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
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.
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.
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).
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.
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. (e–g) Cross sectional view of steps described in b–d.
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. (g–k) Cross sectional view of steps described in b–f.
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. (d–f) Immunofluorescent staining of TY10 microvessels grown under perfusion for adherens and tight junction markers VE-cadherin, claudin-5, and PECAM-1. (g–i) 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).
Abbott NJ, Patabendige AAK, Dolman DEM, Yusof SR, Begley DJ. Structure and function of the blood–brain barrier. Neurobiol Dis. 2010;37:13–25. CrossRef
Wolburg H, Lippoldt A. Tight junctions of the blood–brain barrier: development, composition and regulation. Vasc Pharmacol. 2002;38:323–37. CrossRef
Daneman R. The blood–brain barrier in health and disease. Ann Neurol. 2012;72:648–72. CrossRef
Wevers NR, de Vries HE. Morphogens and blood–brain barrier function in health and disease. Tissue Barriers. 2016;4:e1090524. CrossRef
Pardridge WM. Drug and gene delivery to the brain: the vascular route. Neuron. 2002;36:555–8. CrossRef
Freskgård PO, Urich E. Antibody therapies in CNS diseases. Neuropharmacology. 2017;120:38–55. CrossRef
Niewoehner J, Bohrmann B, Collin L, et al. Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle. Neuron. 2014;81:49–60. CrossRef
Webster CI, Caram-Salas N, Haqqani AS, et al. Brain penetration, target engagement, and disposition of the blood–brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1. FASEB J. 2016;30:1927–40. CrossRef
Zuchero YJY, Chen X, Bien-Ly N, et al. Discovery of novel blood–brain barrier targets to enhance brain uptake of therapeutic antibodies. Neuron. 2016;89:70–82. CrossRef
Yu YJ, Zhang Y, Kenrick M, et al. Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Sci Transl Med. 2011;3:84ra44. CrossRef
Couch JA, Yu YJ, Zhang Y, et al. Addressing safety liabilities of TfR bispecific antibodies that cross the blood–brain barrier. Sci Transl Med. 2013;5:183ra57. CrossRef
Haqqani AS, Delaney CE, Brunette E, et al. Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier. J Cereb Blood Flow Metab. 2018;38:727–40. CrossRef
Thom G, Hatcher J, Hearn A, et al. Isolation of blood–brain barrier-crossing antibodies from a phage display library by competitive elution and their ability to penetrate the central nervous system. MAbs. 2018;10:304–14. CrossRef
Weber F, Bohrmann B, Niewoehner J, et al. Brain shuttle antibody for Alzheimer’s disease with attenuated peripheral effector function due to an inverted binding mode. Cell Rep. 2018;22:149–62. CrossRef
Abbott NJ, Hughes CC, Revest PA, Greenwood J. Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood–brain barrier. J Cell Sci. 1992;103(Pt 1):23–37. PubMed
Biegel D, Pachter JS. Growth of brain microvessel endothelial cells on collagen gels: applications to the study of blood–brain barrier physiology and CNS inflammation. In Vitro Cell Dev Biol Anim. 1994;30:581–8. CrossRef
Gardner TW, Lieth E, Khin SA, et al. Astrocytes increase barrier properties and ZO-1 expression in retinal vascular endothelial cells. Invest Ophthalmol Vis Sci. 1997;38:2423–7. PubMed
Wolff A, Antfolk M, Brodin B, Tenje M. In vitro blood–brain barrier models—an overview of established models and new microfluidic approaches. J Pharm Sci. 2015;104:2727–46. CrossRef
Reichel A, Begley DJ, Abbott NJ. An overview of in vitro techniques for blood–brain barrier studies. Methods Mol Med. 2003;89:307–24. PubMed
van der Meer AD, Orlova VV, ten Dijke P, van den Berg A, Mummery CL. Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device. Lab Chip. 2013;13:3562. CrossRef
Herland A, Van Der Meer AD, FitzGerald EA, Park TE, Sleeboom JJF, Ingber DE. Distinct contributions of astrocytes and pericytes to neuroinflammation identified in a 3D human blood–brain barrier on a chip. PLoS ONE. 2016;11:1–21. CrossRef
