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      Investigating receptor-mediated antibody transcytosis using blood–brain barrier organoid arrays

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          Abstract

          Background

          The pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development.

          Methods

          We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis.

          Results

          BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis.

          Conclusions

          Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

          Supplementary Information

          The online version contains supplementary material available at 10.1186/s12987-021-00276-x.

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          Most cited references46

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          Fiji: an open-source platform for biological-image analysis.

          Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
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            Caveolae as plasma membrane sensors, protectors and organizers.

            Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded. Coat proteins, called cavins, work together with caveolins to regulate the formation of caveolae but also have the potential to dynamically transmit signals that originate in caveolae to various cellular destinations. The importance of caveolae as protective elements in the plasma membrane, and as membrane organizers and sensors, is highlighted by links between caveolae dysfunction and human diseases, including muscular dystrophies and cancer.
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              Mechanisms of clathrin-mediated endocytosis

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                Author and article information

                Contributors
                roberto.villasenor_solorio@roche.com
                Journal
                Fluids Barriers CNS
                Fluids Barriers CNS
                Fluids and Barriers of the CNS
                BioMed Central (London )
                2045-8118
                20 September 2021
                20 September 2021
                2021
                : 18
                Affiliations
                [1 ]GRID grid.417570.0, ISNI 0000 0004 0374 1269, Roche Pharma Research and Early Development (pRED), Pharmaceutical Sciences, , Roche Innovation Center Basel, ; Basel, Switzerland
                [2 ]SUN bioscience, EPFL Innovation Park, Lausanne, Switzerland
                [3 ]Roche Pharma Research and Early Development (pRED), Therapeutic Modalities, Roche Innovation Center Munich, Munich, Germany
                Article
                276
                10.1186/s12987-021-00276-x
                8454074
                babbcace-9f48-4dec-b06d-d6d68c02c3c4
                © The Author(s) 2021

                Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                Funding
                Funded by: Roche Postdoctoral Fellowship
                Award ID: RPFID-491
                Award Recipient :
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                Research
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                © The Author(s) 2021

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