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The intricate architecture of the human central nervous system (CNS) presents significant challenges in neuroscience in developing in vitro models that accurately replicate its structure and function under both physiological and pathological conditions. The brain’s highly organized and layered regions, each characterized by distinct cellular phenotypes and extracellular matrix (ECM) compositions, are particularly difficult to reproduce in terms of compartmentalization and functional connectivity. In vitro neural models are fundamental for understanding CNS function at the microscale. To develop more physiologically relevant models, innovative technologies are being explored to better mimic in vivo cellular environment and the complexity of brain tissue. In this context, 3D bioprinting has emerged as a leading technique to construct tissue-like architectures that support multicellular organization and enable modeling cell-cell and cell-matrix interactions [1]. At the microscopic level, one of the main challenges lies in identifying materials that can mimic the brain’s ECM and providing the mechanical and biochemical cues necessary to support the formation and the maturation of 3D neuronal networks [2].
This study focuses on the development of a chitosan-based bioink suitable for 3D bioprinting of cell-laden constructs embedding different neural cell types to create functional, compartmentalized neuron-glia networks in vitro. Chitosan, a biopolymer known for its similarity to native ECM components, as well as for its low cost and versatility, was selected for its favorable properties in promoting cell adhesion, neuronal growth, and the maturation of neuronal networks derived from human-induced pluripotent stem cells co-cultured with astrocytes [3-4]. The bioink’s rheological and mechanical properties were characterized to ensure optimal performance for extrusion-based 3D printing and to evaluate stiffness. The printing protocol and parameters were optimized to enable efficient bioink deposition, resulting in multicompartment personalized constructs with good resolution and shape fidelity. Complex 3D models were fabricated by embedding human-derived cortical glutamatergic and gabaergic neurons and astrocytes, into spatially defined and personalized arrangements. The constructs were functionally and morphologically characterized using high-density micro-electrode arrays and immunocytochemistry technique, respectively, to assess cell distribution and functional neuronal network maturation.
The developed bioink exhibited excellent printability, with shear-thinning behavior and rapid gelation, that ensured post-printing structural stability and preserved good cell viability. Mechanical testing showed that the printed constructs had stiffness values comparable to those of native brain tissue. The bioprinted constructs maintained high shape fidelity with respect to complex geometries. Functional and morphological analyses revealed homogeneous cell distribution and the formation of functional neuronal networks under long-term culture conditions.
In conclusion, this work demonstrates that the developed chitosan-based bioink and the optimized bioprinting protocol enable the fabrication of functionally compartmentalized 3D neural constructs with precise control over the spatial distribution of neural cells. These models closely mimic the structural and functional organization of brain tissue, offering a promising platform for CNS modeling and in-depth studies of cellular behavior, network connectivity, and intercellular communication.
[1] Wang L. et al., 2025, 032005.
[2] Cadena M. et al., 2021, 2001600.
[3] Di Lisa D. et al., 2020, 104081.
[4] Di Lisa D. et al., 2023, 015011.
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