Background These last decades have seen the emergence and development of cell-based therapies, notably those based on mesenchymal stromal cells (MSCs). The advancement of these promising treatments requires increasing the throughput of processed cell for industrialization in order to reduce production costs. Among the various bioproduction challenges, downstream processing (DSP), including medium exchange, cell washing, cell harvesting and volume reduction, remains a critical step on which improvements are needed. Typically, these processes are performed by centrifugation. However, this approach limits the automation, especially in small batch productions where it is performed manually in open system. Methods An acoustophoresis-based system was developed for cell washing. The cells were transferred from one stream to another via the acoustic forces and were collected in a different medium. The optimal flow rates of the different streams were assessed using Red Blood Cells (RBCs) suspended in an albumin solution. Finally, the impact of acoustic washing on adipose tissue-derived MSCs (AD-MSCs) transcriptome was investigated by RNA-sequencing. Results With a single passage through the acoustic device at input flow rate of 45 mL/h, the albumin removal was up to 90% while recovering 99% of RBCs. To further increase the protein removal, a loop washing in two steps was performed and has allowed an albumin removal ≥99% and a RBCs/AD-MSCs recovery of 99%. After loop washing of AD-MSCs, only 2 genes, HES4 and MIR-3648-1, were differently expressed compared to the input. Conclusion In this study, we developed a continuous cell washing system based on acoustophoresis. The process allows a high cell throughput while inducing little gene expression changes. These results highly suggest that cell washing based on acoustophoresis is a relevant and promising solution for numerous applications in cell manufacturing.