Organs-on-Chips

E18 rat embryo-derived hippocampal neurons were seeded into Culture Channel 1 of NeoBento™, while cortical neurons were seeded into Culture Channel 3, establishing an in vitro co-culture system. The channels were pre-coated with poly-D-lysine (PDL) and laminin to promote neuron adhesion, growth, and the gradual maturation of neural networks. The culture process lasted for 21 days, with the culture medium changed three times a week to maintain an optimal growth environment.

To assess the neuronal state and maturity, β-III-tubulin and DAPI were used for visualizing cell morphology, and MAP2 was employed to label neuronal dendrites to evaluate their maturation. Additionally, electrophysiological recordings using MEA were performed on days 7, 14, and 21 to measure spontaneous neuronal firing activity.

A. Immunofluorescence staining images of rat hippocampal neurons (Culture Channel 1) and cortical neurons (Culture Channel 3). Neurons successfully attached to the culture channels and extended axons. DAPI stains nuclei (blue), β-III-tubulin marks cell bodies and axons (green), and MAP2 labels dendrites (red). Scale bar = 500 µm.
B. Brightfield image of the rat neuron co-culture system on day 15, showing clear axonal extension.
C. Raster plots of the co-culture system on days 7, 14, and 21 (recording duration: 300 seconds). Synchronized firing activity began on day 14 and became more pronounced by day 21, indicating gradual network maturation.
D. Comparison of the mean firing rate (MFR) between the culture channels and connecting microchannel on days 7, 14, and 21. MFR increased significantly over time. On day 14, higher spontaneous activity (0.8 spikes/s) was observed in the microchannel, compared to 0.3 spikes/s in the culture channels, suggesting faster functional development of axonal pathways in the microchannel.