Immunofluorescence Imagery and Characterization in Peer-Reviewed Literature

ACBRI 498 at Passage 3 labeled with antibodies against pericyte markers desmin, PDGFR-b, NG2, and CD13.

From Herland and van der Meer et al. (2016) PLoS ONE 11(3): e0150360.

Fig 2. Co-culture of human brain microvascular endothelial cells (ACBRI 376), pericytes (ACBRI 498) and astrocytes in the 3D BBB chip.

Schematic illustrations of the cells populating the 3D vessel structures for the three experimental set-ups are shown at the top, and fluorescence confocal micrographs of the engineered brain microvessel viewed from the top (A, D, G) or shown in cross-section at either low (B, E, H) or high (C, F, I) magnification (rectangles in lower magnifications images indicate respective areas shown at higher magnification below). The fluorescence micrographs show the cell distributions in 3D BBB chips containing brain microvascular endothelium alone (A-C), endothelium with prior plating of brain pericytes (ACBRI 498) on the surface of the gel in the central lumen (D-F) or endothelium with brain astrocytes embedded in the surrounding gel (G-I). High-magnification cross-sections are projections of confocal stacks (bars, 200 μm in A,B,D,E,G,H and 30 μm in C, F, I). Green indicates F-actin staining, blue represents Hoechst-stained nuclei, and magenta corresponds to VE-Cadherin staining, except for G where morphology and intensity masks were used to discriminate astrocytes (green) from endothelial cells (ACBRI 376) (magenta); original image can be seen in S2 Movie. Arrows indicate contact points between endothelium and pericytes (ACBRI 498) (F) or astrocytes (I).

From Herland and van der Meer et al. (2016) PLoS ONE 11(3): e0150360.

Fig 4. Establishment of a low permeability barrier by the engineered brain microvascular endothelium in the 3D BBB chip.

A) Fluorescence micrograph of a chip containing a cylindrical collagen gel viewed from above with (left) or without (right) a lining endothelial (ACBRI 376) monolayer after five days of culture (left; Cell-lined lumen) compared to a chip with an empty collagen lumen (right). The images were recorded at 0 (top) and 500 (bottom) sec after injection of fluorescently-labeled 3 kDa dextran to analyze the dynamics of dextran diffusion and visualize endothelial barrier function in the 3D BBB chip. Note that the presence of the endothelium significantly restricts dye diffusion compared to gels without cells (left versus right). B) Apparent permeabilities of the endothelium cultured in the 3D BBB chip calculated from the diffusion of 3 kDa dextran with an endothelial (ACBRI 376) monolayer (Endo; n = 6), an endothelial (ACBRI 376) monolayer surrounded by astrocytes (Endo+Astro; n = 3) and an endothelial (ACBRI 376) monolayer surrounded by pericytes (ACBRI 498) (Endo+Peri; n = 3). Error bars indicate S.E. M.; * p<0.05, Student’s t-test.