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Understanding Primary Blast Injury: High Frequency Pressure Acutely Disrupts Neuronal Network Dynamics in Cerebral Organoids.
Blast exposure represents a common occupational risk capable of generating mild to severe traumatic brain injuries (TBI). During blast exposure, a pressure shockwave passes through the skull and exposes brain tissue to complex pressure waveforms. The primary neurophysiological response to blast-induced pressure waveforms remains poorly understood. Here, we use a computer-controlled table-top pressure chamber to expose human stem cell-derived cerebral organoids to varied frequency of pressure waves and characterize the neurophysiological response. Pressure waves that reach a maximum amplitude of 250 kPa were used to model a less severe TBI and 350 kPa for a more severe blast TBI event. With each amplitude, a frequency range of 500 Hz, 3000 Hz, and 5000 Hz was tested. Following the 250 kPa overpressure a multi-electrode array recorded organoid neural activity. We observed an acute suppression neuronal activity in single unit events, population events, and network oscillations that recovered within 24 h. Additionally, we observed a network desynchronization after exposure higher frequency waveforms. Conversely, organoids exposed to higher amplitude pressure (350k Pa) displayed drastic neurophysiological differences that failed to recover within 24 h. Further, lower amplitude "blast" (250 kPa) did not induce cellular damage whereas the higher amplitude "blast" (350 kPa) generated greater apoptosis throughout each organoid. Our data indicate that specific features of pressure waves found intracranially during blast TBI have varied effects on neurophysiological activity that can occur even without cellular damage.
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Patterning Networks of Grade IV Glioblastoma on Silicon Chip.
Glioblastoma (GBM) is the most aggressive high-grade brain cancer with a median survival time of <15 months. Due to GBMs fast and infiltrative growth patient prognosis is poor with recurrence after treatment common. Investigating GBMs ability to communicate, specifically via Ca2+ signaling, within its functional tumour networks may unlock new therapeutics to reduce the rapid infiltration and growth which currently makes treatment ineffective. This work aims to produce patterned networks of GBM cells such that the Ca2+ communication at a network level can be repeatedly and reliably investigated.
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Deciphering pericyte-induced temozolomide resistance in glioblastoma with a 3D microphysiological system mimicking the biomechanical properties of brain tissue.
Glioblastoma (GBM) is a highly aggressive malignancy with a poor prognosis and frequent resistance to temozolomide (TMZ), the standard-of-care chemotherapy. The complex mechanisms underlying GBM chemoresistance, particularly the role of pericytes, remain poorly understood due to the lack of physiologically relevant in vitro models replicating the complex tumor microenvironment (TME). Here, we present a biomimetic 3D GBM microphysiological system that replicates the biomechanical properties of brain tissue (G'∼800Pa, G"∼100Pa) and enables the study of pericyte-mediated TMZ resistance. GBM spheroids (U87, LN229, PDM140) were cultured alone or co-cultured with pericytes in a composite hydrogel for 14 days and remained viable and proliferative. In response to TMZ, PDM140 was the most sensitive (IC50=73μM), followed by LN229 (IC50=278μM) and U87 (IC50=446μM). Co-culture with pericytes significantly increased GBM spheroid viability by 22.7% (PDM140), 32.5% (LN229), and 22.1% (U87), confirming pericyte-induced TMZ resistance. Notably, pericytes exhibited a 160-fold upregulation of C-C motif chemokine ligand 5 (CCL5) upon TMZ treatment, implicating the CCL5-mediated pathway in chemoresistance. This innovative brain-mimicking 3D GBM model provides a physiologically relevant platform for studying tumor-pericyte interactions and testing therapeutic strategies targeting CCL5-mediated resistance mechanisms in GBM. STATEMENT OF SIGNIFICANCE: We developed a multicellular 3D glioblastoma microphysiological system mimicking the physicochemical properties of brain tissues and pericyte-mediated TMZ resistance that can be used to screen for standard-of-care chemotherapy. This advanced hydrogel-based platform demonstrated the critical role of the glioblastoma tumor microenvironment in modulating chemotherapy sensitivity, particularly the pericyte-induced CCL5-CCR5 paracrine axis that can lead to the identification of therapeutic targets within the CCL5-CCR5 pathway toward more effective treatments disrupting these resistance mechanisms. Overall, the proposed 3D glioblastoma microphysiological system can transform drug screening and personalized treatment for GBM by offering ethical and cost-effective alternatives to animal testing and more effective drug screening and discovery efforts, ultimately improving GBM patient outcomes.
