Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model

Abstract

Objectives: In conventional in-vitro blood-brain barrier (BBB) models, primary and immortalized brain microvessel endothelial cell (BMEC) lines are often cultured in a monolayer or indirect coculture or triculture configurations with astrocytes or pericytes, for screening permeation of therapeutic or potentially neurotoxic compounds. In each of these cases, the physiological relevancy associated with the direct contact between the BMECs, pericytes and astrocytes that form the BBB and resulting synergistic interactions are lost. We look to overcome this limitation with a direct contact coculture model.

Methods: We established and optimized a direct interaction coculture system where primary human astrocytes are cultured on the apical surface of a Transwell® filter support and then human cerebral microvessel endothelial cells (hCMEC/D3) seeded directly on the astrocyte lawn.

Key findings: The studies suggest the direct coculture model may provide a more restrictive and physiologically relevant model through a significant reduction in paracellular transport of model compounds in comparison with monoculture and indirect coculture. In comparison with existing methods, the indirect coculture and monoculture models utilized may limit cell-cell signaling between human astrocytes and BMECs that are possible with direct configurations.

Conclusions: Paracellular permeability reductions with the direct coculture system may enhance therapeutic agent and potential neurotoxicant screening for BBB permeability better than the currently available monoculture and indirect coculture in-vitro models.

Keywords: blood-brain barrier coculture; human astrocytes; human cerebral microvessel endothelial cells; paracellular permeability.

Figure 1

Past (indirect) vs current direct contact coculture models. (a) previous direct contact coculture model with BMEC and astrocytes separated by Transwell®-permeable filter support. (b) current direct contact coculture model, with BMEC and astrocytes in direct cell–cell contact. BMEC and astrocytes are shown in red and purple, respectively.

Figure 2

Optimization of direct contact culture using apparent permeability of [¹⁴C]-mannitol. (a) Hydrocortisone added to media at 1.4 μm or 100 nm at the start of human cerebral microvessel endothelial cells plating or 2 days postplating. (b) Lithium chloride at 10 mm compared with control (0 mm) when added at the start of human cerebral microvessel endothelial cells plating or 2 days postplating. (c) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid concentrations of 10, 25 and 50 mm added to media for human cerebral microvessel endothelial cells monolayer in comparison with direct culture. Studies were run in triplicate and subjected to Student's t-test and Mann–Whitney test (a and c) or one-way ANOVA with Bonferroni post-hoc test or Kruskal–Wallis with Dunn's post-hoc test (b). Significant changes are noted with an asterisk (*) for P < 0.05 and (**) for P < 0.01. Significant levels are reported as (t-test, MW) or (one-way ANOVA, KW). Error bars represent 1 standard deviation (n = 3).

Figure 3

Apparent permeability for five paracellular [¹⁴C]-labelled markers of various hydrodynamic radii. Studies were run in triplicate and subjected to Student's t-test or Mann–Whitney test. Significant changes are noted with an asterisk (*) for P < 0.05 and (**) for P < 0.01. Significant levels are reported as (t-test, MW). Error bars represent 1 standard deviation (n = 3).

Figure 4

Apparent permeability of [¹⁴C]-inulin, a paracellular marker, across the direct contact coculture. Studies were subjected to one-way ANOVA with a Bonferroni post-hoc test and Kruskal–Wallis with Dunn's post-hoc test. Significant changes are noted with an asterisk (*) for P < 0.05 and (**) for P < 0.01. Significant levels are reported as (one-way ANOVA, KW). Error bars represent 1 standard deviation (n = 6).

Figure 5

Apparent permeability of [¹⁴C]-mannitol and [¹⁴C]-sucrose across direct and indirect contact cocultures. Studies were run in triplicate and subjected to Student's t-test or Mann–Whitney test. Significant changes are noted with an asterisk (*) for P < 0.05. Significant levels are reported as (t-test, MW). Error bars represent 1 standard deviation (n = 3).

Figure 6

Apparent permeability of [¹⁴C]-propranolol, a passive transcellular permeability marker. Studies were subjected to one-way ANOVA with a Bonferroni post-hoc test and Kruskal–Wallis with Dunn's post-hoc test. Non-significant changes (P > 0.05) were seen between monoculture and coculture. Error bars represent 1 standard deviation (n = 3).

Figure 7

Total percentage of accumulation of rhodamine 123 to show functional expression of efflux transporter P-glycoprotein in human cerebral microvessel endothelial cells monoculture and direct contact coculture. Efflux of P-gp substrate rhodamine 123 was assessed in the presence and absence of P-gp inhibitor verapamil. Studies were subjected to one-way ANOVA with a Bonferroni post-hoc test and Kruskal–Wallis with Dunn's post-hoc test. No significant difference was observed between the presence and absence of inhibitor for either model (P > 0.05). Error bars represent 1 standard deviation (n = 3).