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      Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function and shuttling of drugs and antibodies

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          Abstract

          The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cell-derived human brain microvascular endothelium interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB.

          Abstract

          In vitro blood-brain barrier (BBB) models do not fully recapitulate the in vivo barrier function. Here the authors develop an organ-on-a-chip BBB model using iPS-derived human brain endothelial cells differentiated under hypoxia, primary human pericytes and astrocytes, which maintains in vivo-like BBB barrier and shuttling functions for a week.

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          Most cited references57

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          Blood-brain barrier active efflux transporters: ATP-binding cassette gene family.

          The blood-brain barrier (BBB) contributes to brain homeostasis by protecting the brain from potentially harmful endogenous and exogenous substances. BBB active drug efflux transporters of the ATP-binding cassette (ABC) gene family are increasingly recognized as important determinants of drug distribution to, and elimination from, the CNS. The ABC efflux transporter P-glycoprotein (Pgp) has been demonstrated as a key element of the BBB that can actively transport a huge variety of lipophilic drugs out of the brain capillary endothelial cells that form the BBB. In addition to Pgp, other ABC efflux transporters such as members of the multidrug resistance protein (MRP) family and breast cancer resistance protein (BCRP) seem to contribute to BBB function. Consequences of ABC efflux transporters in the BBB include minimizing or avoiding neurotoxic adverse effects of drugs that otherwise would penetrate into the brain. However, ABC efflux transporters may also limit the central distribution of drugs that are beneficial to treat CNS diseases. Furthermore, neurological disorders such as epilepsy may be associated with overexpression of ABC efflux transporters at the BBB, resulting in pharmacoresistance to therapeutic medication. Therefore, modulation of ABC efflux transporters at the BBB forms a novel strategy to enhance the penetration of drugs into the brain and may yield new therapeutic options for drug-resistant CNS diseases.
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            Microfluidic blood-brain barrier model provides in vivo-like barrier properties for drug permeability screening.

            Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High-fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm(2) on day 3 on chip and were sustained above 2000 Ω · cm(2) up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184-194. © 2016 Wiley Periodicals, Inc.
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              Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector angiopep-2.

              The blood-brain barrier (BBB) restricts the entry of proteins as well as potential drugs to cerebral tissues. We previously reported that a family of Kunitz domain-derived peptides called Angiopeps can be used as a drug delivery system for the brain. Here, we further characterize the transcytosis ability of these peptides using an in vitro model of the BBB and in situ brain perfusion. These peptides, and in particular Angiopep-2, exhibited higher transcytosis capacity and parenchymal accumulation than do transferrin, lactoferrin, and avidin. Angiopep-2 transport and accumulation in brain endothelial cells were unaffected by the P-glycoprotein inhibitor, cyclosporin A, indicating that this peptide is not a substrate for the efflux pump P-glycoprotein. However, competition studies show that activated alpha(2)-macroglobulin, a specific ligand for the low-density lipoprotein receptor-related protein-1 (LRP1) and Angiopep-2 can share the same receptor. In addition, LRP1 was detected in glioblastomas and brain metastases from lung and skin cancers. Fluorescent microscopy also revealed that Alexa488-Angiopep-2 co-localized with LRP1 in brain endothelial cell monolayers. Overall, these results suggest that Angiopep-2 transport across the BBB is, in part, mediated by LRP1.
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                Author and article information

                Contributors
                don.ingber@wyss.harvard.edu
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                13 June 2019
                13 June 2019
                2019
                : 10
                : 2621
                Affiliations
                [1 ]ISNI 000000041936754X, GRID grid.38142.3c, Wyss Institute for Biologically Inspired Engineering at Harvard University, ; Boston, MA 02115 USA
                [2 ]ISNI 0000 0004 0378 8438, GRID grid.2515.3, Department of Neurosurgery, , Boston Children’s Hospital and Harvard Medical School, ; Boston, MA 02115 USA
                [3 ]ISNI 0000 0001 2167 3675, GRID grid.14003.36, Department of Chemical and Biological Engineering, , University of Wisconsin-Madison, ; Madison, WI 53706 USA
                [4 ]ISNI 000000041936754X, GRID grid.38142.3c, Harvard John A. Paulson School of Engineering and Applied Sciences, , Harvard University, ; Cambridge, MA 02138 USA
                [5 ]ISNI 0000 0004 0378 8438, GRID grid.2515.3, Vascular Biology Program and Department of Surgery, , Boston Children’s Hospital and Harvard Medical School, ; Boston, MA 02115 USA
                [6 ]ISNI 0000 0004 0381 814X, GRID grid.42687.3f, Present Address: Ulsan National Institute of Science and Technology (UNIST), ; UNIST-gil 50, Ulsan, 44919 Republic of Korea
                [7 ]ISNI 0000000121581746, GRID grid.5037.1, Division of Micro and Nanosystems, , KTH Royal Institute of Technology, ; Stockholm, Sweden
                [8 ]ISNI 0000 0004 1937 0626, GRID grid.4714.6, Swedish Medical Nanoscience Center, Department of Neuroscience, , Karolinska Institute, ; Stockholm, Sweden
                Author information
                http://orcid.org/0000-0003-3979-5405
                http://orcid.org/0000-0002-0710-8722
                http://orcid.org/0000-0002-0603-1241
                http://orcid.org/0000-0002-8295-4996
                http://orcid.org/0000-0002-2662-719X
                http://orcid.org/0000-0002-4319-6520
                Article
                10588
                10.1038/s41467-019-10588-0
                6565686
                31197168
                933b3134-b3d3-4ff2-a06f-46ab48e5308d
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 16 November 2018
                : 16 May 2019
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100003725, National Research Foundation of Korea (NRF);
                Award ID: NRF-2018R1A5A1024340
                Award ID: 2018K1A4A3A01063890
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/501100004063, Knut och Alice Wallenbergs Stiftelse (Knut and Alice Wallenberg Foundation);
                Award ID: WAF 2015-0178
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100000185, United States Department of Defense | Defense Advanced Research Projects Agency (DARPA);
                Award ID: W911NF-12-2-0036
                Award Recipient :
                Categories
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                Custom metadata
                © The Author(s) 2019

                Uncategorized
                biological techniques,biotechnology,drug discovery,neuroscience,stem cells
                Uncategorized
                biological techniques, biotechnology, drug discovery, neuroscience, stem cells

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