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

Area of Science:

• Neuroscience
• Biomaterials Engineering
• Cell Biology

Background:

• Neuronal circuit impairments are implicated in neurodevelopmental and neurodegenerative disorders.
• Traditional 2D cell cultures lack the microenvironmental complexity of native neural tissue.
• Advanced in vitro models are needed to accurately study neuronal network formation and dysfunction.

Purpose of the Study:

• To develop and characterize a novel 3D cell culture system for studying mouse cortical neurons.
• To assess neuronal viability, network maturation, and electrophysiological properties in a 3D environment.
• To establish a customizable platform for investigating neuronal function under normal and disease conditions.

Main Methods:

• Culturing mouse cortical neurons (embryonic day E17) within a fiber-reinforced hydrogel matrix.
• Reinforcing soft Matrigel (shear modulus 31 ± 5.6 Pa) with scaffolds fabricated via melt electrowriting.
• Assessing neuronal network maturation using dendritic and synaptic staining over 21 days in vitro.
• Utilizing electrophysiological recordings to analyze action potential firing patterns and sodium currents.

Main Results:

• Enhanced cell viability and accelerated neuronal network maturation in 3D cultures compared to 2D.
• Observation of distinct action potential firing patterns in 3D cultures, abolished by tetrodotoxin.
• Characterization of voltage-gated sodium currents with a peak current observed at -25 mV.
• Demonstration of improved mechanical properties and handling of the 3D matrix due to scaffold reinforcement.

Conclusions:

• The developed fiber-reinforced 3D matrix provides a superior microenvironment for neuronal culture.
• This novel 3D model system facilitates faster neuronal network maturation and enables functional electrophysiological studies.
• The customizable nature of this approach makes it a valuable tool for studying neuronal networks in health and disease.