Two-layered microwell-array device for preparation of single-neuron culture samples

Abstract When a single neuron is cultured in isolation from other neurons, its axon connects with its own dendrites to form a simple, independent network with no synaptic inputs from other neurons. This culture system enables detailed analysis of synaptic function and morphology change in neurites at the single-neuron level, which is useful for elucidating the pathogenesis of neurological diseases and for evaluating the efficacy of therapeutic drugs for them. However, there was previously no device technology capable of simultaneously forming multiple single-neuron samples while allowing co-culture with astrocytes, which is essential for culture of a single neuron isolated from other neurons. In this study, we propose a novel microwell-array device for preparing single-neuron samples. The device consists of an upper layer for cell seeding and a lower layer for cell culture. Each layer has 16 × 16 microwells, and the bottom of each well is made of a 1 μm thick silicon nitride membrane. The membrane of the upper well has one microhole for seeding a single neuron, and the lower membrane has multiple microholes for interaction between a single neuron and astrocytes which are co-cultured back-to-back on both sides of the membrane. When neurons are seeded into the upper well, only one of them passes through the microhole in the upper membrane and falls onto the lower membrane. We evaluated a seeding efficiency of single neurons by changing seeding hole diameter and seeding density. The results showed that the yield of more than 20% was obtained regardless of the seeding density when the seeding hole diameter was 13 μm. We also confirmed that single neurons seeded in this manner and co-cultured with astrocytes developed neurites and formed synapses. These results demonstrated the usefulness of this device for the preparation of single-neuron culture samples.