Biology subjects

Smirnova, L.

Publications and source records attributed to Smirnova, L..

9 recordsLinked to original sources

bioRxiv2026

Cryopreservation of brain organoids - a tool for on-demand organoid banking

Cryopreservation offers an option for long-term storage and global distribution of complex in vitro models, yet protocols for multicellular microphysiolgocial systems (MPS) such as brain organoids/spheroids remain limited. Here, we systematically compared three commercially available cryopreservation (mFreSR, CryoStorCS10, and 3dGRO) and two freezing time points, and established a robust workflow for freezing and recovering brain organoids. After defrosting, we assessed morphology and metabolic activity. We also evaluated electrophysiology, calcium transients, and neurite outgrowth. In addition, we measured astrocyte migration, apoptosis, mitochondrial integrity, microglia survival, and neural marker expression. We found that organoids require a 4-week recovery period to regain structural and functional stability. Although organoids frozen at week 6 showed higher metabolic activity after recovery, organoids cryopreserved at week 2 had clearly better functional outcomes. They exhibited stronger spontaneous network firing and maintained calcium transients. Finally, incorporated microglia-like cells survived the freezing and displayed comparable morphology to unfrozen controls. Across the endpoints measured here, 3dGRO showed the most favorable overall performance; formal ranking across media awaits harmonized normalization, single-organoid electrophysiology, and prespecified QC thresholds. Together, these results define a practical and reproducible cryopreservation strategy that preserves key physiological features of brain organoids and supports the establishment of ready-to-use organoid banks. The ability to reliably store and distribute complex brain-like tissues represents an essential step toward global standardization, scalable experimentation, and wider adoption of human-relevant microphysiological systems. Together, these results demonstrate recovery of key physiological features in the subset of organoids that remain viable after thaw and support the feasibility of brain organoid banking.

cell biology↗

bioRxiv2025

Genetic Background and Sex Modulate Androgen Responses in Human Brain Microphysiological System

Sex steroids shape human brain development, yet the cellular and molecular consequences of androgen addition need further exploration. Here, we used a human neural organoid model - brain microphysiological system (bMPS), derived from nine induced pluripotent stem cell (iPSC) lines to model the impact of dihydrotestosterone (DHT), a potent non-aromatizable androgen. All lines differentiated reproducibly into electrically active, neuron-glia-oligodendrocyte organoids expressing robust androgen-signaling components. DHT was bioavailable, elicited nuclear androgen receptor (AR) translocation and increased organoid size in most cell lines, consistent with androgen-responsive mTOR and metabolic pathway activation. Bulk RNA-seq across nine lines revealed that transcriptional responses varied across donor backgrounds, but DHT-responsive genes converged on mitochondrial energetics, lysosomal function, glycoprotein processing, apoptosis, and mTOR signaling. Cell-type expression profiling showed an androgen-driven shift primarily in male lines toward astrocytic profiles with reductions in oligodendrocyte, oligodendrocyte progenitor (OPC), excitatory, and inhibitory neuronal signatures, supported by immunohistochemistry and AR enrichment in astrocytes and OPCs. DHT also altered neurodevelopmental pathways, increasing variation in synaptic pruning and decreasing variation in neuronal migration, with autism spectrum disorder (ASD) diagnosis and seizure status of donors moderating these effects more strongly than sex. Baseline transcriptional differences distinguished iPSC lines which responded more strongly to DHT from weak responders: responders displayed enhanced synaptic maturity and reduced ECM gene expression. Using isogenic XX/XY lines, we found that differences in sex-chromosome expression exceeded DHT-induced changes and that DHT decreased expression of inhibitory neuron genes in males and increased it in females. Finally, DHT induced extensive DNA methylation changes, targeting HOX genes, patterning, and synaptic genes. Collectively, these findings reveal that androgen signaling shapes transcription, cell populations, and epigenetic landscapes in a genetic background-dependent manner. This work contributes to understanding how androgens influence human brain development and highlights how in vitro models can contribute to representing inter-individual variability in neurodevelopment and neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Computational modeling of necrosis in neural organoids

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 Alzheimers disease, microcephaly, and autism. There are many protocols to generate NOs with different complexities and sizes, varying from 400 m 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 O2 starvation-induced necrosis in NOs using the Damkohler 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 m. 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.

bioengineering↗

bioRxiv2025

Modeling Organoid Population Electrophysiology Dynamics

Improving models to investigate neurodegenerative disease, neurodevelopmental disease, neuro-toxicology and neuropharmacology is critical to improve our basic understanding of the human nervous system, as well as to accelerate discovery of interventions and drugs. Improved models of the human central nervous system could enable critical discoveries related to functional changes induced by sensory stimulation or toxic exposures. Neural organoids, complex three-dimensional cell cultures derived from adult human stem cells, have been grown with complex connectivity and neuroanatomy. Moreover, these cultures have been interfaced with bi-directional electrical stimulation and recording, as well as chemical stimulation. This effort sought to develop new computational techniques which could be applied to comparative studies using neural organoids. In particular, we adapted CEBRA, a state of the art model from the in vivo modeling literature, to generate 2D and 3D embeddings (projections into structured low-dimensional spaces) of high dimensional neural organoid electrophysiology data. This can be done in an unsupervised or semi-supervised manner. Results indicate these embeddings can be quickly and reliably generated and serve as a low-dimensional, interpretable embeddings for characterizing changes in neural organoid activity over time, as well as clustering results around known bursting phenomena. Moreover, we demonstrate that mixtures of von Mises-Fisher distributions can be used as a parametric model for these embedding spaces to enable statistics hypothesis testing. This technique may enable new types of comparative studies using neural organoids, and may be critical for creating a representation for quantitative comparison and validation of neural organoid models against human and animal data. Looking ahead, this work could allow the formulation of a new class of experiments investigating the functional impact of toxins, genetic manipulations, or pharmacological interventions on human neurons.

