Immortalized Human Pericytes

HBMVEC Co-Culture with Human Pericytes Increased Barrier Properties

The blood-brain barrier (BBB) limits drug access to brain tumors due to tight junctions (TJs) in endothelial cells. Tumor Treating Fields (TTFields) have been shown to increase BBB permeability in murine models. In this study, Salvador et al. established a 3D co-culture model of the BBB using human brain microvascular endothelial cells (HBMVEC) and pericytes.

They first examined the effects of 100 kHz TTFields on HBMVEC monocultures. After 72 h of treatment, immunofluorescence staining showed a slight change in claudin-5 localization (Fig. 1A). However, compared to cerebEND cells, which exhibited distinct claudin-5 delocalization from cell borders to the cytoplasm, HBMVEC showed weaker staining and lower claudin-5 expression in both treated and untreated conditions (Fig. 1B). They then co-cultured HBMVEC with immortalized human pericytes, which are known to enhance BBB characteristics, including tight junction protein expression. Western blot analysis revealed that pericytes increased ZO-1 and PECAM-1 expression, but not claudin-5 or occludin (Fig. 1C). Additionally, co-culture with pericytes improved barrier integrity, as shown by increased TEER (Fig. 1D). All subsequent experiments used this HBMVEC-pericyte co-culture as a 3D BBB model.

Fig. 1. The addition of pericytes to culture increased HBMVEC barrier properties (Salvador E, Köppl T, et al., 2023).

BBB-on-a-Chip Containing Pericytes to Model Barrier Disruption

The blood–brain barrier (BBB) restricts substances from entering the CNS, and its dysfunction is linked to various neurological diseases. Traditional models like Transwell have limitations, while microfluidic models face challenges in throughput and material properties. In this study, Ohbuchi et al. developed a BBB-on-a-chip using the OrganoPlate platform, incorporating immortalized TY10 brain endothelial cells, pericytes, and astrocytes. They co-cultured these cells in a microfluidic device to mimic the in vivo BBB structure.

Pericytes play an important role in microvasculature integrity under physiological conditions. To develop a BBB-on-a-chip containing pericytes, pericytes were co-cultured in TY10 microvessels by embedding them in the middle ECM (Fig. 2a). Ten days after TY10 and pericyte co-culture, immunostaining for CD31 (endothelia marker) and platelet-derived growth factor receptor (PDGFR; pericyte marker) was performed. In the co-cultured model, microvessels composed of CD31-positive endothelia were formed, as shown in the TY10 monoculture model in Figure 2b. PDGFR-positive pericytes were localized on the basal side, surrounding the TY10 microvasculature (Fig. 2b). These structures closely resembled those in the in vivo BBB. The BBB-on-a-chip model containing pericytes also exhibited a leak-tight barrier function (Fig. 2c and d). The apparent permeability (Papp) was 6–9 × 10⁻⁷ cm/s in the co-culture as compared to 6–7 × 10⁻⁶ cm/s in the monoculture model. The TEER was 15 (AM) to 18 (CB) and 9 (CB) to 11 (AM) Ω·cm², respectively, for the co- and monoculture models (Fig. 2e). These results suggest that the pericytes functionally supported microvascular barrier integrity in our model.

Fig. 2. BBB-on-a-chip model containing TY10 brain endothelia and pericytes (Ohbuchi M, Shibuta M, et al., 2024).