One key application of organ-on-chip systems is the examination of drug transport and absorption through native cell barriers such the blood–brain barrier. To overcome previous hurdles related to the transferability of existing static cell cultivation protocols and polydimethylsiloxane (PDMS) as the construction material, a chip platform with key innovations for practical use in drug-permeation testing is presented. First, the design allows for the transfer of barrier-forming tissue into the microfluidic system after cells have been seeded on porous polymer or Si3N4 membranes. From this, we can follow highly reproducible models and cultivation protocols established for static drug testing, from coating the membrane to seeding the cells and cell analysis. Second, the perfusion system is a microscopable glass chip with two fluid compartments with transparent embedded electrodes separated by the membrane. The reversible closure in a clamping adapter requires only a very thin PDMS sealing with negligible liquid contact, thereby eliminating well-known disadvantages of PDMS, such as its limited usability in the quantitative measurements of hydrophobic drug molecule concentrations. Equipped with tissue transfer capabilities, perfusion chamber inertness and air bubble trapping, and supplemented with automated fluid control, the presented system is a promising platform for studying established in vitro models of tissue barriers under reproducible microfluidic perfusion conditions.

Automated biomimetic systems for the preclinical testing of drugs are of great interest. Here, an in vitro testing platform for in vivo adapted drug absorption studies is presented. It has been designed with a focus on easy handling and the usability of established cell cultivation techniques in standard well plate inserts. The platform consists of a microfluidic device, which accommodates a well plate insert with pre-cultivated cells, and provides a fluid flow with dynamic drug dilution profiles. A low-cost single-board computer with a touchscreen was used as a control unit. This provides a graphical user interface, controls the syringe pump flow rates, and records the transepithelial electrical resistance. It thereby enables automated parallel testing in multiple devices at the same time. To demonstrate functionality, an MDCK cell layer was used as a model for an epithelial barrier for drug permeation testing. This confirms the possibility of performing absorption studies on barrier tissues under conditions close to those in vivo. Therefore, a further reduction in animal experiments can be expected.

The blood–brain barrier (BBB) is the bottleneck in the development of new drugs to reach the brain. Due to the BBB, toxic substances cannot enter the brain, but promising drug candidates also pass the BBB poorly. Suitable in vitro BBB models are therefore of particular importance during the preclinical development process, as they can not only reduce animal testing but also enable new drugs to be developed more quickly. The aim of this study was to isolate cerebral endothelial cells, pericytes, and astrocytes from the porcine brain to produce a primary model of the BBB. Additionally, as primary cells are well suited by their properties but the isolation is complex and better reproducibility with immortalized cells must be ensured, there is a high demand for immortalized cells with suitable properties for use as a BBB model. Thus, isolated primary cells can also serve as the basis for a suitable immortalization technique to generate new cell lines. In this work, cerebral endothelial cells, pericytes, and astrocytes were successfully isolated and expanded using a mechanical/enzymatic method. Furthermore, in a triple coculture model, the cells showed a significant increase in barrier integrity compared with endothelial cell monoculture, as determined by transendothelial electrical resistance measurement and permeation studies using sodium fluorescein. The results demonstrate the opportunity to obtain all three cell types significantly involved in BBB formation from one species, thus providing a suitable tool for testing the permeation properties of new drug candidates. In addition, the protocols are a promising starting point to generate new cell lines of BBB-forming cells as a novel approach for BBB in vitro models.

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.