Abstract

GHz acoustofluidic technology-based acoustic dissociation is a novel non-contact single cell dissociation method, which breaks through the bottleneck of traditional tissue dissociation techniques in organoid construction and significantly improves the success rate of organoid establishment. In this study, a hypersonic vortex acoustic dissociation system was used for tissue dissociation in the pre-preparation stage of organoid culture, and the mouse lung tissue organoid model was taken as the research object to verify the application value of this technology in organoid construction. The system adopts hypersonic vortex non-contact dissociation mode, with the whole process temperature control optional, and matches the innovative cell activity maintenance enzyme formula, which can realize rapid dissociation of 1-100 mg tissue samples within 5-15 min, and the cell yield and activity after dissociation are both over 90%. In the mouse lung tissue organoid culture experiment, the cell yield obtained by acoustic dissociation reached 3.0×10⁷, which was significantly higher than the 1.6×10⁷ of manual dissociation; the cell viability was maintained at nearly 100%, far exceeding the 25%–45% of the manual operation group. The whole process from lung tissue treatment to high-quality single cell suspension acquisition was completed within 30 minutes, and the sorted alveolar type Ⅱ epithelial cells were inoculated into the co-culture system to construct organoids, which significantly improved the stability of subsequent organoid culture. In addition, the acoustic dissociation system can realize the high retention of membrane protein epitopes (>95%) and efficient capture of rare cells, and the survival rate of primary cells cultured after dissociation is increased to more than 85%, which provides a high-quality single cell source for organoid construction and lays a solid foundation for the long-term culture and stable passage of organoids. The hypersonic vortex acoustic dissociation technology has the advantages of high cell activity, high yield, mild dissociation and standardized operation, which can optimize the key link of single cell suspension preparation in organoid construction, effectively improve the success rate and stability of organoid culture, and has broad application prospects in the field of organoid research and regenerative medicine.