Functional assessment of immortalized human brain microvascular endothelial cells with different passage numbers: A case study for a prospective proposal on variability management of in vitro blood-brain barrier models

In vitro blood-brain barrier (BBB) models, primarily consisting of brain microvascular endothelial cells (BMEC), are expected to play pivotal roles in evaluating drug permeability into the brain. However, these models often exhibit functional variability due to various factors, raising practical concerns that can hinder their use in drug development studies. By investigating cell passage numbers as one such factor, we aim to assess how BBB model functionality is affected and to propose a practical strategy for managing this variability. In transwell-BBB models - but not in spheroidal-BBB models - the intercellular barrier integrity was somewhat compromised when higher-passage human immortalized BMEC (HBMEC/ci18) were used. Nonetheless, a clear in vitro-in vivo correlation (IVIVC) curve could still be obtained with these transwell-BBB models, similar to those with lower passage number HBMEC/ci18, presumably allowing for reasonable estimation of in vivo drug permeability. Therefore, changes in functional levels of BBB models do not always significantly diminish their practical value in drug BBB permeability studies. Additionally, the IVIVC curve integrity may serve as an indicator for assessing acceptable BBB model functionality. These findings provide valuable insights for the future application of in vitro human BBB models in drug development studies.