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Establishing a Microfluidic Co-Culture Blood-Brain Barrier Model Using Human Brain Endothelial Cells and Astrocytes.
The blood-brain barrier (BBB) is a highly selective physiological interface that regulates molecular transport between the bloodstream and the central nervous system. In vitro BBB models are valuable tools for studying endothelial barrier formation, cell-cell interactions, and responses to dynamic flow conditions. This protocol describes the establishment of a three-dimensional microfluidic co-culture BBB model using human brain endothelial cells and primary human astrocytes cultured in separate but interconnected compartments of a microfluidic chip. The protocol includes chip preparation, extracellular matrix coating, endothelial and astrocyte seeding, introduction of programmable shear flow, transendothelial electrical resistance (TEER) measurement, fixation, immunofluorescent staining, and confocal imaging. Shear flow is applied to the endothelial compartment using programmable syringe pumps to support endothelial alignment and tight junction formation under dynamic culture conditions. Barrier formation is evaluated using TEER measurements and immunofluorescent characterization of endothelial and astrocytic markers, including cluster of differentiation 31, zonula occludens-1, and glial fibrillary acidic protein. This methodology provides a reproducible workflow for establishing a microfluidic endothelial-astrocyte co-culture model suitable for studying BBB-associated cellular interactions and barrier formation under controlled flow conditions. The protocol is intended to support reproducible implementation of BBB chip workflows by providing detailed guidance for device preparation, cell culture, flow setup, imaging, and data acquisition.
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Retinal organoid chip: engineering a physiomimetic oxygen gradient for optimizing long term culture of human retinal organoids.
An oxygen gradient across the retina plays a crucial role in its development and function. The inner retina resides in a hypoxic environment (2% O2) adjacent to the vitreous cavity. Oxygenation levels rapidly increase towards the outer retina (18% O2) at the choroid. In addition to retinal stratification, oxygen levels are critical for the health of retinal ganglion cells (RGCs), which relay visual information from the retina to the brain. Human stem cell derived retinal organoids are being engineered to mimic the structure and function of human retina for applications such as disease modeling, development of therapeutics, and cell replacement therapies. However, rapid degeneration of the retinal ganglion cell layers are a common limitation of human retinal organoid platforms. We report the design of a novel retinal organoid chip (ROC) that maintains a physiologically relevant oxygen gradient and allows the maturation of inner and outer retinal cell phenotypes for human retinal organoids. Our PDMS-free ROC holds 55 individual retinal organoids that were manually seeded, cultured for extended periods (over 150 days), imaged in situ, and retrieved. ROC was designed from first principles of liquid and gas mass transport, and fabricated from biologically- and chemically inert materials using rapid prototyping techniques such as micromachining, laser cutting, 3D printing and bonding. After computational and experimental validation of oxygen gradients, human induced pluripotent stem cell derived retinal organoids were transferred into the ROC, differentiated, cultured and imaged within the chip. ROCs that maintained active perfusion and stable oxygen gradients were successful in inducing higher viability of RGCs within retinal organoids than static controls, or ROC without oxygen gradients. Our physiologically relevant and higher-throughput retinal organoid culture system is well suited for applications in studying developmental perturbations to primate retinogenesis, including those driven by inherited traits, fetal environmental exposure to toxic agents, or acquired by genetic mutations, such as retinoblastoma.
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Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.
BACKGROUND: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. METHODS: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. RESULTS: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. CONCLUSION: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.
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Establishment of a Novel IDH1 wild type Glioblastoma Cell Line, SHG142, and Its Organoids.
