In Vitro Development of Human iPSC-Derived Functional Neuronal Networks on Laser-Fabricated 3D Scaffolds

Area of Science:

• Neuroscience
• Biomaterials Engineering
• Stem Cell Biology

Background:

• Human induced pluripotent stem cells (hiPSCs) are crucial for brain-on-chip models.
• Current 2D culture methods have limitations in fully maturing neuronal networks.
• Three-dimensional (3D) cultures offer a more physiologically relevant environment for neuronal development.

Purpose of the Study:

• To design and implement a 3D scaffold platform for enhanced neuronal network development.
• To support intricate neuronal network formation and maturation using hiPSC-derived cells.
• To establish a reliable 3D in vitro model for studying cortical circuit development and function.

Main Methods:

• Utilized direct laser writing by two-photon polymerization (2PP) to fabricate 3D scaffolds.
• Employed biocompatible Dental LT Clear (DClear) resin for scaffold construction.
• Cultured hiPSC-derived neural progenitor cells on the 3D scaffolds and monitored network development over 120 days.

Main Results:

• 3D scaffolds successfully supported the development and interconnection of neuronal networks.
• Neurons differentiated into cortical projection neurons (all six layers), inhibitory neurons, and glia.
• Long-term culturing (120 days) was achieved, surpassing 2D culture limitations.
• Calcium imaging revealed spontaneous neuronal activity, indicating functional network development.

Conclusions:

• Advanced microstructured 3D scaffolds provide a reliable platform for 3D in vitro modeling of neuronal functions.
• This technology advances brain-on-chip models for disease research and drug discovery.
• The 3D scaffold system promotes the maturation and long-term viability of hiPSC-derived neuronal networks.