Bang S, Lee SR, Ko J, et al. A low permeability microfluidic blood–brain barrier platform with direct contact between perfusable vascular network and astrocytes. Sci Rep. 2017;7:1–10. CrossRef
Sano Y, Shimizu F, Abe M, et al. Establishment of a new conditionally immortalized human brain microvascular endothelial cell line retaining an in vivo blood–brain barrier function. J Cell Physiol. 2010;225:519–28. CrossRef
Sano Y, Kashiwamura Y, Abe M, et al. Stable human brain microvascular endothelial cell line retaining its barrier-specific nature independent of the passage number. Clin Exp Neuroimmunol. 2013;4:92–103. CrossRef
Shimizu F, Sano Y, Tominaga O, Maeda T, Abe M, Kanda T. Advanced glycation end-products disrupt the blood–brain barrier by stimulating the release of transforming growth factor-β by pericytes and vascular endothelial growth factor and matrix metalloproteinase-2 by endothelial cells in vitro. Neurobiol Aging. 2013;34:1902–12. CrossRef
Haruki H, Sano Y, Shimizu F, et al. NMO sera down-regulate AQP4 in human astrocyte and induce cytotoxicity independent of complement. J Neurol Sci. 2013;331:136–44. CrossRef
Shimizu F, Sano Y, Abe M, et al. Peripheral nerve pericytes modify the blood-nerve barrier function and tight junctional molecules through the secretion of various soluble factors. J Cell Physiol. 2011;226:255–66. CrossRef
Takeshita Y, Obermeier B, Cotleur AC, et al. Effects of neuromyelitis optica-IgG at the blood–brain barrier in vitro. Neurol Neuroimmunol Neuroinflamm. 2017;4:e311. CrossRef
Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. CrossRef
Trietsch SJ, Israëls GD, Joore J, Hankemeier T, Vulto P. Microfluidic titer plate for stratified 3D cell culture. Lab Chip. 2013;13:3548–54. CrossRef
Goldbaum FA, Cauerhff A, Velikovsky CA, Llera AS, Riottot MM, Poljak RJ. Lack of significant differences in association rates and affinities of antibodies from short-term and long-term responses to hen egg lysozyme. J Immunol. 1999;162:6040–5. PubMed
Gumbleton M, Audus KL. Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood–brain barrier. J Pharm Sci. 2001;90:1681–98. CrossRef
Biegel D, Pachter JS. Growth of brain microvessel endothelial cells on collagen gels: applications to the study of blood–brain barrier physiology and CNS inflammation. In Vitro Cell Dev Biol Anim. 1994;30A:581–8. CrossRef
Hopkins AM, DeSimone E, Chwalek K, Kaplan DL. 3D in vitro modeling of the central nervous system. Prog Neurobiol. 2015;125:1–25. CrossRef
Naik P, Cucullo L. In vitro blood–brain barrier models: current and perspective technologies. J Pharm Sci. 2012;101:1337–54. CrossRef
Dewey CF, Bussolari SR, Gimbrone MA, Davies PF. The dynamic response of vascular endothelial cells to fluid shear stress. J Biomech Eng. 1981;103:177. CrossRef
Seebach J, Dieterich P, Luo F, et al. Endothelial barrier function under laminar fluid shear stress. Lab Invest. 2000;80:1819–31. CrossRef
Jefferies WA, Brandon MR, Hunt SV, Williams AF, Gatter KC, Mason DY. Transferrin receptor on endothelium of brain capillaries. Nature. 1984;312:162–3. CrossRef
Johnsen KB, Moos T. Revisiting nanoparticle technology for blood–brain barrier transport: unfolding at the endothelial gate improves the fate of transferrin receptor-targeted liposomes. J Control Release. 2016;222:32–46. CrossRef
Farrington GK, Caram-Salas N, Haqqani AS, et al. A novel platform for engineering blood–brain barrier-crossing bispecific biologics. FASEB J. 2014;28:4764–78. CrossRef
Ober RJ, Radu CG, Ghetie V, Ward ES. Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies. Int Immunol. 2001;13:1551–9. CrossRef
Moos T, Morgan EH. Restricted transport of anti-transferrin receptor antibody (OX26) through the blood–brain barrier in the rat. J Neurochem. 2001;79:119–29. CrossRef
- A perfused human blood–brain barrier on-a-chip for high-throughput assessment of barrier function and antibody transport
Nienke R. Wevers
Dhanesh G. Kasi
Karlijn J. Wilschut
Remko van Vught
Sebastiaan J. Trietsch
Henriëtte L. Lanz
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II