Cortical Neurons form a Functional Neuronal Network in a 3D Printed Reinforced Matrix

Abstract

Impairments in neuronal circuits underly multiple neurodevelopmental and neurodegenerative disorders. 3D cell culture models enhance the complexity of in vitro systems and provide a microenvironment closer to the native situation than with 2D cultures. Such novel model systems will allow the assessment of neuronal network formation and their dysfunction under disease conditions. Here, mouse cortical neurons are cultured from embryonic day E17 within in a fiber‐reinforced matrix. A soft Matrigel with a shear modulus of 31 ± 5.6 Pa is reinforced with scaffolds created by melt electrowriting, improving its mechanical properties and facilitating the handling. Cortical neurons display enhance cell viability and the neuronal network maturation in 3D, estimated by staining of dendrites and synapses over 21 days in vitro, is faster in 3D compared to 2D cultures. Using functional readouts with electrophysiological recordings, different firing patterns of action potentials are observed, which are absent in the presence of the sodium channel blocker, tetrodotoxin. Voltage‐gated sodium currents display a current–voltage relationship with a maximum peak current at −25 mV. With its high customizability in terms of scaffold reinforcement and soft matrix formulation, this approach represents a new tool to study neuronal networks in 3D under normal and, potentially, disease conditions.

Polymers for Melt Electrowriting

“…This is one approach to shift the biofabrication window to softer hydrogels and matrices while retaining handling or biomechanical requirements. [35,38,42,57] Such composite approaches, reinforcing MEW-fabricated PCL scaffolds into hydrogels that include gelatin methacryloyl (GelMA), show promising results regarding the stiffness increasing by up to 54-fold. [42] In this fiber-reinforced composite study, embedded human chondrocytes showed good viability and retained their roundish morphology, showing compatibility in terms of stiffness and biological cues for cartilage applications.…”

“…Finally, to provide 3D neuronal cell culture models with a weak matrix microenvironment closer to the native tissue, MEW fabricated scaffolds were embedded into Matrigel. [38,41] Those reinforced scaffolds within the Matrigel improved the mechanical properties and therefore the handling of the delicate constructs. [38,41] Another approach to mimic native tissue and its composition was demonstrated by using orthogonal microfiber scaffolds embedded into compressed collagen to design an artificial cornea.…”

“…[38,41] Those reinforced scaffolds within the Matrigel improved the mechanical properties and therefore the handling of the delicate constructs. [38,41] Another approach to mimic native tissue and its composition was demonstrated by using orthogonal microfiber scaffolds embedded into compressed collagen to design an artificial cornea. [56] Furthermore, the PCL scaffold served as an interface between the compressed collagen and a PHEMA hydrogel resulting in a coreskirt design ( Figure 6A).…”

“…Additionally, the hydrogel can provide an improved biological environment for cells. [35,37,38,41,42] However, embedding the MEW scaffold into a gel prevents an accurate and controlled placement of the cells within the constructs. Therefore, de Ruijter et al [43] used a simultaneous micropatterning approach by combining extrusion-based bioprinting and MEW.…”

Microphysiological Systems for Neurodegenerative Diseases in Central Nervous System

“…The laminin-rich property can fulfill an essential component of the brain. Laminin is one of the abundant proteins in the brain ECM; it promotes cell proliferation and differentiation, and supports neural cell attachment [ 101 , 102 , 103 ].…”