Brain organoids are important models for mimicking some three-dimensional (3D) cytoarchitectural and functional aspects of the brain. Multielectrode arrays (MEAs) that enable recording and stimulation of activity from electrogenic cells offer notable potential for interrogating brain organoids. However, conventional MEAs, initially designed for monolayer cultures, offer limited recording contact area restricted to the bottom of the 3D organoids. Inspired by the shape of electroencephalography caps, we developed miniaturized wafer-integrated MEA caps for organoids. The optically transparent shells are composed of self-folding polymer leaflets with conductive polymer–coated metal electrodes. Tunable folding of the minicaps’ polymer leaflets guided by mechanics simulations enables versatile recording from organoids of different sizes, and we validate the feasibility of electrophysiology recording from 400- to 600-μm-sized organoids for up to 4 weeks and in response to glutamate stimulation. Our studies suggest that 3D shell MEAs offer great potential for high signal-to-noise ratio and 3D spatiotemporal brain organoid recording.

Brain microphysiological systems (bMPS) recapitulate human brain cellular architecture and functionality more closely than traditional monolayer cultures and have become increasingly relevant for the study of neurological function in health and disease. Existing 3D brain models vary in reflecting the relative populations of different cell types present in the human brain. Most models consist mainly of neurons, while glial cells represent a smaller portion of the cell populations. Here, by means of a chemically defined glial‐enriched medium (GEM), an improved method to expand the population of astrocytes and oligodendrocytes without compromising neuronal differentiation in bMPS, is presented. An important finding is that astrocytes also change in morphology when cultured in GEM, more closely recapitulating primary culture astrocytes. GEM bMPS are electro‐chemically active and show different patterns of calcium staining and flux. Synaptic vesicles and terminals observed by electron microscopy are also present. No significant changes in neuronal differentiation are observed by gene expression, however, GEM enhanced neurite outgrowth and cell migration, and differentially modulated neuronal maturation in two different cell lines. These results have the potential to significantly improve functionality of bMPS for the study of neurological diseases and drug discovery, contributing to the unmet need for safe human models.

It took decades for the neuron-centric approach in neuroscience to appreciate the critical role that astrocytes play in health and disease. Now recognized as key regulators of brain function 1,2 , astrocytes' multifaceted roles mediate cellular crosstalk with neurons 3 , oligodendrocytes 4 , and microglia 5 , actively influencing human learning and behavior 2,6 . Underlying astrocytes' capacity to maintain balance and respond promptly is a flexible system of gene regulation, driven by a dynamic epigenetic landscape that adapts to immediate needs and shifts across a lifespan 7 . Dysregulation of these malleable epigenetic programs may predispose the brain to pathological states. An emerging body of research suggests astrocytes play a central causative role in the pathology of neurodevelopmental and neurodegenerative disorders 8 . In conditions such as autism spectrum disorder and Rett syndrome, alterations in astrocytic DNA methylation and histone modifications impact neuronal circuitry and plasticity 9 . Similarly, in neurodegenerative diseases, shifts in astrocytic chromatin led to inflammatory and metabolic dysfunction 8,10 .We propose that astrocyte epigenetics offers a unifying framework in neuroscience; one that connects brain development, plasticity, and disease vulnerability, while highlighting upstream and potentially reversible mechanisms that could inform future therapeutic strategies.Astrocytes are architects of brain development and function 11,12 . They integrate developmental cues with adaptive responses, positioning them as central modulators of neural circuit formation and long-term brain function, contributing directly to the plasticity of learning and regenerative capacity 13,14 .Astrocyte diversity may be best understood as a purposeful organizational feature of the CNS rather than a structural coincidence. The fact that protoplasmic and fibrous astrocytes occupy distinct gray and white matter domains, while specialized forms such as radial, Bergmann, Müller, velate, surface-associated, Gomori, and pituicytes arise in specific regions, suggests that astrocytes are tailored to the unique physiological demands of their local environments 15,16 . The discovery of additional region-specific molecular subtypes through single-cell transcriptomic profiling strengthens this view, showing that astrocytes are not uniform support cells but exhibit spatially patterned gene expression programs that likely correspond to functional specialization 1,[17][18][19] .Given this deep functional integration, we propose that astrocyte epigenetics be prioritized as a central regulatory layer shaping developmental trajectories and cognitive outcomes.3.1. Epigenetic immune memory Astrocytic epigenetic immune memory is the ability of astrocytes to "remember" immune challenges, leading to chronic reactive states that amplify neuroinflammatory responses upon reexposure 20 . These states accelerate chronic neuroinflammation and disease progression in conditions such as AD, PD, ALS, and MS 8,20,21 . This immune memory is encoded in epigenetic mechanisms that alter chromatin structure and gene expression. Multiple lines of evidence from single-cell genomics, functional perturbation experiments, and in vivo disease models converge to demonstrate that astrocytes acquire persistent reactive states encoding prior inflammatory exposures 20 . Reactive astrocytes acquire a memory of inflammatory insults through ACLY-and p300-mediated DNA methylation changes. A consistent feature is cytokine-driven chromatin rewiring, particularly through IL-1a, TNF, and C1q, which reshapes astrocytic identity and function 20,[22][23][24][25] . These epigenetic changes profoundly alter astrocytic homeostasis, disrupting transitions into and out of reactive states, thereby contributing to disease progression 20,[26][27][28][29] . For example, cell-specific CRISPR/Cas9 targeting ACLY+p300 in astrocytes in the EAE animal model of MS significantly reduced disease severity 20 . CRISPRi (CRISPR interference) screens in human iPSC-derived astrocytes identified canonical NF-kB activation as a driver of inflammatory states, followed by IL-6 and interferon signaling 30 . Similarly, viewing reactivity as a spectrum rather than a binary A1/A2 switch better captures how astrocytes tune transcriptional programs to inflammatory cues 31,32 . This plasticity is rooted in development, as astrocytes transition from radial glia via gliogenic signals and intrinsic epigenetic programs, including DNMT1-mediated repression and later STAT3-p300/CBP-dependent activation of differentiation 32 . In mature astrocytes, cytokine-driven increase in MAFG homodimers, and MAT2A-dependent DNA methylation represses NRF2 signaling and stabilizes NF-kB-associated inflammatory phenotypes, supporting inflammatory memory 31 . Thus, astrocyte identity is continuously shaped by developmental history and environmental experience, enabling functional diversity and persistent shifts towards reactive states when inflammation is sustained 1,18 .Collectively, these findings show that persistent reactive states are not incidental, but encoded, reversible, and therapeutically actionable, arguing for systematic efforts to map, modulate, and reset epigenetic memory as a strategy to halt chronic neuroinflammation.Neurodevelopmental disorders are characterized by impaired brain maturation, disrupted neural connectivity, and cognitive and behavioral deficits. Although neurons have long dominated mechanistic discussions, astrocytes are key contributors to disease pathogenesis. Astrocyte development is orchestrated by dynamic chromatin remodeling, DNA methylation, and histone modifications that establish cell identity and regional specialization. Disrupting these epigenetic programs produces lasting alteration in astrocyte function 33,34 . Proper maturation requires the sequential activation of transcription factors that drive the transition from an immature to a mature...

