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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.

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such an endothelial compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays. (c) 2022 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro., Previous approaches to derive embryoids either lack physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here the authors use a high-throughput approach to induce mouse embryonic stem cells into epiblast-like aggregates, which are then co-cultured with mouse trophoblast stem cell aggregates, to yield embryoids with axial morphogenesis and anterior development.

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such a vascular compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behaviorin vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays.

Immunotherapy has shown promise as an approach to fight cancer by harnessing the immune system. However, due to the lack of biomarkers to guide treatment regimens and predict response rates, there is an unmet need for more robust ex vivo and in vitro systems that recapitulate patient-specific tumor biology and enable response prediction for immune therapies in an autologous setting. To address this issue, we developed a high-content screening-compatible assay based on microcavity arrays to study tumor-infiltrating lymphocyte (TIL) functionality on 3D tumoroid models. We validated our system using the pmel-1 activated T cell mouse model to assess both cancer immunogenicity and T cell functionality. To demonstrate the translational potential of the platform, we used it to evaluate the response of patient-derived TILs to autologous human colorectal cancer (CRC) tumoroids. Using a combination of imaging and flow cytometry, we determined several features of the antitumor activity of TILs, including the extent of tumoroid killing and secretion of cytokines. We then used the approach to identify responders to immunotherapy, such as the immune checkpoint blockade (ICB) agent Nivolumab (PD-1 inhibitor) and Ipilimumab (CTLA-4 inhibitor). Our system allows not only the identification of immunogenic tumors, but also the testing of patients for response to immunomodulators, enabling personalized immuno-oncology.

BackgroundThe 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.MethodsWe 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.ResultsBBB organoid arrays allowed the simultaneous growth of more than 5000 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.ConclusionsHuman 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.

Organoids derived from epithelial stem cells have emerged as powerful platforms to model development and disease in a dish1-3. However, the current mismatch in anatomy, lifespan and size between native organs and their in vitro counterparts severely limits their applicability4. In particular, the closed, cystic architecture of most epithelial stem cell-derived organoids makes experimental manipulation and assay development cumbersome. Here we describe how tissue engineering and cellular self-organization can be combined to guide in vitro organogenesis into openly accessible, functional intestinal tubes termed ‘mini-guts’. Intestinal stem cells (ISCs) rapidly generate simple columnar epithelia when propagated inside basal lamina-like hydrogel scaffolds that feature a tubular and crypt-containing, in vivo-like anatomical structure. Using a microfluidic perfusion system, dead cells shed into the lumen can be continuously removed from the mini-guts. This increases tissue lifespan to months, establishing a homeostatic organoid culture system in which cell proliferation (in crypts) is balanced with cell death (in villus-like domains). The approach developed here can be extended to generate functional tissue/organ models from other epithelial cell types, including primary human stem/progenitor cells from the small intestine, colon or airway, permitting reconstitution of complex organ-level physiology and disease in a personalized manner.

The development of improved methods to culture retinal organoids is relevant for the investigation of mechanisms of retinal development under pathophysiological conditions, for screening of neuroprotective compounds, and for providing a cellular source for clinical transplantation. We report a tissue-engineering approach to accelerate and standardize the production of retinal organoids by culturing mouse embryonic stem cells (mESC) in optimal physico-chemical microenvironments. Arrayed round-bottom milliwells composed of biomimetic hydrogels, combined with an optimized medium formulation, promoted the rapid generation of retina-like tissue from mESC aggregates in a highly efficient and stereotypical manner: ∼93% of the aggregates contained retinal organoid structures. 26 day-old retinal organoids were composed of ∼80% of photoreceptors, of which ∼22% are GNAT2-positive cones, an important and rare sensory cell type that is difficult to study in rodent models. The compartmentalization of retinal organoids into predefined locations on a two-dimensional array not only allowed us to derive almost all aggregates into retinal organoids, but also to reliably capture the dynamics of individual organoids, an advantageous requirement for high-throughput experimentation. Our improved retinal organoid culture system should be useful for applications that require scalability and single-organoid traceability.

