Mass Production of Uniform Embryoid Bodies by Acoustic Standing Waves.

Embryoid bodies (EBs) derived from human induced pluripotent stem cells (hiPSCs) provide the basis to obtain any type of organotypic cells and even complex multicellular organoids in 3D cell culture. However, traditional methods for generating EBs are labor-intensive and often lack control over size uniformity. Here, this work presents a novel method for generating thousands of uniform EBs using acoustic standing waves in a single step. By utilizing piezoelectric ceramics to create controlled acoustic fields, this work achieves rapid, scaffold-free aggregation of hiPSCs. This work demonstrates precise control of EB size by adjusting the ultrasound frequency and the cell seeding density, resulting in EB diameters ranging from 70 to 320 microns. This method enables the simultaneous formation of up to 28 000 EBs with more uniform size compared to those formed by the established ultra-low-attachment plate method. The generated EBs maintain pluripotency after 24 h of ultrasound exposure, as indicated by successful staining of key pluripotency markers. The EBs are successfully differentiated into functional, spontaneously contracting cardiomyocyte clusters. This novel method offers a low-cost, scalable and efficient approach to produce a large amount of functional and uniform EBs serving as a starting material to produce cell clusters and organoids in suspension cultures or bioreactors.