Motivation The brain is formed by an intricate assembly of cellular networks, where neurons are embedded in an extracellular matrix (ECM) consisting of an intricate three-dimensional (3D) mesh of proteins that provides complex chemical, electrical and mechanical signalling.1 Given this complexity as well as the limitations of in vivo studies,2 it is important to develop in vitro models able to recapitulate the brain connectivity at various levels and ultimately, provide a mimic of the human brain suitable for preclinical applications.3 By reproducing cell to cell and cell to ECM interactions so to mimic the in vivo microenvironment, 3D tissue engineering promotes more physiological responses than conventional 2D cultures.4 Toward this goal, several 3D supporting materials or scaffolds have been developed, tested and applied.5 Among them, emerging carbon-based materials, such as carbon nanotubes (CNTs) 6, graphene oxide 7 and graphene foam (GF) 8 have high mechanical stability, high porosity and dense interconnectivity, providing a 3D microenvironment beneficial for cell growth and interaction.9 My Work In my Ph.D., I aimed to construct 3D neural scaffolds based on carbon materials especially graphene and carbon nanotubes (CNTs) and explore the regulation of these scaffolds for specific application in neural cultures. To achieve these goals, I combined chemical vapor deposition (CVD) and nano-fabrication for the preparation of different kinds of scaffolds and then used these scaffolds for the neural cultures. In the characterization of neural culture part, I mainly used optical imaging methods, particularly immunochemistry and calcium imaging, to investigate the neuronal network morphology and electrical dynamics of reconstructed 3D primary cultures from rats. These are my main results: 1) By using Fe nanoparticles confined to the interlamination of graphite as catalyst, we have obtained a fully 3D interconnected CNT web through the pores of graphene foam (GCNT web) by in situ chemical vapor deposition. This 3D GCNT web has a thickness up to 1.5 mm and a completely geometric, mechanical and electrical interconnectivity. Dissociated cortical cells cultured inside the GCNT web form a functional 3D cortex-like network exhibiting a spontaneous electrical activity that is closer to what is observed in vivo. Moreover, we have explored the application of this functional 3D cortex-like network: 2) By co-culturing and fluorescently labelling glioma and healthy cortical cells with different colours, a new in vitro model is obtained to investigate malignant glioma infiltration. This model allows reconstruction of the 3D trajectories and velocity distribution of individual infiltrating glioma with an unprecedented precision. The model is cost-effective and allows a quantitative and rigorous screening of anti-cancer drugs. 3) We have fabricated a 3D free-standing ordered graphene (3D-OG) network with the pore size of 20 μm, the skeleton width of 20 μm and an exact 90° orientation angle between the building blocks. Extensive interconnectivity of graphene sheets allows 3D-OG scaffolds to be free-standing and to be easily manipulated. When primary cortical cells are cultured on 3D-OG scaffolds, the cells form well-defined 3D connections with a cellular density similar to that observed when cells were cultured on 2D coverslip. In contrast to the 2D coverslips culture, astrocytes cultured on 3D-OG scaffolds did not have a flat morphology but had a more ramified shape similar to that seen in vivo conditions. Moreover, neurons on 3D-OG scaffolds had axons and dendrites aligned along the graphene skeleton allowing the formation of neuronal networks with highly controlled connections. Neuronal networks grown on 3D-OG scaffolds had a higher electrical activity with functional signaling over a long distance. 4) We have constructed a novel scaffold of three-dimensional bacterial cellulose-graphene foam (3D-BC/G) for neural stem cells (NSCs) in vitro, which was prepared via in situ bacterial cellulose interfacial polymerization on the skeleton surface of porous graphene foam. We found that 3D-BC/G can not only support NSCs growth and adhesion, but also keep NSCs stemness and enhanced its proliferative capacity. Further phenotypic analysis indicated that 3D-BC/G can induce NSCs selectively to differentiate into neurons, forming a neural network in short time. It was also meanwhile demonstrated to have good biocompatibility for primary cortical neurons and enhanced neuronal network activities by measuring calcium transient.

Construction of carbon-based three-dimensional neural scaffolds and their structural regulation / Xiao, Miao. - (2020 Feb 04).

