Parametric Study to Optimize the Cultivation Conditions of Blood–Brain Barrier Models for In Vitro Drug Testing

Background/Objectives: The permeation behavior of new drug candidates for central nervous system (CNS) diseases is a major bottleneck in drug development. Thus, using a meaningful in vitro blood–brain barrier (BBB) model in the preclinical phase to determine cerebral drug bioavailability is crucial. However, due to the large number of heterogeneous in vitro BBB models and the resulting lack of comparability caused by different cultivation conditions, this study aimed to develop an optimized test system by systematically varying relevant cultivation parameters. Methods: An in vitro BBB model comprising primary porcine brain microvascular endothelial cells, pericytes, and astrocytes was optimized by evaluating extracellular matrix coatings, differentiation media, and co-culture conditions. Barrier integrity was assessed by transendothelial electrical resistance (TEER), permeability studies, and BBB-associated transporter gene expression. A chimeric BBB model incorporating immortalized porcine pericytes and human astrocytes was established to evaluate protocol transferability. Results: The optimal culture conditions comprised a collagen IV, fibronectin, and laminin coating, followed by a three-day triple co-culture and 24 h of incubation in differentiation medium supplemented with hydrocortisone, pCPT-cAMP, and Ro-20-1724. Under these conditions, the triple co-culture model reached a maximum TEER of 1692 ± 99 Ω·cm2, significantly exceeding the endothelial monoculture (1144 ± 43 Ω·cm2). The optimized BBB model discriminated between hydrophilic and lipophilic marker compounds, as well as molecules of different molecular sizes. Co-culture significantly increased the endothelial gene expression of MRP5, SLC29A1, OATP1A2, and OATP2B1 compared to endothelial monocultures. The chimeric BBB model exhibited TEER values and permeability characteristics comparable to those of the primary model, although some differences were observed in transporter gene expression. Conclusions: Stepwise optimization of key culture parameters enhanced the barrier integrity and physiological relevance of the in vitro BBB model, providing a robust platform for preclinical BBB transport studies. The successful establishment of a chimeric BBB model further demonstrates protocol transferability and supports the development of more standardized and reproducible in vitro BBB models.