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01.12.2018 | Research | Ausgabe 1/2018 Open Access

Fluids and Barriers of the CNS 1/2018

A perfused human blood–brain barrier on-a-chip for high-throughput assessment of barrier function and antibody transport

Zeitschrift:
Fluids and Barriers of the CNS > Ausgabe 1/2018
Autoren:
Nienke R. Wevers, Dhanesh G. Kasi, Taylor Gray, Karlijn J. Wilschut, Benjamin Smith, Remko van Vught, Fumitaka Shimizu, Yasuteru Sano, Takashi Kanda, Graham Marsh, Sebastiaan J. Trietsch, Paul Vulto, Henriëtte L. Lanz, Birgit Obermeier
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12987-018-0108-3) contains supplementary material, which is available to authorized users.
Henriëtte L. Lanz and Birgit Obermeier contributed equally to this work

Abstract

Background

Receptor-mediated transcytosis is one of the major routes for drug delivery of large molecules into the brain. The aim of this study was to develop a novel model of the human blood–brain barrier (BBB) in a high-throughput microfluidic device. This model can be used to assess passage of large biopharmaceuticals, such as therapeutic antibodies, across the BBB.

Methods

The model comprises human cell lines of brain endothelial cells, astrocytes, and pericytes in a two-lane or three-lane microfluidic platform that harbors 96 or 40 chips, respectively, in a 384-well plate format. In each chip, a perfused vessel of brain endothelial cells was grown against an extracellular matrix gel, which was patterned by means of surface tension techniques. Astrocytes and pericytes were added on the other side of the gel to complete the BBB on-a-chip model. Barrier function of the model was studied using fluorescent barrier integrity assays. To test antibody transcytosis, the lumen of the model’s endothelial vessel was perfused with an anti-transferrin receptor antibody or with a control antibody. The levels of antibody that penetrated to the basal compartment were quantified using a mesoscale discovery assay.

Results

The perfused BBB on-a-chip model shows presence of adherens and tight junctions and severely limits the passage of a 20 kDa FITC-dextran dye. Penetration of the antibody targeting the human transferrin receptor (MEM-189) was markedly higher than penetration of the control antibody (apparent permeability of 2.9 × 10−5 versus 1.6 × 10−5 cm/min, respectively).

Conclusions

We demonstrate successful integration of a human BBB microfluidic model in a high-throughput plate-based format that can be used for drug screening purposes. This in vitro model shows sufficient barrier function to study the passage of large molecules and is sensitive to differences in antibody penetration, which could support discovery and engineering of BBB-shuttle technologies.
Zusatzmaterial
Additional file 1. Endothelial microvessel seeding in the two-lane OrganoPlate. (a) Schematic representation of one chip of a two-lane OrganoPlate. (b) An ECM gel is seeded in the gel channel, after which endothelial cells are seeded in the medium channel. (c) Endothelial cells attach to the ECM gel and perfusion is started by placing the OrganoPlate on a rocker platform. (d) A microvessel of endothelial cells is formed. (eg) Cross sectional view of steps described in bd.
Additional file 2. BBB co-culture seeding in the three-lane OrganoPlate®. (a) Schematic representation of one chip of a three-lane OrganoPlate. (b) ECM gel is seeded in the middle gel of the chip, after which endothelial cells (TY10) are seeded in the top channel. (c) Endothelial cells attach to the ECM and perfusion is started by placing the plate on a rocking platform. (d) A microvessel of endothelial cells forms in the top channel, against the ECM gel. (e) Astrocytes (hAst) and pericytes (hBPCTs) are seeded in the bottom channel. (f) hAst and hBPCT cells attach and a BBB co-culture is established. (gk) Cross sectional view of steps described in bf.
Additional file 3. Comparing perfused and static culture of TY10 microvessels. (a, b) Phase contrast images of TY10 microvessels grown in the two-lane OrganoPlate under perfused or static conditions (day 7). Scale bar is 100 µm. (c) Microvessels grown under perfused or static conditions were fixed and nuclei were stained with Hoechst. The average number of nuclei was counted in both conditions and normalized to the perfused condition. n = 6, Student’s t-test p < 0.05. (df) Immunofluorescent staining of TY10 microvessels grown under perfusion for adherens and tight junction markers VE-cadherin, claudin-5, and PECAM-1. (gi) Immunofluorescent staining of TY10 microvessels grown static for adherens and tight junction markers VE-cadherin, claudin-5, and PECAM-1. Scale bar is 100 µm.
Additional file 4. Characterization of the human transferrin receptor in TY10 endothelial cells. (a) Immunofluorescent staining of the hTfR in TY10 endothelial cells. Scale bar is 50 µm. (b) Flow cytometry analysis of cell surface binding of anti-TfR MEM-189 to TY10 endothelial cells in the presence and absence of transferrin (25 µg/mL), EC50 = 0.44 ± 0.09 nM (−Tf); 0.5 ± 0.1 nM (+Tf).
Literatur
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