Constructive Neuroengineering of Crossing Multi-Neurite Wiring Using Modifiable Agarose Gel Platforms
Constructing stable and flexible neuronal networks with multi-neurite wiring is essential for the in vitro modeling of brain function, connectivity, and neuroplasticity. However, most existing neuroengineering platforms rely on static microfabrication techniques, which limit the ability to dynamical...
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MDPI AG
2025-05-01
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| author | Soya Hagiwara Kazuhiro Tsuneishi Naoya Takada Kenji Yasuda |
| author_facet | Soya Hagiwara Kazuhiro Tsuneishi Naoya Takada Kenji Yasuda |
| author_sort | Soya Hagiwara |
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| description | Constructing stable and flexible neuronal networks with multi-neurite wiring is essential for the in vitro modeling of brain function, connectivity, and neuroplasticity. However, most existing neuroengineering platforms rely on static microfabrication techniques, which limit the ability to dynamically control circuit architecture during cultivation. In this study, we developed a modifiable agarose gel-based platform that enables real-time microstructure fabrication using an infrared (IR) laser system under live-cell conditions. This approach allows for the stepwise construction of directional neurite paths, including sequential microchannel formation, cell chamber fabrication, and controlled neurite–neurite crossings. To support long-term neuronal health and network integrity in agarose microstructures, we incorporated direct glial co-culture into the system. A comparative analysis showed that co-culture significantly enhanced neuronal adhesion, neurite outgrowth, and survival over several weeks. The feeder layer configuration provided localized trophic support while maintaining a clear separation between glial and neuronal populations. Dynamic wiring experiments further confirmed the platform’s precision and compatibility. Neurites extended through newly fabricated channels and crossed pre-existing neurites without morphological damage, even when laser fabrication occurred after initial outgrowth. Time-lapse imaging showed a temporary growth cone stalling at crossing points, followed by successful elongation in all tested samples. Furthermore, the direct laser irradiation of extending neurites during microstructure modification did not visibly impair neurite elongation, suggesting minimal morphological damage under the applied conditions. However, potential effects on molecular signaling and electrophysiological function remain to be evaluated in future studies. Together, these findings establish a powerful, flexible system for constructive neuroengineering. The platform supports long-term culture, real-time modification, and multidirectional wiring, offering new opportunities for studying neural development, synaptic integration, and regeneration in vitro. |
| format | Article |
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| spelling | doaj-art-2ff08095f20c4867bae3a98c6a48d76d2025-06-25T13:53:47ZengMDPI AGGels2310-28612025-05-0111641910.3390/gels11060419Constructive Neuroengineering of Crossing Multi-Neurite Wiring Using Modifiable Agarose Gel PlatformsSoya Hagiwara0Kazuhiro Tsuneishi1Naoya Takada2Kenji Yasuda3Department of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku 169-8555, Tokyo, JapanDepartment of Physics, School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku 169-8555, Tokyo, JapanDepartment of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku 169-8555, Tokyo, JapanDepartment of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku 169-8555, Tokyo, JapanConstructing stable and flexible neuronal networks with multi-neurite wiring is essential for the in vitro modeling of brain function, connectivity, and neuroplasticity. However, most existing neuroengineering platforms rely on static microfabrication techniques, which limit the ability to dynamically control circuit architecture during cultivation. In this study, we developed a modifiable agarose gel-based platform that enables real-time microstructure fabrication using an infrared (IR) laser system under live-cell conditions. This approach allows for the stepwise construction of directional neurite paths, including sequential microchannel formation, cell chamber fabrication, and controlled neurite–neurite crossings. To support long-term neuronal health and network integrity in agarose microstructures, we incorporated direct glial co-culture into the system. A comparative analysis showed that co-culture significantly enhanced neuronal adhesion, neurite outgrowth, and survival over several weeks. The feeder layer configuration provided localized trophic support while maintaining a clear separation between glial and neuronal populations. Dynamic wiring experiments further confirmed the platform’s precision and compatibility. Neurites extended through newly fabricated channels and crossed pre-existing neurites without morphological damage, even when laser fabrication occurred after initial outgrowth. Time-lapse imaging showed a temporary growth cone stalling at crossing points, followed by successful elongation in all tested samples. Furthermore, the direct laser irradiation of extending neurites during microstructure modification did not visibly impair neurite elongation, suggesting minimal morphological damage under the applied conditions. However, potential effects on molecular signaling and electrophysiological function remain to be evaluated in future studies. Together, these findings establish a powerful, flexible system for constructive neuroengineering. The platform supports long-term culture, real-time modification, and multidirectional wiring, offering new opportunities for studying neural development, synaptic integration, and regeneration in vitro.https://www.mdpi.com/2310-2861/11/6/419neuronhippocampal cellneurite elongation controlagarose gel micropatternbending microchannel arraybending angle |
| spellingShingle | Soya Hagiwara Kazuhiro Tsuneishi Naoya Takada Kenji Yasuda Constructive Neuroengineering of Crossing Multi-Neurite Wiring Using Modifiable Agarose Gel Platforms Gels neuron hippocampal cell neurite elongation control agarose gel micropattern bending microchannel array bending angle |
| title | Constructive Neuroengineering of Crossing Multi-Neurite Wiring Using Modifiable Agarose Gel Platforms |
| title_full | Constructive Neuroengineering of Crossing Multi-Neurite Wiring Using Modifiable Agarose Gel Platforms |
| title_fullStr | Constructive Neuroengineering of Crossing Multi-Neurite Wiring Using Modifiable Agarose Gel Platforms |
| title_full_unstemmed | Constructive Neuroengineering of Crossing Multi-Neurite Wiring Using Modifiable Agarose Gel Platforms |
| title_short | Constructive Neuroengineering of Crossing Multi-Neurite Wiring Using Modifiable Agarose Gel Platforms |
| title_sort | constructive neuroengineering of crossing multi neurite wiring using modifiable agarose gel platforms |
| topic | neuron hippocampal cell neurite elongation control agarose gel micropattern bending microchannel array bending angle |
| url | https://www.mdpi.com/2310-2861/11/6/419 |
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