neuroscience↗

bioRxiv2025

Neuromodulation in neural organoids with shell MEAs

Neural organoids (NOs) have emerged as important tissue engineering models for brain sciences and biocomputing. Establishing reliable relationships between stimulation and recording traces of electrical activity is essential to monitor the functionality of NOs, especially as it relates to realizing biocomputing paradigms such as reinforcement learning or stimulus discrimination. While researchers have demonstrated neuromodulation in NOs, they have primarily used 2D microelectrode arrays (MEAs) with limited access to the entire 3D contour of the NOs. Here, we report neuromodulation using tiny mimics of macroscale EEG caps or shell MEAs. Specifically, we observe that stimulating current within a specific range (20 to 30 {micro}A) induced a statistically significant increase in neuron firing rate when comparing the activity five seconds before and after stimulation. We observed neuromodulatory behavior using both three- and 16-electrode shells and could generate 3D spatiotemporal maps of neuromodulatory activity around the surface of the NO. Our studies demonstrate a methodology for investigating 3D spatiotemporal neuromodulation in organoids of broad relevance to biomedical engineering and biocomputing. One-Sentence SummaryNeuromodulation, an essential intelligence feature, was observed using 3D stimulation and recording from neural organoids.

bioengineering↗

bioRxiv2024

Human Neural Organoid Microphysiological Systems Show the Building Blocks Necessary for Basic Learning and Memory

Brain Microphysiological Systems including neural organoids derived from human induced pluripotent stem cells offer a unique lens to study the intricate workings of the human brain. This paper investigates the foundational elements of learning and memory in neural organoids, also known as Organoid Intelligence by quantifying immediate early gene expression, synaptic plasticity, neuronal network dynamics, and criticality to demonstrate the utility of these organoids in basic science research. Neural organoids showed synapse formation, glutamatergic and GABAergic receptor expression, immediate early gene expression basally and evoked, functional connectivity, criticality, and synaptic plasticity in response to theta-burst stimulation. In addition, pharmacological interventions on GABAergic and glutamatergic receptors, and input specific theta-burst stimulation further shed light on the capacity of neural organoids to mirror synaptic modulation and short-term potentiation, demonstrating their potential as tools for studying neurophysiological and neurological processes and informing therapeutic strategies for diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/613333v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@50a256org.highwire.dtl.DTLVardef@1d1f8c9org.highwire.dtl.DTLVardef@24688borg.highwire.dtl.DTLVardef@4ba3b4_HPS_FORMAT_FIGEXP M_FIG C_FIG Overview of the main components of the experiments conducted. Figure created using BioRender.com.

neuroscience↗

bioRxiv2024

Glia-enriched stem-cell 3D model of the human brain mimics the glial-immune neurodegenerative phenotypes of multiple sclerosis

The role of central nervous system (CNS) glia in sustaining self-autonomous inflammation and driving clinical progression in multiple sclerosis (MS) is gaining scientific interest. We applied a single transcription factor (SOX10)-based protocol to accelerate oligodendrocyte differentiation from hiPSC-derived neural precursor cells, generating self-organizing forebrain organoids. These organoids include neurons, astrocytes, oligodendroglia, and hiPSC-derived microglia to achieve immunocompetence. Over 8 weeks, organoids reproducibly generated mature CNS cell types, exhibiting single-cell transcriptional profiles similar to the adult human brain. Exposed to inflamed cerebrospinal fluid (CSF) from MS patients, organoids properly mimic macroglia-microglia neuro-degenerative phenotypes and intercellular communication seen in chronic active MS. Oligodendrocyte vulnerability emerged by day 6 post-MS-CSF exposure, with nearly 50% reduction. Temporally-resolved organoid data support and expand on the role of soluble CSF mediators in sustaining downstream events leading to oligodendrocyte death and inflammatory neurodegeneration. Such findings support implementing this organoid model for drug screening to halt inflammatory neurodegeneration.

neuroscience↗

bioRxiv2023

A novel approach to increase glial cell populations in brain microphysiological systems

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.

cell biology↗

bioRxiv2022

Shell Microelectrode Arrays (MEAs) for brain organoids

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 are widely utilized in biomedical engineering. 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 (EEG) caps, we developed miniaturized chip-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 400-600 {micro}m sized organoids for up to four weeks and in response to glutamate stimulation. Our studies suggest that 3D shell MEAs offer significant potential for high signal-to-noise and 3D spatiotemporal brain organoid recording.

bioengineering↗