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Human brain organoids present a new paradigm for modeling human brain organogenesis, providing unprecedented insight to the molecular and cellular processes of brain development and maturation. Other potential applications include in vitro models of disease and tissue trauma, as well as three-dimensional (3D) clinically relevant tissues for pharmaceuticals development and cell or tissue replacement. A key requirement for this emerging technology in both research and medicine is the simple, scalable, and reproducible generation of organoids using reliable, economical, and high-throughput culture platforms. Here we describe such a platform using a defined, clinically compliant, and readily available hydrogel generated from gelatin methacrylate (GelMA). We demonstrate the efficient production of organoids on GelMA from human induced pluripotent stem cells (iPSCs), with scalable production attained using 3D printed GelMA-based multiwell arrays. The differentiation of iPSCs was systematic, rapid, and direct to enable iPSCs to form organoids in their original position following seeding on GelMA, thereby avoiding further cell and organoid disruption. Early neural precursors formed by day 5, neural rosettes and early-stage neurons by day 14, and organoids with cellular and regional heterogeneity, including mature and electrophysiologically active neurons, by day 28. The optimised method provides a simplified and well-defined platform for both research and translation of iPSCs and derivative brain organoids, enabling reliable 3D in vitro modelling and experimentation, as well as the provision of clinically relevant cells and tissues for future therapeutics.

To characterize the growth of brain organoids (BOs), cultures that replicate some early physiological or pathological developments of the human brain are usually manually extracted. Due to their novelty, only small datasets of these images are available, but segmenting the organoid shape automatically with deep learning (DL) tools requires a larger number of images. Light U-Net segmentation architectures, which reduce the training time while increasing the sensitivity under small input datasets, have recently emerged. We further reduce the U-Net architecture and compare the proposed architecture (MU-Net) with U-Net and UNet-Mini on bright-field images of BOs using several data augmentation strategies. In each case, we perform leave-one-out cross-validation on 40 original and 40 synthesized images with an optimized adversarial autoencoder (AAE) or on 40 transformed images. The best results are achieved with U-Net segmentation trained on optimized augmentation. However, our novel method, MU-Net, is more robust: it achieves nearly as accurate segmentation results regardless of the dataset used for training (various AAEs or a transformation augmentation). In this study, we confirm that small datasets of BOs can be segmented with a light U-Net method almost as accurately as with the original method.

Nanoparticles (NPs) are the focus of research efforts that aim to develop successful drug delivery systems for the brain. Polypeptide nanocarriers are versatile platforms and combine high functionality with good biocompatibility and biodegradability. The key to the efficient brain delivery of NPs is the specific targeting of cerebral endothelial cells that form the blood–brain barrier (BBB). We have previously discovered that the combination of two different ligands of BBB nutrient transporters, alanine and glutathione, increases the permeability of vesicular NPs across the BBB. Our aim here was to investigate whether the combination of these molecules can also promote the efficient transfer of 3-armed poly(l-glutamic acid) NPs across a human endothelial cell and brain pericyte BBB co-culture model. Alanine and glutathione dual-targeted polypeptide NPs showed good cytocompatibility and elevated cellular uptake in a time-dependent and active manner. Targeted NPs had a higher permeability across the BBB model and could subsequently enter midbrain-like organoids derived from healthy and Parkinson’s disease patient-specific stem cells. These results indicate that poly(l-glutamic acid) NPs can be used as nanocarriers for nervous system application and that the right combination of molecules that target cerebral endothelial cells, in this case alanine and glutathione, can facilitate drug delivery to the brain.

