Cell lines of brain endothelial cells, pericytes, and astrocytes were provided by Yamaguchi University, Japan, and originate from the following human primary cell sources: human brain microvascular endothelial cells (TY10 cell line) were isolated from normal brain tissue from a patient with meningioma. Human brain pericytes (hBPCT cell line) were derived from brain tissue of a patient that died from a heart attack. Human astrocytes (hAst cell line) were generated from human primary astrocytes distributed by Lonza (Basel, Switzerland). All three cell types were immortalized with retroviral vectors harboring a SV40 large T antigen gene that is engineered to drive proliferation at 33 °C [30,31,32,33,34] and have been used to model the BBB in previous studies [35,36,37,38]. Cells were cultured at 33 °C, 5% CO2 to allow optimal cell expansion in T75 flasks (734-2705, Corning, NY, USA), which were pre-coated with 50 µg/mL collagen-I (Cultrex 3D collagen-I Rat Tail, 5 mg/mL, 3447-020-01, AMSbio, Abingdon, UK) in 1% acetic acid (A6283, Sigma, St. Louis, MO, USA) in water. TY10 cells were used between passage 17–25 and cultured in ScienCell endothelial cell medium (#1001, Sciencell, Carlsbad, CA, USA). The hAst cells were used between passage 7–12 and cultured in ScienCell astrocyte medium (#1018, Sciencell). The hBPCT cells were used between passage 14–25 and cultured in ScienCell pericyte medium (#1012, Sciencell). Cells were routinely tested for mycoplasma contamination and found negative.
Culture of TY10 microvessels in the two-lane OrganoPlate
Two-lane OrganoPlates (Mimetas BV, the Netherlands) with 400 µm × 220 µm (w × h) channels were employed. Phaseguides had dimensions of 100 µm × 55 µm (w × h) and 2 µL of gel composed of 4 mg/mL collagen-I (Cultrex 3D collagen-I Rat Tail, 5 mg/mL, 3447-020-01, AMSbio), 100 mM HEPES (15630-122, Thermo Fisher, Waltham, MA, USA) and 3.7 mg/mL NaHCO3 (S5761, Sigma) was dispensed in the gel inlet and the OrganoPlate was incubated for 15 min at 33 °C. After plate incubation, 25 µL of PBS was added to the gel inlet to prevent the gel from drying out. A TY10 cell suspension of 1.5 × 107 cells/mL was prepared and 2 µL was seeded in the medium inlet. 50 µL of medium was added to the medium inlet and PBS was aspirated from the gel inlet. The plate was incubated on the side for 3 h in the incubator to allow the cells to sediment against the collagen-I gel and attach. After incubation, 50 µL of medium was added to the medium outlet. The OrganoPlate was placed on an interval rocker switching between a + 7° and − 7° inclination every 8 min (Mimetas Rocker Mini, Mimetas BV), allowing bidirectional flow. Cells were cultured at 33 °C (and 5% CO2) to allow full cell coverage of the ECM gel. Medium was refreshed every 2–3 days. A schematic representation of all steps is shown in Additional file 1. The following media were used to assess their influence on barrier function: ScienCell endothelial cell medium (#1001, Sciencell), Cell Biologics endothelial cell medium (#H1168, Cell Biologics, Chicago, IL, USA), MV2 medium (C-22121, Bioconnect, Huissen, the Netherlands), and EBM-2 medium (cc-3156, Lonza).
BBB co-culture in the three-lane OrganoPlate
OrganoPlate BBB co-culture was performed using three-lane OrganoPlates with 400 µm × 220 µm (w × h) channels (Mimetas BV). Phaseguides had dimensions of 100 µm × 55 µm (w × h). To establish a BBB co-culture, a collagen-I gel was dispensed in the gel inlet of the chips and filled the middle channel. TY10 cells were seeded and cultured in the top channel as described in the previous section. After 3 days, 2 µL of a 7 × 106 cells/mL cell suspension of hAst and hBPCT cells (1:3 ratio) was seeded in the bottom channel. The plate was incubated on the side for 1.5 h to allow the hAst and hBPCT cells to attach to the collagen-I gel. After incubation, fresh Sciencell astrocyte medium is added to the inlet and outlets of the top channel (50 µL in each), after which perfusion is reinstated by placing the plate on the rocker platform. During the entire culture period, only the top channel, which contains the TY10 microvessel, is perfused to allow optimal endothelial barrier strength. Medium was refreshed every 2–3 days. Assays were performed at day 7. A schematic representation of all steps is shown in Additional file 2.
For the images shown in Fig. 3f, g, hBPCT cells were labeled with Calcein red™ AM (21900, AAT Bioquest, Sunnyvale, CA, USA) and hAst cells were labeled with green-fluorescent calcein-AM (C3099, Thermo Fisher) before seeding in the OrganoPlate.
Cultures in the OrganoPlate were fixed with 100% methanol (− 20 °C, 494437, Sigma) and permeabilized with 0.3% Triton X-100 (T8787, Sigma). Cells were incubated with blocking solution (2% FCS, 2% bovine serum albumin (BSA, A2153, Sigma), 0.1% Tween-20 (P9416, Sigma)) for 45 min. Primary antibody was incubated for 1–2 h, after which secondary antibody was incubated for 30 min. The following antibodies were used: anti-claudin-5 (35-2500, Thermo Fisher), anti-VE-cadherin (ab33168, Abcam), anti-PECAM-1 (M0823, Dako), goat anti-rabbit AlexaFluor 488 (A11008, Thermo Fisher), goat anti-rabbit AlexaFluor 555 (A21428, Thermo Fisher), goat anti-mouse AlexaFluor 488 (A11001, Thermo Fisher), goat anti-mouse AlexaFluor 555 (A21422, Thermo Fisher), and donkey anti-mouse AlexaFluor 647 (A31571, Thermo Fisher). Nuclei were stained using Hoechst (H3570, Thermo Fisher). All steps were performed at room temperature (RT). Cells were imaged with ImageXpress Micro XLS and Micro XLS-C HCI Systems (Molecular Devices, San Jose, CA, USA).
