A cryopreserved and in vivo-in vitro validated human induced pluripotent stem cell blood-brain barrier model for reliable neurotoxicity assessment

• Human blood-brain barrier (BBB) model enables prediction of in vivo permeability. • High correlation of in vitro permeability to human clinical brain penetration data. • Commercially available, cryopreserved BBB models can be set up within five days. • Cryopreserved human induced pluripotent stem cells facilitate model standardization. Brain exposure to toxic agents is a key condition for neurotoxicity hazards in the human central nervous system (CNS). Therefore, reliable prediction of brain penetration is needed to confirm the toxicological safety of compounds to be developed. However, current regulatory risk assessments of neurotoxicity are mainly performed in rodents, which entail several critical issues associated with animal studies. Consequently, authorities worldwide have highlighted the importance of harmonized, human-relevant new approach methodologies (NAMs) such as human induced pluripotent stem cell (hiPSC)-derived models to estimate substrate permeabilities concerning the blood-brain barrier (BBB). This study aimed to redesign a commercially available human BBB model and permeability testing method for future regulatory neurotoxicity studies, considering the in vivo architecture and functionality as well as potential for standardization and higher throughput. Model validation should be achieved by correlation of in vitro permeability to human clinical positron emission tomography (PET) data to enable reliable prediction of in vivo brain penetration. Accordingly, cryopreserved hiPSC-derived BBB models were set up within five days in 96-transwells®, quality-controlled for in vivo barrier properties and exposed to seven pharmaceuticals analogously to clinical PET studies (exposure concentrations: 5 µM to 10 µM for 60 min). The results indicated that the redesigned in vitro permeability assessment model allows to emulate human in vivo brain penetration (Spearman rank correlation coefficient: 0.964). This suggests that utilizing novel commercially available, cryopreserved hiPSC-derived BBB cells could be an important starting point for a standardizable NAMs application to be implemented in regulatory neurotoxicity hazard assessment. A cryopreserved and in vivo-in vitro validated human induced pluripotent stem cell blood-brain barrier model for reliable neurotoxicity assessment. Created with Biorender.com