diseases and develop potential cures. [1] Developments across the fields of biotechnology, tissue engineering, biomaterials, and microtechnology, have led to in vitro models ranging from multilayer 3D cell cultures [2] to small self-standing cell aggregates called spheroids, [3] up to complex brain organoids derived from human pluripotent stem (hPS) cells. [4] Albeit grown in an artificial in vitro environment, the shift from conventional 2D neural cultures to 3D models was shown to better mimic the complexity of intertwined 3D networks found in the brain. [5] hPS-derived brain organoids can indeed recapitulate several aspects specific to human brain development at the level of gene expression, [6] cell-type differentiation and network formation, [7] and can express phenotypes of human brain diseases when generated from patient-derived hPS-cells. [4a,8] Following these results, 3D neural cell assemblies have raised a large interest for the study of human brain diseases and therapies. Furthermore, these models can overcome certain limitations of currently used animal models, such as low experimental accessibility for functional studies, [9] low sample size, low reproducibility and, above all, poor translational relevance of screening results to humans. [5] The routine experimental use of 3D brain tissue models, however, remains largely unpractical for applications in drugdiscovery. On one side, intermodel variability and unmonitored cellular viability can affect the reliable generation of complex 3D brain tissue model systems. For instance, as 3D models become critically sized, the low diffusion of nutrients and oxygen tends to induce the formation of a necrotic core, [1b,4a,5] with consequent losses in cellular viability. On the other side, available biosensing technologies are not yet adapted for the routine monitoring of biosignals such as neural activity inside individual 3D models. This hinders studies aiming toward a better understanding of the emergence of spontaneous neural activity in these models as well as their optimization to reliably generate electrically active brain tissue models for functional assays. Over the last few years, researchers have been working on protocols for culturing organoids with minimal variability, mainly focusing on homogenizing morphologies. [4c] As far as monitoring brain organoids, current major biosensing Brain organoids is an exciting technology proposed to advance studies on human brain development, diseases, and possible therapies. Establishing and applying such models, however, is hindered by the lack of technologies to chronically monitor neural activity. A promising new approach comprising selfstanding biosensing microdevices capable of achieving seamless tissue integration during cell aggregation and culture. To date, there is little information on how to control the aggregation of such bioartificial 3D neural assemblies. Here, the growth of hybrid neurospheroids obtained by the aggregation of silicon sham microchips (100 × 100 × 50 μm 3) with primary cortical cells ...
Visual information processing in the retina requires the rhythmic expression of clock genes. The intrinsic retinal circadian clock is independent of the master clock located in the hypothalamic suprachiasmatic nucleus and emerges from retinal cells, including glia. Less clear is how glial oscillators influence the daily regulation of visual information processing in the mouse retina. Here, we demonstrate that the adult conditional deletion of the gene Bmal1 in GLAST-positive glial cells alters retinal physiology. Specifically, such deletion was sufficient to lower the amplitude of the electroretinogram b-wave recorded under light-adapted conditions. Furthermore, recordings from > 20,000 retinal ganglion cells (RGCs), the retina output, showed a non-uniform effect on RGCs activity in response to light across different cell types and over a 24-h period. Overall, our results suggest a new role of a glial circadian gene in adjusting mammalian retinal output throughout the night-day cycle.
Advancements in stem cell technology together with an improved understanding of in vitro organogenesis have enabled new routes that exploit cell-autonomous self-organization responses of adult stem cells (ASCs) and homogenous pluripotent stem cells (PSCs) to grow complex, three-dimensional (3D), mini-organ like structures on demand, the so-called organoids. Conventional optical and electrical neurophysiological techniques to acquire functional data from brain organoids, however, are not adequate for chronic recordings of neural activity from these model systems, and are not ideal approaches for throughput screenings applied to drug discovery. To overcome these issues, new emerging approaches aim at fusing sensing mechanisms and/or actuating artificial devices within organoids. Here we introduce and develop the concept of the Lab-in-Organoid (LIO) technology for in-tissue sensing and actuation within 3D cell aggregates. This challenging technology grounds on the self-aggregation of brain cells and on integrated bioelectronic micro-scale devices to provide an advanced tool for generating 3D biological brain models with in-tissue artificial functionalities adapted for routine, label-free functional measurements and for assay’s development. We complete previously reported results on the implementation of the integrated self-standing wireless silicon micro-devices with experiments aiming at investigating the impact on neuronal spheroids of sinusoidal electro-magnetic fields as those required for wireless power and data transmission. Finally, we discuss the technology headway and future perspectives.
In mammals, the suprachiasmatic nucleus of the hypothalamus is the master circadian pacemaker that synchronizes the clocks in the central nervous system and periphery, thus orchestrating rhythms throughout the body. However, little is known about how so many cellular clocks within and across brain circuits can be effectively synchronized. In this work, we investigated the implication of two possible pathways: (i) astrocytes-mediated synchronization and (ii) neuronal paracrine factors-mediated synchronization. By taking advantage of a lab-on-a-chip microfluidic device developed in our laboratory, here we report that both pathways are involved. We found the paracrine factors-mediated synchronization of molecular clocks is diffusion-limited and, in our device, effective only in case of a short distance between neuronal populations. Interestingly, interconnecting astrocytes define an active signaling channel that can synchronize molecular clocks of neuronal populations also at longer distances. At mechanism level, we found that astrocytes-mediated synchronization involves both GABA and glutamate, while neuronal paracrine factors-mediated synchronization occurs through GABA signaling. These findings identify a previously unknown role of astrocytes as active cells that might distribute long-range signals to synchronize the brain clocks, thus further strengthening the importance of reciprocal interactions between glial and neuronal cells in the context of circadian circuitry.
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