An enzyme-free, cold-process acoustic method for gentle and effective tissue dissociation

As biological advances continue to improve the resolution of genomic and proteomic studies, the quality of single cell suspensions is becoming increasingly important. While conventional approaches use enzymes which may require heat Abbreviations: Bulk Lateral Ultrasound (BLU) activation to break down extracellular tissue matrices and gain access to single cells, recent studies have suggested that these harsh biochemical and heat-based treatments may result in genomic and proteomic modulation. To minimize these dissociation artifacts, we have developed an instrument for dissociating cells from various tissue matrices using Bulk Lateral Ultrasound (BLU™) energy. This enzyme-free, gentle mechanical dissociation maintains sample temperatures below 8°C for the duration of processing, resulting in high-fidelity single cell suspensions with comparable viability and live cell counts to those obtained with conventional enzymatic dissociations. Here, in murine-derived brain, heart, lung, and B16 melanoma tumor tissue dissociated by either BLU or by a commercially available dissociation kit which uses enzymes and heat, we compare cell viability and expression of population-specific immunological markers. The dramatic differences observed in cell surface expression suggest that cells dissociated using enzymes and heat may be experiencing stress-induced changes post-harvest that could impact conclusions and impede research progress. Alternatively, using gentle mechanical dissociation with BLU, we demonstrate the preservation of these markers and enable a minimally invasive alternative to obtaining high integrity single cell suspensions. Highlights ### Competing Interest Statement This work was funded by Cellsonics, an acoustic energy based tissue dissociation device development company. M.A.M, S.Q., and B.Q. are or were at the time of research employed by Cellsonics. V.V. and S.L. have partial ownership of Cellsonics.