A Simple Three-Dimensional Compartmentalized Co-Culture Model for Basal Forebrain and Hippocampal Neurons

The basal forebrain (BF)-hippocampus (HPC) circuit is indispensable for learning and memory, and in vitro models are essential for dissecting its age-related decline. Nonetheless, current culture methods endure brief survival or confine cells to two dimensions, leaving the circuit's progressive degeneration refractory to long-term investigation. Here, we developed a simple, three-dimensional (3D) compartmentalized co-culture model that mimics the anatomical organization of BF and HPC neurons. Results demonstrate that basal forebrain cholinergic neurons (BFCNs) co-cultured with primary HPC neurons remain viable for more than two months without exogenous growth factors, significantly promoting BFCNs growth, polarity development, and functional maturation. In this system, BFCNs somata were confined within the hydrogel, whereas cholinergic axons extended toward adjacent hippocampal area, reaching 1681.9 ± 351.8 μm by week 5-significantly longer than in BFCNs monocultures. This model can successfully recapitulate age-dependent progressive neuronal degeneration during long-term culture, validating this long-term co-culture as a platform for studying circuit aging and degeneration. Therefore, this low-cost and highly physiological platform provides a new avenue for in-depth investigations into the mechanisms of neurodegenerative diseases.