BACKGROUND: Glioblastoma (GBM) is the most aggressive primary malignant brain tumor and is associated with limited treatment options and poor prognosis. Conventional GBM cell lines undergo genetic drift and progressive divergence from patient-relevant molecular features over long-term culture, limiting their translational relevance. This study aimed to establish and characterize a novel isocitrate dehydrogenase 1 (IDH1)-wildtype GBM cell line, SHG142, along with its associated glioma stem-like cells (GSCs) and organoid models, to advance GBM biology and therapeutic research. METHODS: Primary tumor tissue from a 70-year-old female patient was cultured under standard conditions to generate the SHG142 cell line, which was authenticated by short tandem repeat (STR) profiling. Phenotypic and genetic features were evaluated using immunofluorescence (IF), karyotyping, and whole-exome sequencing (WES) including a concordance analysis between an early bulk primary culture (P2) and late-passage SHG142 (P50). GSCs were isolated via serum-free culture and magnetic-activated cell sorting (MACS). Tumorigenicity and invasiveness were assessed through intracranial xenografts in nude mice (n = 6/group) and coculture with brain organoids derived from human embryonic stem cells (n = 4 independent organoids). RESULTS: SHG142 cells exhibited stable morphology and proliferation over more than 50 passages, retained key GBM markers (e.g., Nestin, Glial Fibrillary Acidic Protein (GFAP), O6-methylguanine-DNA methyltransferase (MGMT), Ki-67, P53, S-100, CD34), and exhibited chromosomal instability (+7, -10, and t(9;13)). WES revealed mutations in the TERT promoter, TP53 and NF1 and supported lineage continuity from P2 to P50 through shared SNVs and conserved driver events. CD133+ SHG142 GSCs demonstrated stem-like properties and invasive behavior in vivo and in organoid coculture. Compared with xenografts derived from SHG142 cells, GSC-derived xenografts were associated with increased malignancy and shorter survival (median survival 25 vs. 31.5 days, p = 0.025). The organoid coculture model confirmed deep infiltration by proliferative, stem-like tumor cells. CONCLUSIONS: SHG142 and its GSCs represent a phenotypically stable, genetically defined GBM model with evidence of lineage continuity and retention of key driver events during in vitro propagation. Their tumorigenic and invasive properties make them valuable tools for mechanistic studies and preclinical therapeutic screening, supported by initial two-dimensional and three-dimensional drug response readouts that enable first-pass evaluation of therapeutic response.
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Protruding cantilever microelectrode array to monitor the inner electrical activity of cerebral organoids.
Stem cell-derived cerebral organoids are artificially grown miniature organ-like structures mimicking embryonic brain architecture. They are composed of multiple neural cell types with 3D cell layer organization exhibiting local field potential. Measuring the extracellular electrical activity by means of conventional planar microelectrode arrays is particularly challenging due to the 3D architecture of organoids. In order to monitor the intra-organoid electrical activity of thick spheroid-shaped samples, we developed long protruding microelectrode arrays able to penetrate the inner regions of cerebral organoids to measure the local potential of neurons within the organoids. A new microfabrication process has been developed which, thanks to the relaxation of internal stresses of a stack of materials deposited over a sacrificial layer, allows one to build a protruding cantilever microelectrode array placed at the apex of beams which rise vertically, over two hundred microns. These slender beams inserted deeply into the organoids give access to the recording of local field potential from neurons buried inside the organoid. This novel device shall provide valuable tools to study neural functions in greater detail.
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A compatible gravity-driven organoid perfusion (GDOP) platform for drug screening with sensitivity and toxicity process evaluation.
High-throughput experiments, unidirectional fluid replacement, real-time process monitoring, and simultaneous drug sensitivity and toxicity tests are hard to achieve on most existing tumor organoid chips. Here, we developed a gravity-driven organoid perfusion (GDOP) platform facilitating scalable throughput and supporting drug sensitivity and toxicity assessment on organoids. The unidirectional perfusion capability and optimized operational parameters of the GDOP chip were validated through fluid dynamics simulations. Using this platform, we successfully established uniform on-chip triple-negative breast cancer (TNBC) organoids, with endpoint detection results aligning closely with clinical diagnosis. Throughout the drug treatment process, we monitored and then analyzed the morphological and grayscale changes of the organoids. The sensitivity and toxicity tests revealed the optimal concentration range for the 3 chemotherapeutic drugs. In addition, on-chip brain organoids were established, which lays a feasible foundation for future drug toxicity tests of complex organoids. The GDOP platform, combined with its integrated evaluation method, provides a powerful and reliable approach for advancing organoid-based researches.
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Organ-on-a-Chip Approach for Accelerating Blood-Brain Barrier Nanoshuttle Discovery.