Brain organoids are invaluable tools for pathophysiological studies or drug screening, but there are still challenges to overcome in making them more reproducible and relevant. Recent advances in three-dimensional (3D) bioprinting of human neural organoids is an emerging approach that may overcome the limitations of self-organized organoids. It requires the development of optimal hydrogels, and a wealth of research has improved our knowledge about biomaterials both in terms of their intrinsic properties and their relevance on 3D culture of brain cells and tissue. Although biomaterials are rarely biologically neutral, few articles have reviewed their roles on neural cells. We here review the current knowledge on unmodified biomaterials amenable to support 3D bioprinting of neural organoids with a particular interest in their impact on cell homeostasis. Alginate is a particularly suitable bioink base for cell encapsulation. Gelatine is a valuable helper agent for 3D bioprinting due to its viscosity. Collagen, fibrin, hyaluronic acid and laminin provide biological support to adhesion, motility, differentiation or synaptogenesis and optimize the 3D culture of neural cells. Optimization of specialized hydrogels to direct differentiation of stem cells together with an increased resolution in phenotype analysis will further extend the spectrum of possible bioprinted brain disease models.

Amyloid-β (Aβ)-peptide production or deposition in the neuropathology of Alzheimer’s disease (AD) was shown to be caused by chronic inflammation that may be induced by infection, but the role of pathogenic-bacteria-related AD-associated Aβ is not yet clearly understood. In this study, we validated the hypothesis that there is a correlation between the Aβ-protein load and bacterial infection and that there are effects of bacteria, Staphylococcus aureus (S. aureus), on the Aβ load in the inflammatory environment of human tonsils. Here, we detected Aβ-peptide deposits in human tonsil tissue as well as tissue similar to tonsilloliths found in the olfactory cleft. Interestingly, we demonstrated for the first time the presence of Staphylococcus aureus (S. aureus) clustered around or embedded in the Aβ deposits. Notably, we showed that treatment with S. aureus upregulated the Aβ-protein load in cultures of human tonsil organoids and brain organoids, showing the new role of S. aureus in Aβ-protein aggregation. These findings suggest that a reservoir of Aβ and pathogenic bacteria may be a possible therapeutic target in human tonsils, supporting the treatment of antibiotics to prevent the deposition of Aβ peptides via the removal of pathogens in the intervention of AD pathogenesis.

Finding curable therapies for neurodegenerative disease (ND) is still a worldwide medical and clinical challenge. Recently, investigations have been made into the development of novel therapeutic techniques, and examples include the remote stimulation of nanocarriers to deliver neuroprotective drugs, genes, growth factors, and antibodies using a magnetic field and/or low-power lights. Among these potential nanocarriers, magneto-plasmonic nanoparticles possess obvious advantages, such as the functional restoration of ND models, due to their unique nanostructure and physiochemical properties. In this review, we provide an overview of the latest advances in magneto-plasmonic nanoparticles, and the associated therapeutic approaches to repair and restore brain tissues. We have reviewed their potential as smart nanocarriers, including their unique responsivity under remote magnetic and light stimulation for the controlled and sustained drug delivery for reversing neurodegenerations, as well as the utilization of brain organoids in studying the interaction between NPs and neuronal tissue. This review aims to provide a comprehensive summary of the current progress, opportunities, and challenges of using these smart nanocarriers for programmable therapeutics to treat ND, and predict the mechanism and future directions.

Background and Objectives: Brain organoids are self-assembled, three-dimensional (3D) aggregates generated from pluripotent stem cells. These models are useful for experimental studies on human brain development and function and are therefore increasingly used for research worldwide. As their increasing use raises several ethical questions, we aimed to assess the current state of the press on brain organoid research using a cross-sectional database to understand the extent of discussion of this subject in the public. Materials and Methods: We conducted a descriptive analysis of news reports obtained from the Nexis Uni database, searched in April 2020. After extracting the news reports, the number of published reports in each year and the included terms were analyzed. Results: Up to April 2020, 332 news reports had been published, with over half of them published in the United States and the United Kingdom, with the numbers gradually increasing every year. In total, 113 (34.0%) news reports included ethics-related keywords, and the ratio of studies before and after the study-period midpoint was significantly increased (21.0% (2013–2016) vs. 38.2% (2017–2020); p = 0.0066, Chi-square test with Yates’ continuity correction). Conclusions: Although news reports on the ethical aspects of brain organoid research have been increasing gradually, there was a bias in the region of publication. Additional studies focusing on the ethical aspects of brain organoid research should strive to assess the public perception on the subject in different parts of the world.

