AbstractThis article describes two types of flow‐through cell retention devices based on the concept of layered piezoelectric resonators. A single‐chamber device is compared to a novel optimized steam‐sterilizable prototype ultrasonic cell separator with improved acoustic design and an integrated cooling circuit, eliminating the problem of local temperature increase caused by the high amplitudes necessary to achieve the separation of animal cells with low acoustic contrast. This setup yields highly reproducible results and is ideal for studying the long‐term effects of ultrasonic sound fields and separation efficiency. The novel two‐chamber system has the potential for scaleability due to the reduction in thermal and acoustic flow, increased field stability, and separation efficiency. Finally, the effect of power input on separation and cell viability is reported. Such flow‐through cell retention systems could be used as systems to retain biomass within the fermentor or as a substitute for centrifugation, with the major advantage of eliminating high‐speed rota
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