Existing three-dimensional (3D) neuronal culture technology has limitations in brain research due to the difficulty of precisely replicating the brain’s complex multilayered structure and the lack of a platform that can simultaneously analyze both structure and function. A KAIST research team has successfully developed an integrated platform that can implement brain-like layered neuronal structures using 3D printing technology and precisely measure neuronal activity within them.

A joint research team led by Professors Je-Kyun Park and Yoonkey Nam from the Department of Bio and Brain Engineering at KAIST has developed an integrated platform capable of fabricating high-resolution 3D multilayer neuronal networks using low-viscosity natural hydrogels with mechanical properties similar to brain tissue, and simultaneously analyzing their structural and functional connectivity. The study is published in Biosensors and Bioelectronics.

Conventional bioprinting technology uses high-viscosity bioinks for structural stability, but this limits neuronal proliferation and neurite growth. Conversely, neural cell-friendly low-viscosity hydrogels are difficult to precisely pattern, leading to a fundamental trade-off between structural stability and biological function.

The research team completed a sophisticated and stable brain-mimicking platform by combining three key technologies that enable the precise creation of brain structure with dilute gels, accurate alignment between layers, and simultaneous observation of neuronal activity.

The three core technologies are: capillary pinning effect technology, which enables the dilute gel (hydrogel) to adhere firmly to a stainless steel mesh (micromesh) to prevent it from flowing, thereby reproducing brain structures with six times greater precision (resolution of 500 μm or less) than conventional methods; the 3D printing aligner, a cylindrical design that ensures the printed layers are precisely stacked without misalignment, guaranteeing the accurate assembly of multilayer structures and stable integration with microelectrode chips; and dual-mode analysis system technology, which simultaneously measures electrical signals from below and observes cell activity with light (calcium imaging) from above, allowing for the simultaneous verification of the functional operation of interlayer connections through multiple methods.

The research team successfully implemented a three-layered mini-brain structure using 3D printing with a fibrin hydrogel, which has elastic properties similar to those of the brain, and experimentally verified the process of actual neural cells transmitting and receiving signals within it.

Cortical neurons were placed in the upper and lower layers, while the middle layer was left empty but designed to allow neurons to penetrate and connect through it. Electrical signals were measured from the lower layer using a microsensor (electrode chip), and cell activity was observed from the upper layer using light (calcium imaging).

The results showed that when electrical stimulation was applied, neural cells in both upper and lower layers responded simultaneously. When a synapse-blocking agent (synaptic blocker) was introduced, the response decreased, proving that the neural cells were genuinely connected and transmitting signals.

Professor Park explained, “This research is a joint development achievement of an integrated platform that can simultaneously reproduce the complex multilayered structure and function of brain tissue. Compared to existing technologies where signal measurement was impossible for more than 14 days, this platform maintains a stable microelectrode chip interface for over 27 days, allowing the real-time analysis of structure-function relationships.

“It can be utilized in various brain research fields, such as neurological disease modeling, brain function research, neurotoxicity assessment, and neuroprotective drug screening in the future.”