Long-termin vitroculture of 3D brain tissue model based on chitosan thermogel.

Methods for studying brain function and disease heavily rely onin vivoanimal models,ex-vivotissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects ofin vivohuman brain are needed as additional preclinical tools. In this context, 3D hydrogel-basedin vitromodels of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-ß-glycerophosphate (CHITO-ß-GP) thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that CHITO-ß-GP thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells (h-iPSCs) co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from h-iPSCs were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 d. In conclusion, CHITO-ß-GP thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity toward nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.

Human-Derived Cortical Neurospheroids Coupled to Passive, High-Density and 3D MEAs: A Valid Platform for Functional Tests.

With the advent of human-induced pluripotent stem cells (hiPSCs) and differentiation protocols, methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality (3D) would make them more representative of an in-vivo environment. Thus, human-derived 3D structures are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, methodologies to create 3D structures with controlled cellular density and composition and platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapidly generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by using micro-electrode arrays (MEAs) with different types (i.e., passive, C-MOS, and 3D) and number of electrodes. Neurospheroids grown in free culture and transferred on MEAs exhibited functional activity that can be chemically and electrically modulated. Our results indicate that this model holds great potential for an in-depth study of signal transmission to drug screening and disease modeling and offers a platform for in-vitro functional testing.

On the way back from 3D to 2D: Chitosan promotes adhesion and development of neuronal networks onto culture supports.

Most in vitro functional and morphological studies for developing nervous system have been performed using traditional monolayer cultures onto supports modified by extracellular matrix components or synthetic biopolymers. These biomolecules act as adhesion factors essential for neuronal growth and differentiation. In this study, the use of chitosan as adhesion factor was investigated. Primary rat neurons and neurons differentiated from human induced pluripotent stem cells were cultured onto chitosan and standard adhesion factors modified supports. The initiation, elongation and branching of neuritic processes, synaptogenesis and electrophysiological behavior were studied. The biopolymers affected neurites outgrowth in a time dependent manner; in particular, chitosan promoted neuronal polarity in both cell cultures. These results indicate chitosan as a valid adhesion factor alternative to the standard ones, with the advantage that it can be used both in 2D and 3D cultures, acting as a bridge between these in vitro models.

Neuroprotective effect of hypoxic preconditioning and neuronal activation in a in vitro human model of the ischemic penumbra.

OBJECTIVE: In ischemic stroke, treatments to protect neurons from irreversible damage are urgently needed. Studies in animal models have shown that neuroprotective treatments targeting neuronal silencing improve brain recovery, but in clinical trials none of these were effective in patients. This failure of translation poses doubts on the real efficacy of treatments tested and on the validity of animal models for human stroke. Here, we established a human neuronal model of the ischemic penumbra by using human induced pluripotent stem cells and we provided an in-depth characterization of neuronal responses to hypoxia and treatment strategies at the network level. APPROACH: We generated neurons from induced pluripotent stem cells derived from healthy donor and we cultured them on micro-electrode arrays. We measured the electrophysiological activity of human neuronal networks under controlled hypoxic conditions. We tested the effect of different treatment strategies on neuronal network functionality. MAIN RESULTS: Human neuronal networks are vulnerable to hypoxia reflected by a decrease in activity and synchronicity under low oxygen conditions. We observe that full, partial or absent recovery depend on the timing of re-oxygenation and we provide a critical time threshold that, if crossed, is associated with irreversible impairments. We found that hypoxic preconditioning improves resistance to a second hypoxic insult. Finally, in contrast to previously tested, ineffective treatments, we show that stimulatory treatments counteracting neuronal silencing during hypoxia, such as optogenetic stimulation, are neuroprotective. SIGNIFICANCE: We presented a human neuronal model of the ischemic penumbra and we provided insights that may offer the basis for novel therapeutic approaches for patients after stroke. The use of human neurons might improve drug discovery and translation of findings to patients and might open new perspectives for personalized investigations.

Assembly of chitosan-graphite oxide nanoplatelets core shell microparticles for advanced 3D scaffolds supporting neuronal networks growth.

This manuscript reports the development of functional 3D scaffolds based on chitosan (CHI) and graphite oxide nanoplatelets (GO) for neuronal network growth. To this aim, CHI microparticles, produced by alkaline gelation method, were coated with GO exploiting a simple template-assisted assembly based on the electrostatic attraction in an aqueous medium. The optimal deposition conditions were evaluated by optical microscopy and studied by quartz crystal microbalance. FE-SEM observations highlight the formation of a core-shell structure where the porous chitosan core is completely wrapped by a uniform GO layer. This outer shell protects the inner chitosan from enzymatic degradation thus potentially extending the scaffold viability for in vivo applications. The presence of hydrophilic oxygen-containing functionalities on the outermost layer of GO and its inner conductive graphitic core maintained the bioactivity of the scaffold and promoted neuronal cell adhesion and growth. The proposed approach to modify the surface of CHI microparticles makes it possible for the design of 3D scaffolds for advanced neuronal tissue engineering applications.

Brain-on-a-Chip: A Human 3D Model for Clinical Application.

The main goal of this research is to design, develop and implement an efficient protocol to generate 3D neural cultures derived from human induced Pluripotent Stem Cells (hiPSCs) coupled to Micro Electrode Arrays (MEA) in order to obtain an engineered and controlled brain-on-a-chip model. The use of patient specific iPSCs may offer novel insights into the pathophysiology of a large variety of disorders, including numerous neurodevelopmental and late-onset neurodegenerative conditions. With these in vitro patient specific models, we may have the possibility to test drugs and find ad hoc therapies in the direction of precision medicine.

Mild stimulation improves neuronal survival in an in vitro model of the ischemic penumbra.

OBJECTIVE: In the core of a brain infarct, characterized by severely reduced blood supply, loss of neuronal function is rapidly followed by neuronal death. In peripheral areas of the infarct, the penumbra, damage is initially reversible, and neuronal activity is typically reduced due to ischemia-induced synaptic failure. There is limited understanding of factors governing neuronal recovery or the transition to irreversible damage. Neuronal activity has been shown to be crucial for survival. Consequently, hypoxia induced neuronal inactivity may contribute to cell death, and activation of penumbral neurons possibly improves survival. Adversely, activation increases ATP demand, and a balance should be found between the available energy and sufficient activity. APPROACH: We monitored activity and viability of neurons in an in vitro model of the penumbra, consisting of (rat) neuronal networks on micro electrode arrays (MEAs) under controlled hypoxic conditions. We tested effects of optogenetic and electrical activation during hypoxia. MAIN RESULTS: Mild stimulation yielded significantly better recovery of activity immediately after re-oxygenation, compared with no stimulation, and a 60%-70% higher survival rate after 5 d. Stronger stimulation was not associated with better recovery than no stimulation, suggesting that beneficial effects depend on a delicate balance between sufficient activity and available energy. SIGNIFICANCE: We show that mild activation during hypoxia/ischemia is beneficial for cell survival in an in vitro model of the penumbra. This finding opposes the current common belief that suppression of neuronal activity is the cornerstone of neuroprotection during cerebral ischemia, and may open new possibilities for the treatment of secondary brain damage after stroke.