Construction of carbon-based three-dimensional neural scaffolds and their structural regulation

Xiao, Miao
2020-02-04

Abstract

Motivation The brain is formed by an intricate assembly of cellular networks, where neurons are embedded in an extracellular matrix (ECM) consisting of an intricate three-dimensional (3D) mesh of proteins that provides complex chemical, electrical and mechanical signalling.1 Given this complexity as well as the limitations of in vivo studies,2 it is important to develop in vitro models able to recapitulate the brain connectivity at various levels and ultimately, provide a mimic of the human brain suitable for preclinical applications.3 By reproducing cell to cell and cell to ECM interactions so to mimic the in vivo microenvironment, 3D tissue engineering promotes more physiological responses than conventional 2D cultures.4 Toward this goal, several 3D supporting materials or scaffolds have been developed, tested and applied.5 Among them, emerging carbon-based materials, such as carbon nanotubes (CNTs) 6, graphene oxide 7 and graphene foam (GF) 8 have high mechanical stability, high porosity and dense interconnectivity, providing a 3D microenvironment beneficial for cell growth and interaction.9 My Work In my Ph.D., I aimed to construct 3D neural scaffolds based on carbon materials especially graphene and carbon nanotubes (CNTs) and explore the regulation of these scaffolds for specific application in neural cultures. To achieve these goals, I combined chemical vapor deposition (CVD) and nano-fabrication for the preparation of different kinds of scaffolds and then used these scaffolds for the neural cultures. In the characterization of neural culture part, I mainly used optical imaging methods, particularly immunochemistry and calcium imaging, to investigate the neuronal network morphology and electrical dynamics of reconstructed 3D primary cultures from rats. These are my main results: 1) By using Fe nanoparticles confined to the interlamination of graphite as catalyst, we have obtained a fully 3D interconnected CNT web through the pores of graphene foam (GCNT web) by in situ chemical vapor deposition. This 3D GCNT web has a thickness up to 1.5 mm and a completely geometric, mechanical and electrical interconnectivity. Dissociated cortical cells cultured inside the GCNT web form a functional 3D cortex-like network exhibiting a spontaneous electrical activity that is closer to what is observed in vivo. Moreover, we have explored the application of this functional 3D cortex-like network: 2) By co-culturing and fluorescently labelling glioma and healthy cortical cells with different colours, a new in vitro model is obtained to investigate malignant glioma infiltration. This model allows reconstruction of the 3D trajectories and velocity distribution of individual infiltrating glioma with an unprecedented precision. The model is cost-effective and allows a quantitative and rigorous screening of anti-cancer drugs. 3) We have fabricated a 3D free-standing ordered graphene (3D-OG) network with the pore size of 20 μm, the skeleton width of 20 μm and an exact 90° orientation angle between the building blocks. Extensive interconnectivity of graphene sheets allows 3D-OG scaffolds to be free-standing and to be easily manipulated. When primary cortical cells are cultured on 3D-OG scaffolds, the cells form well-defined 3D connections with a cellular density similar to that observed when cells were cultured on 2D coverslip. In contrast to the 2D coverslips culture, astrocytes cultured on 3D-OG scaffolds did not have a flat morphology but had a more ramified shape similar to that seen in vivo conditions. Moreover, neurons on 3D-OG scaffolds had axons and dendrites aligned along the graphene skeleton allowing the formation of neuronal networks with highly controlled connections. Neuronal networks grown on 3D-OG scaffolds had a higher electrical activity with functional signaling over a long distance. 4) We have constructed a novel scaffold of three-dimensional bacterial cellulose-graphene foam (3D-BC/G) for neural stem cells (NSCs) in vitro, which was prepared via in situ bacterial cellulose interfacial polymerization on the skeleton surface of porous graphene foam. We found that 3D-BC/G can not only support NSCs growth and adhesion, but also keep NSCs stemness and enhanced its proliferative capacity. Further phenotypic analysis indicated that 3D-BC/G can induce NSCs selectively to differentiate into neurons, forming a neural network in short time. It was also meanwhile demonstrated to have good biocompatibility for primary cortical neurons and enhanced neuronal network activities by measuring calcium transient.
4-feb-2020
Torre, Vincent
Xiao, Miao
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11767/108831
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