3D brain angiogenesis model to reconstitute maturation of functional human blood-brain barrier in vitro

Abstract

The human central nervous system (CNS) vasculature expresses a distinctive barrier phenotype, the blood–brain barrier (BBB). As the BBB contributes to low efficiency in CNS pharmacotherapy by restricting drug transport, the development of an in vitro human BBB model has been in demand. Here, we present a microfluidic model of CNS angiogenesis having three‐dimensional (3D) lumenized vasculature in concert with perivascular cells. We confirmed the necessity of the angiogenic tri‐culture system (brain endothelium in direct interaction with pericytes and astrocytes) to attain essential phenotypes of BBB vasculature, such as minimized vessel diameter and maximized junction expression. In addition, lower vascular permeability is achieved in the tri‐culture condition compared to the monoculture condition. Notably, we focussed on reconstituting the functional efflux transporter system, including p‐glycoprotein (p‐gp), which is highly responsible for restrictive drug transport. By conducting the calcein‐AM efflux assay on our 3D perfusable vasculature after treatment of efflux transporter inhibitors, we confirmed the higher efflux property and prominent effect of inhibitors in the tri‐culture model. Taken together, we designed a 3D human BBB model with functional barrier properties based on a developmentally inspired CNS angiogenesis protocol. We expect the model to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

Impact of Pericytes on the Stabilisation of Microvascular Networks in Microfluidic Systems in Response to Nanotoxicity

“…Therefore, we next investigated the impact of pericytes on vessel permeability in our system at this time point. Using protocols comparable to those reported by others, we perfused 70 kDa FITC-dextran through the vessel network [15, 21, 23, 33, 50]. Quantitative analysis of diffusion to interstitial gel areas indicated no change significant difference in the cross-endothelial diffusion of this dye, when comparing HUVEC monocultures and HUVEC/ pericyte co-cultures (Figure 4).…”

“…Similar recruitment patterns were observed for VE-cadherin and β-catenin. Expression of these tight and adherens junction proteins are routinely demonstrated to indicate vessels which have reached maturation [15, 16, 22, 23, 46]. Accordingly, in our system endothelial cells have differentiated into mature vessels following 10-days culture.…”

“…Researchers have developed various iterations of a 'vasculature-on-a-chip' [3,[13][14][15][16][17][18][19][20]. These models generally use PDMS chips which are manufactured using photo-and soft-lithography.…”

“…This could limit long-term culture and the use of these systems for embedding spheroids and organoids for the development of advanced tissue models. To overcome this issue, endothelial cells can be cultured with pericytes [15][16][17]. Pericytes are abluminally located mesenchymal cells that have the capacity to differentiate in many types of cells [25] Due to their heterogeneity, the identification of specific markers has been disputed [26], but platelet-derived growth factor receptor-β (PDGFRβ) and neural/glial antigen-2 (NG2) are generally accepted markers associated with these cells [27,28] Pericytes typically interact with a number of endothelial cells, with primary cytoplasmic processes running along the abluminal surface of the endothelium and secondary processes running perpendicularly, enclosing the endothelial tube [29].…”

“…Pericytes are abluminally located mesenchymal cells that have the capacity to differentiate in many types of cells [25] Due to their heterogeneity, the identification of specific markers has been disputed [26], but platelet-derived growth factor receptor-β (PDGFRβ) and neural/glial antigen-2 (NG2) are generally accepted markers associated with these cells [27,28] Pericytes typically interact with a number of endothelial cells, with primary cytoplasmic processes running along the abluminal surface of the endothelium and secondary processes running perpendicularly, enclosing the endothelial tube [29]. Co-culturing endothelial cells with pericytes inhibits vessel hyperplasia and endothelial cell proliferation, and promotes vessel barrier function and endothelial cell survival [15,17,[30][31][32].…”