCMEC/D3 and HBEC-5i, were compared in terms of morphology, angiogeneic potential and barrier integrity using a Transwell model to identify a suitable system for drug permeability assessment. The phenotypic and transcriptomic response of these cells to growth factor withdrawal, serum reduction and the supplementation of dexamethasone were also examined. hCMEC/D3 cells in standard EGM-2 medium exhibited a BBB phenotype and transcriptomic profile that more closely aligned with human endothelial cells from surgical samples, which were further enhanced by the modified medium. In contrast, the HBEC-5i cells lacked expression of BMEC markers and displayed signs of mesenchymal-like transition. Attempts to establish a 3D hCMEC/D3 barrier using the MIMETAS OrganoPlate 3-Lane 40 were unsuccessful, however, a Transwell model enabled acute and long-term cytotoxicity to assess the permeability and cytotoxicity of the in-house compounds. While the permeability quantification method requires further optimisation, this work provides a comparative framework for evaluating human BBB models and highlights hCMEC/D3 cells as more physiologically relevant for drug permeability studies.

Date of Award	Nov 2025
Original language	English
Awarding Institution	Aston University
Supervisor	Philip Kitchen (Supervisor) & Roslyn Bill (Supervisor)