Neural organoids (NOs), also known as brain organoids, are derived from human-induced pluripotent stem cells and are Microphysiological Systems (MPS) of the brain that can recapitulate key aspects of neurodevelopment. They enable in vitro studies of brain development and disease mechanisms, providing disease models for various neurodegenerative or neurodevelopmental/degenerative disorders like Alzheimer’s disease, microcephaly, and autism. There are many protocols to generate NOs with different complexities and sizes, varying from 400 μm to several mm in diameter, with a starvation-induced necrotic core eventually forming depending on the diameter and culture conditions. Thus, they can benefit from vascularization and more optimal culture conditions. There have been several attempts to decrease necrosis while growing larger NOs, such as by using orbital shaking or 2D/3D microfluidic chips, but only with limited success. In this study, we describe a 3D finite element model to simulate O ₂ starvation-induced necrosis in NOs using the Damköhler Number ( Da ) and the Michaelis-Menten kinetics. We measured the necrotic areas in NOs using fluorescent imaging and used them to calibrate the model with a specific Da . Using these calibrated values, we systematically compared simulations of different NO culture methods—static, orbital shaking, and microfluidic flow around organoids—highlighting their relative impacts on nutrient diffusion and necrosis. We observed that these culture strategies cannot prevent necrosis beyond a diameter of ∼800 μm. Based on these findings, we propose that 3D spatial perfusion, achieved through uniformly distributed fluidic capillaries within the NO, could significantly reduce necrosis. We conducted parametric studies on capillary spacing, density, and layout. Our calibrated model offers insights for designing next-generation microfabricated bioreactors and culture devices, not just for NOs but also for all 3D tissue engineering and organoid research.

Brain microphysiological systems (bMPS), which recapitulate human brain cellular architecture and functionality more closely than traditional monolayer cultures, have become a practical, non-invasive, and increasingly relevant platform for the study of neurological function in health and disease. These models include 3D spheroids and organoids as well as organ-on-chip models. Currently, however, existing 3D brain models vary in reflecting the relative populations of the different cell types present in the human brain. Most of the models consist mainly of neurons, while glial cells represent a smaller portion of the cell populations. Here, by means of a chemically defined glial-enriched medium (GEM), we present an improved method to expand the population of astrocytes and oligodendrocytes without compromising neuronal differentiation in bMPS. An important finding is that astrocytes not only increased in number but also changed in morphology when cultured in GEM, more closely recapitulating primary culture astrocytes. We demonstrate oligodendrocyte and astrocyte enrichment in GEM bMPS using a variety of complementary methods. We found that GEM bMPS are electro-chemically active and showed different patterns of Ca ⁺² staining and flux. Synaptic vesicles and terminals observed by electron microscopy were also present. No significant changes in neuronal differentiation were observed by gene expression, however, GEM enhanced neurite outgrowth and cell migration, and differentially modulated neuronal maturation in two different iPSC lines. Our results have the potential to significantly improve in vivo-like functionality of bMPS for the study of neurological diseases and drug discovery, contributing to the unmet need for safe human models.