Supplementary Figure 1 Diagram of BBB and brain chips. (a) Schematic of the BBB Chip demonstrates the 3 parts of the chip, Top PDMS channel, membrane and Bottom PDMS channel; (b) Image of 2 BBB Chips, the red color represents the bottom (Endo) channel while the blue represents the top (Pericytes/Astrocytes) channel, (c) Schematic of the Brain Chip demonstrates the different parts of the chip, (from top to bottom) the manifold which hold the chip and enables perfusion of the chip, Brain Chip, PDMS gasket, Topas substrate and PC base which hold the chip; (d) Image of Brain Chips, top tube represents the inlet and outlet of the Brain Chips. The orange triangles represent the location of the human neuronal culture. (e) The diagram represents the experimental setup. The red line represents the endothelial media (ablood) whereas the blue line represents the neuronal media (acsf).
Supplementary Figure 2 Immunohistochemical characterization of human neural cell cultures in the brain chip and the neurovascular cells in the BBB chip. (a, b) Confocal images of astrocytes stained for glial fibrillary acidic protein (GFAP, red), neurons stained for β-iii-tubulin (βiii, green) and cell nuclei stained with 4,6-diamidino-2phenylindole (DAPI, blue). (c, d) Confocal images of neurons (βiii, green), synapses (synaptophysin, red) and cell nuclei (DAPI, blue). (e, f) Confocal images of dopaminergic neurons stained for tyrosine hydroxylase (TH, green), astrocytes (GFAP, red) and cell nuclei (DAPI, blue). (g, h) Confocal images of neurons (βiii, green), GABAergic neurons stained for vesicular gamma-aminobutiric acid transporter (VGAT, red) and cell nuclei (DAPI, blue). (i, j) Confocal micrographs of neurons (βiii, green), glutamatergic neurons stained for glutamate decarboxylase (GAD, red) and GABAergic neurons (VGAT, blue). (a, c, e, g, i) scale bars = 3 mm; (b, d, f, h, j) scale bars = 100 μm. (k, l) Demonstration of different cell types that comprise the BBB: (k) Endothelial cells (ZO-1, green, nuclei, blue), (l) Pericytes (Phalloidin, green) and Astrocytes (GFAP, red) and nuclei, blue. Scale bar = 50 μm.(m) Endothelial cells (Occludin, green, nuclei, blue), (n) Endothelial cells (Claudin-5, green, nuclei, blue). More than 10 cultures have been replicated and characterized with immunofluorescence for each condition with similar results.
Supplementary Figure 3 Flow velocity in the model system (a) Simulation of flow distribution in the coupled chip setup (COMSOL ). Model illustrates precise control of fluidic flow using our microfluidic platform to accurately mimic the differential flow velocities found throughout the BBB-Brain-BBB Chips. Flow distribution confirms our coupled system follows laminar flow with lower velocities applied to the apical channels of the BBB Chip and Brain Chip with a higher velocity found in the basal or vascular component of the BBB Chip.
Supplementary Figure 4 Permeability measurements in the empty BBB-brain-BBB system. (a) Mean fluorescence intensity measurement of cascade blue-containing medium at different points of the system: vascular component of influx BBB, inlet of Brain Chip, inlet of the brain side of efflux BBB and outlet of the vascular side of efflux BBB. Values are presented as percentage of cascade blue (CB) fluorescence intensity of medium flowing into the system at the inlet vascular part of the influx BBB. N=5 for Vessel 1 and N= 3 for Perivasc.1, Brain and Vessel 2, N representing independent NVU systems, (p values: SI Notes, Supplementary Table 1b.) (b) Mean fluorescence intensity measurement of Alexa-555 labeled BSA (BSA-555) at different points throughout the system. Values are presented as percentage of the BSA-555 intensity values of the medium flowing into the system at the inlet vascular component of the influx BBB microfluidic device (p values: SI Notes, Supplementary Table 1b.). Error bars are SEM, N=5 for Vessel 1 and N= 3 for Perivasc.1, Brain and Vessel 2, significance calculated with one-way ANOVA, Bonferroni post-test.
Supplementary Figure 5 Protein cluster density maps for GEDI Figure 2 and breakdown of the biological processes associated with NVU compartment proteomes. (a) Mosaics representing the number of proteins that were assigned to each cluster (i.e. tile) in the mosaic after SOM training. These mosaics illustrate the distribution of proteins across the global expression profile GEDI maps shown in Fig. 2. The data are reduced dimensionality by classifying proteins with similar expression profiles into discrete groups that are organized into distinct twodimensional mosaics. Each tile of these mosaics represents a cluster of proteins and the mapping of proteins to these tiles is conserved across samples to facilitate comparison. Thus, the tiles represent the amount of proteins that have the same expression profile, ranging from low numbers illustrated in blue and high numbers in red. The protein expression for each of the NVU compartments (Endo, Peri/Astro and Neurons) was compared between the uncoupled and coupled conditions. (b). Proteomaps illustrate the KEGG Orthology biological process terms associated with low and high abundance proteins observed in the expression profiles of fluidically uncoupled chips compartments of endothelial cells, pericyte/astrocyte and neuronal/astrocyte cultures contrasted with expression values observed in coupled BBB-Brain-BBB Chips. In these Voronoi diagrams, each individual polygon represents one protein, wherein polygon area is a function of mass abundance. Each polygon is color-coded according to the KEGG Orthology term associated with it, and polygons representing the same KEGG Orthology term are clustered into larger polygons to form the map (Supplementary Video 3-8). The percentage of the protein expression profiles represented by each KEGG Orthology term shown in the Proteomaps is presented in Fig. 2b. (c) Principle component analysis (PCA) was used to assess the variability in global protein expression observed in the coupled and uncoupled cell populations for each compartment of the BBB and Brain Chips N=3 representing independent NVU systems.
