Methods, systems, and apparatus for acoustic separation of cellular supporting materials such as microcarriers or microbubbles from cell culture are provided. In one aspect, a method includes flowing a fluid containing the cellular supporting material and the cells through a flow chamber; driving at least one acoustic transducer to launch an acoustic wave from the acoustic transducer positioned on a first wall of the flow chamber to a reflector positioned on a second wall of the flow chamber to create, in the flow chamber, a multi-dimensional field that includes first spatial locales where acoustic pressure amplitude is elevated from a baseline level when the acoustic transducer is turned off, and second spatial locales where acoustic pressure amplitude is substantially identical to the baseline level when the acoustic transducer is turned off, wherein the first wall is opposite to the second wall; preferentially trapping, and gravity separating, the cellular supporting material or the cells at the first or second spatial locales inside the flow chamber; and collecting cells separated from the cellular supporting material using a first collection duct coupled to the flow chamber.