We developed a new ODT microscope for large-sample imaging which incorporates a custom sample holder and two complementary scanning modalities. We measured for the first time the 3D refractive index of an unlabeled unmodified stem-cell-derived organoid.

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.

The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver.

Cystic fibrosis (CF) is a genetic disease caused by a bi-allelic mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. When the diagnosis cannot be confirmed by a positive sweat test or/and the identification of two CF-causing variants, international guidelines recommend the use of CFTR functional assays. These tests assess whether CFTR activity is normal or diminished/absent through measurement of CFTR-mediated chloride secretion/absorption., CFTR functional assays are not only useful for diagnostic purposes but can also serve as a surrogate outcome for clinical trials of CFTR modulators, which are emerging therapeutic agents designed to correct the malfunctioning protein. In the near future they could also be used as precision-medicine techniques, to help guidance and optimisation of treatment., Until now, sweat testing has been the only CFTR functional assay available in Switzerland. Since 2020, the Centre Hospitalier Universitaire Vaudois (CHUV) at Lausanne and the Lucerne Children's Hospital perform nasal potential difference measurement. Moreover, The Ecole Poly-technique Federale de Lausanne (EPFL) established a reliable procedure to generate adult intestinal organoids, i.e., stem cell-derived in-vitro grown mini tissues, extracted from rectal biopsies, which can be used to assess CFTR function in vitro., This narrative review describes the most popular CFTR functional assays, as well as their indications, limitations and availability in Switzerland.

Focalized short-pulsed lasers have sufficient power to generate micrometer-sized cavities in various hydrogels. An in situ technique based on laser ablation to fabricate intricate microfluidic networks in biocompatible gels without manual handling is presented. This method is fully compatible with 3D cell culture and opens up unprecedented opportunities for cell biology, developmental biology, and stem-cell-based tissue engineering.

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology(1-4). However, the approaches that are used at present to derive these organoids in three-dimensional matrices(5,6)result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.

Building the neural tube The development of the neural tube is regulated by a pair of morphogens acting in opposing gradients. The mature neural tube is built from a variety of different cell types organized in a consistent dorsal-ventral pattern. Zagorski et al. asked how this pattern is defined in a reproducible way from individual to individual. The morphogens define positions most accurately toward the top of their respective gradients, but things get a bit messy in the middle. Modeling the gene regulatory network's response as a maximum likelihood estimation from the combined input of both morphogens, however, succeeds at defining even the intermediate positions. Thus, the computation of position by the gene regulatory network establishes accurate tissue patterning despite messy inputs. Science , this issue p. 1379

Quantitative phase imaging (QPI) is an emerging label-free technique that produces images containing morphological and dynamical information without contrast agents. Unfortunately, the phase is wrapped in most imaging system. Phase unwrapping is the computational process that recovers a more informative image. It is particularly challenging with thick and complex samples such as organoids. Recent works that rely on supervised training show that deep learning is a powerful method to unwrap the phase; however, supervised approaches require large and representative datasets which are difficult to obtain for complex biological samples. Inspired by the concept of deep image priors, we propose a deep-learning-based method that does not need any training set. Our framework relies on an untrained convolutional neural network to accurately unwrap the phase while ensuring the consistency of the measurements. We experimentally demonstrate that the proposed method faithfully recovers the phase of complex samples on both real and simulated data. Our work paves the way to reliable phase imaging of thick and complex samples with QPI.

The fabrication of microfluidic devices is often still a time-consuming and costly process. Here we introduce a very simple and cheap microfabrication process based on "razor writing", also termed xurography, for the ultra-rapid prototyping of microfluidic devices. Thin poly(dimethylsiloxane) (PDMS) membranes are spin-coated on flexible plastic foil and cut into user-defined shapes with a bench-top cutter plotter. The PDMS membranes can then be assembled into desirable microdevices via plasma bonding. The plastic foil allows manipulation of exceptionally thin (30-300 mu m) PDMS layers and can be readily peeled after fabrication. This versatile technique can be used to produce a wide variety of microfluidic device prototypes within just a few hours.