Supplementary Figure 6 GluR2 penetration across the barrier after Meth addition, schematic of experiment shown in Figure 3. (a) The BBB Chip influx recapitulates tight barrier preventing penetration of an antibody (anti-glur2, pink y-shaped objects) from the vascular to the perivascular compartment during control conditions. (b) 24 hrs of Meth addition compromises the barrier, allowing the anti-glur2 to stain the neurons. Due to the low concentration of Meth reaching the BBB Chip efflux, the barrier stayed intact. (c) 24 hrs after Meth withdrawal resulted in recovery of the Chip influx while anti-glur2 remains attached to the neurons.
Supplementary Figure 7 Effect of Meth on the barrier properties of the BBB. (a-b) Meth dose response of TEER values for a BBB Transwell model. Mean values are represented as percentage of control (untreated well). (a) Meth concentrations were applied 24hrs and (b) recovery for 24 hrs after Meth removal. Error bars are SEM, N=2. (c) Effect of Meth on neuronal viability in the Brain Chip. Cell viability of neurons treated with 1 mm Meth. Dead cells were stained with ethidium homodimer while all cells were stained with DAPI and images were analyzed with a custom MATLAB script. Results are presented as percentage of live and dead cells, (p values: for each group, the p values for the live vs. dead was <0.0001, SI Notes, Supplementary Table 1b.). Error bars are SEM, N=3 neuronal cultures, were 17-24 field of views were taken for each culture, in order to calculate the mean value for each setup. significance calculated with one-way ANOVA, Bonferroni post-test.
Supplementary Figure 8 Mass balance of Meth in the coupled NVU microfluidic system and Meth uptake by cells in well plates. (a) Characterization of Meth dilution in BBB-Brain-BBB Chips. Mass spectrometry analysis of the concentration of Meth in different compartments throughout the system: vascular component of the influx BBB (Vessel 1), inlet of the Brain Chip (Perivasc. 1), outlet of the CSF (Brain) and vascular part of the efflux BBB (Vessel 2). Mean values are represented as percentage of the Meth dose flowed into the system (vascular part of the influx BBB), (p values <0.0001 SI Notes, Supplementary Table 1b.). N=3 representing independent NVU systems, significance calculated with one-way ANOVA, Bonferroni post-test (b) Mean values of Meth uptake by the neurovascular cell types in well-plates after 24hrs incubation N=3 representing independent NVU systems.
Supplementary Figure 9 Perivasculature response to Meth. Immunofluorescence microscopic views of the different cell populations within the influx and efflux BBB Chip in the fluidically coupled NVU system. Perivascular cells stained with astrocyte specific glial fibrillary associated protein (GFAP) and general F-actin staining with Phalloidin demonstrated consistent morphology in the influx and efflux BBB. The cell morphology was compared between steady state (a, b), addition of Meth (c, d), and drug removal (e, f). Administration of Meth for 24 hrs demonstrated only a minor change in perivascular cell morphology of mostly GFAP-negative cells (pericytes and GFAP-negative astrocytes) in the influx BBB (c), whereas efflux BBB maintained the control morphology (d). 24 hrs recovery restored the morphology of the influx BBB Chip (e) which is now identical to the efflux BBB (f). The experiment represented by the immunofluorescence micrographs was repeated 3 times with 2-4 individual NVU systems for each repeat.
Supplementary Figure 10 Untargeted metabolism. Potential biochemical pathways associated with significant metabolic changes identified by MS analysis (Compound Discoverer) within cells in the vascular compartment of the influx or efflux BBB Chips (Vessel 1 or 2, respectively), perivascular compartments of the influx or efflux BBB Chips (Perivasc 1 or 2, respectively), or the lower compartment of the Brain Chip (Brain) within the coupled NVU system under control conditions (b) or exposed to Meth for 24 hrs (c). Vessel 1 (Blue), Vessel 2 (Magenta), Perivasc 1 (Green), Perivasc 2 (Yellow) and Brain (Black). (c) IPA was used to identify the significant Disease Pathways together with Biofunction and their regulation which change due to Meth administration, using Z-score, (Z-score scale: red, high Z-score, pathway is upregulated, blue, low Z-score, pathway is downregulated)
Supplementary Figure 11 Metabolic cycle of labelled glucose. (a) Exploded illustration of the glycolysis and TCA cycles highlighting the modifications to each carbon entity during all steps in the pathway. In our system, we used all carbons C 13 labeled glucose (referred to as 6C 13 ; i.e, when a molecule had 2 carbons C 13 it was referred as 2C 13 ) to illustrate the glycolysis processes of each carbon. (b) Distribution of the number of C 13 -labelled carbons in glucose and glutamate in the fluidically coupled NVU system. (c) Distribution of the number of C 13 -labelled carbons in glucose and glutamate in the uncoupled Brain Chip as it was perfused with C 13 -labeled glucose, lactate and pyruvate.
Supplementary Figure 12 Theoretical metabolic flux balance analysis. Theoretical metabolic flux balance analysis of the GABA production in synaptic cleft as the neurons can uptake exogenous pyruvate and lactate without the astrocytes contribution (while Fig. 6d display the exogenous uptake by the astrocytes and directly supplying to neurons). The GABA exchange is shown as a function of the uptake of glucose or equal amounts of lactate and pyruvate in the presence or absence of metabolites in parentheses. All fluxes are reported as µmol g wet brain -1 min -1.