Figure 7.

A) Spheroid diameters at the same total cell numbers and different seeding ratios of hA, hP, and hCMEC/D3 after 6 d postseeding, n = 6–9 ± SD. Statistical analysis was performed using the mixed‐effects model. B) Fluorescence plot profiles of 4 kDa FITC‐dextran (FD4) treated different‐sized BBB spheroids after one hour, seeded at a cell ratio of 1:1:3. Scale bar, 1 cm. C) Mean fluorescence intensities of triple‐culture spheroids of different sizes and cell ratios after 4 h of cultivation with 10 × 10⁻⁶ m FD4, n = 9–12 ± SD. Statistical analysis was performed using Dunnett's multiple comparisons test. (*p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001). D) Immunofluorescence staining of tight‐junction associated protein ZO‐1 (orange) of large (900 µm) and small (300 µm) BBB spheroids of various cell seeding ratios. Cell nuclei were stained with DAPI (blue). Scale bar, 50 µm.