TY10 cells grown in a collagen-I coated 24-well glass bottom plate (P24-0-N, Cellvis, Mountain View, CA, USA) were fixed with 4% paraformaldehyde (50-980-487, Thermo Fisher) for 10 min and incubated with primary antibody against the human transferrin receptor (A11130, Thermo Fisher) for 3 h, followed by 1 h incubation with goat anti-mouse Alexa Fluor 488 (A11001, Thermo Fisher). Nuclei were stained with Hoechst and cells were imaged using a Zeiss LSM 710 Confocal Microscope (Zeiss, Oberkochen, Germany).
Barrier integrity assay
Chips were washed with culture medium (25 µL on all inlets and outlets, 1 × 5 min) to ensure proper flow profiles during the subsequent barrier integrity assay. Next, all medium was aspirated from the chips and 20 µL of medium without fluorescent compound was added to the basal side of the chips (for the two-lane OrganoPlate this is the gel inlet, for the three-lane OrganoPlate these are the gel inlets and outlets and bottom medium inlets and outlets). Medium containing 0.1 mg/mL FITC-dextran (20 kDa, FD20S, Sigma) was added to the top channel, which contained the TY10 microvessel (40 µL on inlet, 30 µL on outlet) and image acquisition was started. Leakage of the fluorescent molecule from the lumen of the microvessel into the adjacent gel channel was automatically imaged using an ImageXpress XLS Micro HCI system (molecular devices). The ratio between the fluorescent signal in the basal and apical region of the tube was analyzed using FiJi . Graphs were plotted using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA).
Analysis of cell surface binding of anti-hTfR MEM-189
TY10 cells were cultured to confluency and lifted with accutase for 1 h. 2.5 × 105 cells/mL were mixed with antibody in PBS with 0.5% BSA and incubated for 1 h at 4 °C. Cells were washed 3× and incubated with 3 µg/mL PE-goat anti-mIgG (115-116-146, Jackson ImmunoResearch, Cambridgeshire, UK) for 1 h at 4 °C, then washed and fixed with 1% paraformaldehyde for 10 min at RT. Cell fluorescence intensity was analyzed by flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA) and data was analyzed in FlowJo v10 (FlowJo LLC, Ashland, OR, USA) and GraphPad Prism software.
Antibody transcytosis assay
Anti-human transferrin receptor mouse monoclonal antibody MEM-189 mIgG1 (MA1-21562, Thermo Fisher, 10 × 0.1 mg) was dialyzed into pyrogen free PBS to remove the azide in the supplied storage solution. For negative control, an anti-hen egg lysozyme (anti-HEL) antibody (F10.6.6, Genbank AF110316 VH and AY277254.1 VL) was expressed as a mouse IgG1 antibody in CHO cells and purified by recombinant protein A Sepharose (GE) affinity chromatography and size exclusion chromatography (superdex 200).
Chips were washed once with medium to ensure proper flow profiles (as described for the barrier integrity assay). Next, medium was aspirated from the chips, after which 20 µL of fresh medium without antibody was added to the gel inlets and outlets and the bottom medium inlets and outlets. A total of 70 µL of antibody dilution (1.25 µM in medium) was added to the top channel and the OrganoPlate was incubated on the rocker platform in the incubator for 1 h after which basal samples were taken, which consisted of the full contents of the gel inlet and outlet and bottom medium inlet and outlet.
Analysis of antibody contents in basal samples
A Meso Scale Discovery (MSD) platform based quantitative immunoassay was used to determine the concentration of antibodies in basal samples collected from the OrganoPlate. Multi-Array 96-well MSD high-binding plates (L15XB-3/L11XB-3, Meso Scale Discovery, Rockville, MD, USA) were coated overnight at 4 °C with 5 µg/mL capturing agent AffiniPure goat anti-mouse IgG, Fcγ fragment specific (115-005-071, Jackson ImmunoResearch). Plates were blocked (1% BSA in PBS) for 2 h, after which 100 µL/well standards (generated from 1.25 µM antibody stock dilutions) or samples were added to the plate and incubated for 1.5–2 h. Plates were incubated with 100 µL/well 0.25 µg/mL primary detection agent Biotin-SP-conjugated AffiniPure goat-anti-mouse IgG (115-005-071, Jackson ImmunoResearch) for 45 min, followed by 100 µL/well 0.75 µg/mL secondary detection agent MSD Sulfo-TAG StreptAvidin (R32AD-1, Meso Scale Discovery) for 30 min. Between each step, the plates were washed 4× with wash buffer (PBST: 1× PBS with 0.05% Tween-20, 28352, Thermo Fisher). Immediately before MSD read, 2× Read Buffer (MSD Read Buffer T (4×), R92TD-2, diluted 1:2 in diH2O) was added to the plates. Plates were read on a MSD QuickPlex SQ 120 in automatic read mode, then a Sigmoidal, 4PL, X = log(concentration) interpolation was used to determine antibody concentration in GraphPad Prism 7.02 (GraphPad Software). The apparent permeability (Papp) of both antibodies was determined using the following formula: Papp = [ΔCreceiver/Δt] × [Vreceiver/(Abarrier × Cdonor, initial)], in which ΔCreceiver/Δt is the change in antibody concentration in the receiving compartment over time, Vreceiver is the volume of the receiving compartment, Abarrier is the surface area of the barrier, and Cdonor, initial is the initial antibody concentration of the donor compartment.