At the gliovascular interface, reciprocal inductive influences between brain microvascular endothelial cells (BMVEC) and astrocytes occur. Most of the knowledge in this area of research is derived from in vitro eo-culture models in which astrocytes are cultured on a stiff, two-dimensional (2D) surface. Three-dimensional (3D) culture models closely mimic the in lJZVO cellular architecture and they bridge the gap between 2D culture models and animal models. Hence, an in vitro 3D eo-culture model was developed and characterised, to study the interactions between BMVEC and astrocytes. In this model, human astrocytes (HA) were seeded inside a collagen type--I gel while human immortalised cerebral microvascular endothelial cells (hCMEC/D3) were cultured on the gel surface. Both cell types were of human origin to improve the translatability of findings to humans in vivo. Additional important features of the model are the culture of endothelial cells on a soft matrix, and the simulation of the geometric relationship that exists in vivo i.e., the interaction of astrocytes with BMVEC from their ab luminal side. To determine the effect of the 3D environment on the HA, the proliferation rate and expression of four molecules namely, glial fibrillary acidic protein (GF AP), aquaporin-4 (AQP4), endothelin-l (Et-l) and endothelin receptor type-B (EDNRB), were compared between 3D and 2D cultured HA. The decreased expression of AQP4 and EDNRB and the much-decreased proliferation rate of 3D HA suggested their reduced reactivity and a similarity to their in lJiVO counterparts. However, 3D HA did not differ from the 2D HA in their ability to release soluble factors that induce barrier properties on BMVEC, as observed by similar levels of three expression markers of barrier phenotype on hCMEC/D3 cells namely, zonula occludens-l, claudin-5, and P-glycoprotein and similar paracellular permeability coefficients to fluorescent-dextrans (70 kDa).Using the developed model, the effect of endothelial cells on the AQP4 expression in HA was investigated. While the localisation of astrocytic AQP4 did not change, its total, cellular expression levels were decreased as analysed by flow cytometry and fluorescence microscopy. The results could not be explained by physical-contact and therefore may be mediated by soluble factors released by the endothelial cells. Further investigation showed that Et-l, at least by itself, is not the mediator of this effect. The endothelial-astrocyte physical contact points were too few, as revealed by transmission electron microscopy, to study its effect on the localisation of AQP4 in HA. The developed 3D co-culture model is usable and amenable to various analytical techniques such as fluorescence microscopy, electron microscopy, flow cytometry, and enzyme linked immunosorbent assay. The two-step collagenase digestion method developed in this study further broadens the model's utility to standard molecular and cellular biology techniques. The model has some advantages as well as limitations compared to the existing 2D and 3D co-culture models. The model may be used as an in vitro BBB model to study the transcytosis of nanoparticles, leukocytes, and possibly pathogens across the endothelial barrier and their interactions with astrocytes. In addition, the alterations in phenotype of astrocytes and endothelium in situ, in response to the soluble factors released by the other cell type can be studied. Further work is needed to validate some of the supposed unique features of the model. With the proposed modifications, it can be made physiologically more relevant, and the usability can be widened