Organ-on-a-chip, which recapitulates the dynamics of in vivo vasculature, has emerged as a promising platform for studying organ-specific vascular beds. However, its practical advantages in identifying vascular-targeted drug delivery systems (DDS) over traditional in vitro models remain underexplored. This study demonstrates the reliability and efficacy of the organ-on-a-chip in screening efficient DDS by comparing its performance with that of a conventional transwell, both designed to simulate the blood-brain barrier (BBB). The BBB nanoshuttles discovered through BBB Chip-based screening demonstrated superior functionality in vivo compared to those identified using transwell methods. This enhanced effectiveness is attributed to the BBB Chip's accurate replication of the structure and dynamics of the endothelial glycocalyx, a crucial protective layer within blood vessels, especially under shear stress. This capability of the BBB Chip has enabled the identification of molecular shuttles that efficiently exploit the endothelial glycocalyx, thereby enhancing transendothelial transport efficacy. Our findings suggest that organ-on-a-chip technology holds considerable promise for advancing research in vascular-targeted DDS due to its accurate simulation of molecular transport within endothelial systems.
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Perfused brain slice-on-chip platforms: a systematic review of long-term organotypic hippocampal tissue culture.
Organ-on-chip (OoC) platforms are increasingly used to replicate structural, functional and molecular features of native tissue within controlled microenvironments. While most current brain-on-chip (BoC) systems rely on 2D cultures or 3D stem cell-derived constructs, the integration of intact brain tissue slices-particularly organotypic explants of the central nervous system-offers distinct advantages by preserving native cytoarchitecture, synaptic connectivity, and regional specificity. This systematic review aimed to identify and critically assess OoC platforms that incorporate ex vivo brain tissue slices maintained under dynamic perfusion for extended periods (≥10 days in vitro). A structured PubMed search conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines identified 2796 records, of which 7 studies met the predefined inclusion criteria. All included studies employed organotypic brain slices derived from early postnatal rodents and integrated them into perfused microfluidic systems. Most platforms combined air-liquid interface culture with low-volume perfusion to support prolonged tissue viability and partial functional maintenance, including electrophysiological activity, preserved structural integrity, and molecular homeostasis. Notably, none of the identified studies reported the successful long-term cultivation of adult rodent or human brain tissue in a comparable perfused OoC configuration, underscoring a major limitation of current approaches. Moreover, assessment of tissue viability and function was heterogeneous and frequently relied on descriptive or insufficiently sensitive readouts, limiting cross-platform comparisons and translational relevance. Future development of BoC technologies should prioritize improved microenvironmental control, the integration of suitable biomaterials, and embedded monitoring strategies capable of assessing metabolic state and circuit-level function. Addressing these challenges will be essential for advancing OoC platforms towards physiologically meaningful and translationally relevant applications in neuroscience.
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Generation of Neural Organoids and Their Application in Disease Modeling and Regenerative Medicine.
The complexity and precision of the human nervous system have posed significant challenges for researchers seeking suitable models to elucidate refractory neural disorders. Traditional approaches, including monolayer cell cultures and animal models, often fail to replicate the intricacies of human neural tissue. The advent of organoid technology derived from stem cells has addressed many of these limitations, providing highly representative platforms for studying the structure and function of the human embryonic brain and spinal cord. Researchers have induced neural organoids with regional characteristics by mimicking morphogen gradients in neural development. Recent advancements have demonstrated the utility of neural organoids in disease modeling, offering insights into the pathophysiology of various neural disorders, as well as in the field of neural regeneration. Developmental defects in neural organoids due to the lack of microglia or vascular systems are addressed. In addition to induction methods, microfluidics is used to simulate the dynamic physiological environment; bio-manufacturing technologies are employed to regulate physical signaling and shape the structure of complex organs. These technologies further expand the construction strategies and application scope of neural organoids. With the emergence of new material paradigms and advances in AI, new possibilities in the realm of neural organoids are witnessed.
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Engineering vascularized inner ear organoids: Challenges and advances in recapitulating the cochlear microenvironment.
The inner ear is a highly specialized structure responsible for auditory and vestibular functions; however, research progress has been limited by its deep anatomical location and the difficulty of obtaining human tissue samples. Recent advances in inner ear organoids (IEOs) and organoid-on-a-chip technologies offer powerful platforms for studying hearing loss mechanisms and testing therapies. This review outlines progress in generating IEOs from human pluripotent stem cells, including differentiation into hair cells and spiral ganglion neuron-like cells, and improvements in 3D culture, co-culture, and microfluidics. Special attention is given to the reconstruction of the blood-labyrinth barrier (BLB) within organoids, focusing on vascular formation, barrier integrity, and drug delivery limitations. Drawing from advances in brain and kidney organoid models, we explore strategies to improve vascularization using microfluidic technologies. Ultimately, building vascularized IEOs that recapitulate the cochlear microenvironment offers significant potential for drug screening, disease modeling, and regenerative therapies.