(G-241) Integration of reconfigurable microchannels into aligned three-dimensional neural networks for spatially controllable neuromodulation

Introduction:: Anisotropically organized neural networks are indispensable routes for functional connectivity in the brain, which remains largely unknown. While prevailing animal models require additional preparation and stimulation-applying devices and have exhibited limited capabilities regarding localized stimulation, no in vitro platform exists that permits spatiotemporal control of chemo-stimulation in anisotropic 3D neural networks. We present the integration of microchannels seamlessly into a fibril-aligned 3D scaffold by adapting a single fabrication principle. We investigated the underlying physics of elastic microchannels’ ridges and interfacial sol-gel transition of collagen under compression to determine a critical window of geometry and strain. For the first time, we demonstrated the spatiotemporally resolved neuromodulation in an aligned 3D neural network by local deliveries of KCl and Ca²⁺-signal inhibitors such as Tetrodotoxin (TTX), Nifedipine, and Mibefradil, and also visualized Ca²⁺ signal propagation with a speed of ~3.7 μm/s. We anticipate that our technology will pave the way to elucidate functional connectivity and neurological diseases associated with trans-synaptic propagation.


Materials and Methods:: We used a single fabrication principle based on compression-induced sol-gel transition of collagen and hyperelastic properties of the PDMS. We optimized the geometry and strain of the microchannels to achieve seamless integration with 3D aligned collagen scaffold. Then we cultured rat cortical neurons in the scaffold and observed directional alignment and network formation. Then we delivered KCl and Ca2+ signal inhibitors through the microchannels and measured the Ca2+ signal propagation in the neural network. We found that the speed of signal propagation was about 3.7 μm/s and that the local stimulation could modulate the network activity.


Results, Conclusions, and Discussions:: We fabricated a PDMS based 3D scaffold platform with integrated reconfigurable microchannels which by applying compressive stress to the platform, we can form a fibril-aligned 3D collagen scaffold with embedded microchannels that can deliver different neuromodulators to specific regions of the neural network. We first set design parameters of the microchannels’ dimensions where it could not disturb the formation of the aligned 3D scaffold and remain as an intact microchannel after the compression. The parameters were investigated through simulation and fluorescence imaging. Then, through the optimized platform design, we demonstrated the alignment of the collagen fibrils within the 3D scaffold and by incorporating cortical neurons of a SD rat embryos, observed the formation of aligned 3D neural networks within our platform. We then demonstrated the feasibility and functionality of our platform by applying KCl and Ca 2+ signal inhibitors (tetrodotoxin, nifedipine, and mibefradil) to the microchannels and observing the changes in Ca 2+ signal propagation along the aligned 3D neural network. We found that the speed of Ca 2+ signal propagation was about ~3.7 μm/s and that it could be modulated by the local delivery of neuromodulators. We concluded that our technology could provide a new way to investigate functional connectivity and neurological diseases associated with transsynaptic propagation in anisotropic 3D neural networks.


Acknowledgements (Optional): : We acknowledge support from the Ministry of Science and ICT through the National Research Foundation of Korea (NRF), the KIST Young Fellow project, the KIST institutional projects, and the KU-KIST Graduate School of Converging Science and Technology Program

References (Optional): : S. Jeong, H. W. Kang, S. H. Kim, S. Bang, S. Chung, H. N. Kim, N. Choi, Integration of reconfigurable microchannels into aligned three-dimensional neural networks for spatially controllable neuromodulation. Science Advances 9, (2023).

S. H. Kim, S.-K. Im, S.-J. Oh, S. Jeong, E.-S. Yoon, C. J. Lee, N. Choi, E.-M. Hur, Anisotropically organized three-dimensional
culture platform for reconstruction of a hippocampal neural network. Nature Communications 8, 1-16 (2017).