Brain-on-Chip (BoC) biotechnology is emerging as a promising tool for biomedical and pharmaceutical research applied to the neurosciences. At the convergence between lab-on-chip and cell biology, BoC couples in vitro three-dimensional brain-like systems to an engineered microfluidics platform designed to provide an in vivo-like extrinsic microenvironment with the aim of replicating tissue- or organ-level physiological functions. BoC therefore offers the advantage of an in vitro reproduction of brain structures that is more faithful to the native correlate than what is obtained with conventional cell culture techniques. As brain function ultimately results in the generation of electrical signals, electrophysiology techniques are paramount for studying brain activity in health and disease. However, as BoC is still in its infancy, the availability of combined BoC–electrophysiology platforms is still limited. Here, we summarize the available biological substrates for BoC, starting with a historical perspective. We then describe the available tools enabling BoC electrophysiology studies, detailing their fabrication process and technical features, along with their advantages and limitations. We discuss the current and future applications of BoC electrophysiology, also expanding to complementary approaches. We conclude with an evaluation of the potential translational applications and prospective technology developments.

Since the global outbreak of SARS-CoV-2 (COVID-19), infections of diverse human organs along with multiple symptoms continue to be reported. However, the susceptibility of the brain to SARS-CoV-2, and the mechanisms underlying neurological infection are still elusive. Here, we utilized human embryonic stem cell-derived brain organoids and monolayer cortical neurons to investigate infection of brain with pseudotyped SARS-CoV-2 viral particles. Spike-containing SARS-CoV-2 pseudovirus infected neural layers within brain organoids. The expression of ACE2, a host cell receptor for SARS-CoV-2, was sustained during the development of brain organoids, especially in the somas of mature neurons, while remaining rare in neural stem cells. However, pseudotyped SARS-CoV-2 was observed in the axon of neurons, which lack ACE2. Neural infectivity of SARS-CoV-2 pseudovirus did not increase in proportion to viral load, but only 10% of neurons were infected. Our findings demonstrate that brain organoids provide a useful model for investigating SARS-CoV-2 entry into the human brain and elucidating the susceptibility of the brain to SARS-CoV-2.

To characterize the growth of brain organoids (BOs), cultures that replicate some early physiological or pathological developments of the human brain are usually manually extracted. Due to their novelty, only small datasets of these images are available, but segmenting the organoid shape automatically with deep learning (DL) tools requires a larger number of images. Light U-Net segmentation architectures, which reduce the training time while increasing the sensitivity under small input datasets, have recently emerged. We further reduce the U-Net architecture and compare the proposed architecture (MU-Net) with U-Net and UNet-Mini on bright-field images of BOs using several data augmentation strategies. In each case, we perform leave-one-out cross-validation on 40 original and 40 synthesized images with an optimized adversarial autoencoder (AAE) or on 40 transformed images. The best results are achieved with U-Net segmentation trained on optimized augmentation. However, our novel method, MU-Net, is more robust: it achieves nearly as accurate segmentation results regardless of the dataset used for training (various AAEs or a transformation augmentation). In this study, we confirm that small datasets of BOs can be segmented with a light U-Net method almost as accurately as with the original method.

Finding curable therapies for neurodegenerative disease (ND) is still a worldwide medical and clinical challenge. Recently, investigations have been made into the development of novel therapeutic techniques, and examples include the remote stimulation of nanocarriers to deliver neuroprotective drugs, genes, growth factors, and antibodies using a magnetic field and/or low-power lights. Among these potential nanocarriers, magneto-plasmonic nanoparticles possess obvious advantages, such as the functional restoration of ND models, due to their unique nanostructure and physiochemical properties. In this review, we provide an overview of the latest advances in magneto-plasmonic nanoparticles, and the associated therapeutic approaches to repair and restore brain tissues. We have reviewed their potential as smart nanocarriers, including their unique responsivity under remote magnetic and light stimulation for the controlled and sustained drug delivery for reversing neurodegenerations, as well as the utilization of brain organoids in studying the interaction between NPs and neuronal tissue. This review aims to provide a comprehensive summary of the current progress, opportunities, and challenges of using these smart nanocarriers for programmable therapeutics to treat ND, and predict the mechanism and future directions.