Establishment of murinein vitroblood-brain barrier models using immortalized cell lines: co-cultures of brain endothelial cells, astrocytes, and neurons

Abstract

Blood-brain barrier (BBB) is a selective barrier formed by the endothelial cells that line cerebral microvessels. It serves as a physical barrier due to the presence of complex tight junctions between adjacent endothelial cells which limits the paracellular movement of most molecules across the BBB. Many in vitro models of the BBB have been established to mimic these in vivo properties with limited success. In this study, we described the properties of a cell-based murine in vitro BBB model in five configurations constructed using immortalized cell lines in a 12-well format Transwell system: murine brain endothelial cells (bEnd.3) grown in a monoculture, or as co-culture in contact with astrocytes, or without contact with astrocytes or neurons, and triple coculture combining the three cell lines. We found that only contact and triple co-culture model closely mimic the in vivo BBB tightness as evaluated by apparent permeability (Papp) of sucrose and albumin producing the lowest Papp values of 0.56 ± 0.16 x 10 -6 cms -1 and 3.30 ± 0.51 x 10 -6 cms -1 , respectively, obtained in triple co-culture model. Co-culturing of bEnd.3 with astrocytes increased the expression of occludin as shown by western blot analysis, and immunohistochemistry showed an increase in peripheral localization of occludin and claudin-5.In addition, we found conditioned media were able to increase in vitro BBB model tightness through the modulation of tight junction proteins localization. We conclude that the presence of astrocytes and neurons in close proximity to brain endothelial cells is essential to produce a tight in vitro BBB model.

Anin-vitroBBB-on-a-chip open model of human blood-brain barrier enabling advanced optical imaging

“…Nonetheless, certain cell lines may still exhibit the required properties for some pathophysiological and medicinal applications in a fit-for-purpose approach. We therefore chose immortalized human brain derived TY10 capillary endothelial cells that have been used to model the human BBB in a number of previous studies (Takeshita et al, 2014;Spampinato et al, 2015;Idris et al, 2018;Wevers et al, 2018).…”

Advancing brain barriers RNA sequencing: guidelines from experimental design to publication

“…In both cases, the presence of additional BBB cell types found in vivo is not always modeled in the in vitro systems, such as for example the presence and anatomical disposition of the astrocytic endfeet found in the NVU in vivo. To overcome these limitations, sophisticated co- or tri-cultures of brain endothelial cells together with pericytes or astrocytes from different sources have been established and, to a certain degree, mimic the in vivo NVU structure [ 94 – 96 ].…”

Vascularizing the brain in vitro

“…For the co-culture of BMECs and ACs, BMECs are cultivated in the top layer of the Transwell facing the upper chamber whereas ACs can either be in the bottom of the lower chamber (non-contact approach) or on the opposite side of the Transwell membrane, which will allow crosstalk with BMECs (contact approach). The non-contact approach is more doable, yet the contact approach could exhibit an increase in the expression of the gene encoding the TJ proteins claudin-5, occluding, and ZO-1, which then tightens the barrier ( Idris et al., 2018 ). Therefore, the contact co-culture of BMECs and ACs is preferred to increase the BBB integrity.…”