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Individualized patient tumor organoids faithfully preserve human brain tumor ecosystems and predict patient response to therapy.
Tumor organoids are important tools for cancer research, but current models have drawbacks that limit their applications for predicting response to therapy. Here, we developed a fast, efficient, and complex culture system (IPTO, individualized patient tumor organoid) that accurately recapitulates the cellular and molecular pathology of human brain tumors. Patient-derived tumor explants were cultured in induced pluripotent stem cell (iPSC)-derived cerebral organoids, thus enabling culture of a wide range of human tumors in the central nervous system (CNS), including adult, pediatric, and metastatic brain cancers. Histopathological, genomic, epigenomic, and single-cell RNA sequencing (scRNA-seq) analyses demonstrated that the IPTO model recapitulates cellular heterogeneity and molecular features of original tumors. Crucially, we showed that the IPTO model predicts patient-specific drug responses, including resistance mechanisms, in a prospective patient cohort. Collectively, the IPTO model represents a major breakthrough in preclinical modeling of human cancers, which provides a path toward personalized cancer therapy.
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Translational applicability of human blood-brain barrier spheroid models for the development of brain-penetrant therapeutic antibodies.
Drug delivery systems play a key role in the development of new therapeutic strategies for brain diseases. Antibodies capable of crossing the blood–brain barrier (BBB) have attracted considerable attention as promising drug carriers. To facilitate their development, we previously established human multi-cellular spheroidal (hMCS)-BBB models as microphysiological system-based platforms. In this study, we aimed to enhance the applicability of these hMCS-BBB models for the development of brain-permeable antibodies using four anti-human transferrin receptor monoclonal antibodies (hTfRMAbs). Permeability assays using hMCS-BBB models revealed BBB permeability in three out of four hTfRMAbs, with two antibodies exhibiting Km values of 6.5 and 12.0 µg/mL, respectively. Importantly, differences in the BBB permeability of tested hTfRMAbs were consistent with the brain permeability profiles reported in monkeys in vivo. Moreover, these differences were not detected via simple uptake assays using monoculture brain microvascular endothelial cells, highlighting the superior evaluation potential of hMCS-BBB models. Lysosome co-localization assays using hMCS-BBB models revealed the distinct intracellular localization patterns of BBB-permeable and non-permeable hTfRMAbs with lysosomes, presumably explaining their different permeability properties. Overall, hMCS-BBB models effectively characterized the BBB permeability of hTfRMAbs, demonstrated strong potential for in vivo extrapolation, and proved suitable for mechanistic studies of the cellular pathways regulating their BBB permeability. Therefore, hMCS-BBB models are considered promising tools for advancing the development of brain-permeable therapeutic antibodies.
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Development of Colonic Organoids Containing Enteric Nerves or Blood Vessels from Human Embryonic Stem Cells.
The increased interest in organoid research in recent years has contributed to an improved understanding of diseases that are currently untreatable. Various organoids, including kidney, brain, retina, liver, and spinal cord, have been successfully developed and serve as potential sources for regenerative medicine studies. However, the application of organoids has been limited by their lack of tissue components such as nerve and blood vessels that are essential to organ physiology. In this study, we used three-dimensional co-culture methods to develop colonic organoids that contained enteric nerves and blood vessels. The development of enteric nerves and blood vessels was confirmed phenotypically and genetically by the use of immunofluorescent staining and Western blotting. Colonic organoids that contain essential tissue components could serve as a useful model for the study of colon diseases and help to overcome current bottlenecks in colon disease research.
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Microfluidic organoids-on-a-chip: The future of human models.
Microfluidics opened the possibility to model the physiological environment by controlling fluids flows, and therefore nutrients supply. It allows to integrate external stimuli such as electricals or mechanicals and in situ monitoring important parameters such as pH, oxygen and metabolite concentrations. Organoids are self-organized 3D organ-like clusters, which allow to closely model original organ functionalities. Applying microfluidics to organoids allows to generate powerful human models for studying organ development, diseases, and drug testing. In this review, after a brief introduction on microfluidics, organoids and organoids-on-a-chip are described by organs (brain, heart, gastrointestinal tract, liver, pancreas) highlighting the microfluidic approaches since this point of view was overlooked in previously published reviews. Indeed, the review aims to discuss from a different point of view, primary microfluidics, the available literature on organoids-on-a-chip, standing out from the published literature by focusing on each specific organ.
