Fig. 3

BBB co-cultures of brain endothelium, astrocytes, and pericytes in the three-lane OrganoPlate. a Picture shows the three-lane OrganoPlate. The plate combines a 384-well microtiter plate on the top with microfluidic channels on the bottom that make up 40 tissue culture chips. b Zoom-in of the bottom of the three-lane OrganoPlate, showing one tissue culture chip that consists of three channels. c 3D artist impression of the center of a chip. ECM gel is added to the gel channel and two phaseguides (white rims) prevent it from flowing into the adjacent medium channels. After gelation of the ECM gel, TY10 cells are added to one of the medium channels and allowed to form a vessel structure. The microvessel has a lumen at its apical side that is perfused. Next, astrocytes (hAst cells) and pericytes (hBPCT cells) are added to the other medium channel to establish a BBB co-culture. d, e Phase contrast images of a TY10 monoculture (top image) and a BBB co-culture (bottom image) in the three-lane OrganoPlate, day 7. Scale bar is 100 µm. f, g 3D reconstruction of a confocal z-stack showing the organization of the three cell types in a BBB co-culture. The hAst and hBPCT cells were labeled with green-fluorescent calcein-AM and calcein red™ AM, respectively, here depicted in the colors green and magenta. Tight junctions of the TY10 microvessel are shown by a PECAM-1 staining (red). h Images acquired of chips with a leaktight barrier (top), a leaky barrier (middle), or TY10-free control (gel with hAst and hBPCT, bottom) during a barrier integrity assay (for FITC-dextran, 20 kDa, t = 12 min). i Assessment of barrier function (for FITC-dextran, 20 kDa) at different time points for TY10 monocultures and BBB co-cultures cultured under perfusion in the three-lane OrganoPlate. n = 3 for TY10-free controls and TY10 monocultures and n = 6 for BBB co-cultures. Error bars show standard deviation of the mean