Multifrequency control of Faraday wave bioassembly for constructing multiscale hPSC-derived neuronal networks

Bioassembly is recently regarded as a critical alternative biofabrication technical route to bioprinting since it can directly manipulate millions of live cells to form multicellular structures with close intercellular proximity, improving contact-dependent cell communication and promoting the emergence of tissue-specific functions. However, acoustic bioassembly techniques are currently limited to generating cytoarchitecture with a single characteristic length which cannot faithfully mimic the multiscale cellular structures in native tissues. To overcome this challenge, herein we report a novel acoustic bioassembly technique that employs multifrequency control of Faraday waves to form multiscale cellular structures. By superimposing multiple sine wave signals with proper amplitude ratios, Faraday waves containing multiple wavelengths can be induced and enabled to generate multiscale structures in few seconds. Using this technique, we construct functional neuronal networks with multiscale connectivity that display spontaneous neuroelectrical activities. We anticipate this technique will find wide applications in tissue engineering and regenerative medicine.