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Investigating the FLASH Effect in a Rat Brain Organotypic Model With a Novel High-Energy Electron Beam.
PURPOSE: Ultrahigh dose rate (FLASH) radiation therapy is reported to reduce normal tissue toxicity while maintaining tumor control; however, mechanism(s) remain obscure. To study FLASH mechanisms in brain tissue, we developed a novel experimental platform featuring a specialized high-energy electron linear accelerator, High Intensity Gamma Ray Source (HIGS), paired with an organotypic ex vivo brain metastasis model. METHODS AND MATERIALS: We varied interpulse spacing to modulate the mean dose rate (MDR) of our unique 35 MeV electron beam, while maintaining extremely high instantaneous dose rate (IDR). We characterized dosimetry and targeting accuracy of the FLASH beam with film dosimetry. We combined this FLASH beam with an organotypic rat brain slice/breast carcinoma coculture model of brain metastasis to assess effects on normal and neoplastic tissues. Live-cell and bioluminescence imaging demonstrated cancer cell growth effects, whereas normal tissue responses and immune activation were assessed using live-cell imaging, cytokine profiles, and confocal microscopy. We performed comparison experiments with 20 MeV electrons from a Varian clinical linear accelerator (VCLA) using conventional dose rates. RESULTS: The highest IDR of the FLASH beam to date was 20.7 ± 0.6 MGy/s, with maximum MDR of 20.7 MGy/s delivered in 1 pulse of 1 µs duration. Beam targeting was accurate to <1 mm and reproducible. HIGS-FLASH and VCLA dose rates equivalently decreased cancer cell growth. HIGS-FLASH irradiation significantly increased tumor necrosis factor α and fractalkine levels and confocal microscopy revealed distinct changes in microglial morphology slices suggesting microglia activation. CONCLUSIONS: Our novel experimental platform produces extremely high dose rates and rapid normal/neoplastic tissue readouts for mechanistic research into the effects of FLASH radiation in the brain. HIGS-FLASH irradiation induces comparable cancer cell growth inhibition but differential effects on cytokines and microglial morphology, suggesting that acute innate immune responses may be involved in FLASH normal tissue effects in the brain.
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Organotypic culture of post-mortem adult human brain explants exhibits synaptic plasticity.
BACKGROUND: Synaptic plasticity is an essential process encoding fine-tuned brain functions, but models to study this process in adult human systems are lacking. OBJECTIVE: We aim to test whether ex vivo organotypic culture of post-mortem adult brain explants (OPABs) retain synaptic plasticity. METHODS: OPABs were seeded on 3D microelectrode arrays to measure local field potential (LFP). Paired stimulation of distant electrodes was performed over three days to investigate our capacity to modulate specific neuronal connections. RESULTS: Long-term potentiation (LTP) or depression (LTD) did not occur within a single day. In contrast, after two and three days of training, OPABs showed a significant modulation of the paired electrodes' response compared to the non-paired electrodes from the same array. This response was alleviated upon treatment with dopamine. CONCLUSION: Our work highlights that adult human brain explants retain synaptic plasticity, offering novel approaches to neural circuitry in animal-free models.
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Single Cell-Seeded Human Intestinal Organoids for Organoid Research.
Recent developments in organoid technology have enabled the creation of patient-derived intestinal organoids (PDIOs) that recapitulate the structural, functional, genetic, and epigenetic features of their original tissues. However, conventional passage-derived organoids inevitably yield heterogeneous populations in size and number, leading to inconsistent results even under identical conditions. To address this, a standardized approach, referred to here as "single cell-seeded PDIOs," was established. In this method, mature PDIOs were enzymatically dissociated into single cells and seeded at a defined number into individual wells of a 96-well plate. This controlled seeding normalized the size and number of PDIOs. Compared with passage-derived organoids, single cell-seeded PDIOs displayed reduced inter-well variability in organoid numbers and intra-well variability in organoid sizes, which enables the determination of generation efficiency and improves the reproducibility of viability assays. Moreover, this platform is compatible with downstream analysis, including transcriptomic analysis and protein expression profiling. Collectively, this approach may enhance experimental consistency and provide a practical foundation for reproducible PDIO-based studies.