Acoustic droplet ejection enables the transfer of induced pluripotent stem cells in ultra-high concentrations while preserving their pluripotency

Acoustic droplet ejection (ADE) has been widely used for the precise transfer of liquids, hydrogels, and cell suspensions in different applications. One interesting advantage of this technique is its nozzle-less nature, allowing for low-shear droplet generation without the risk of clogging. This is especially relevant for printing biomimetic constructs with cell densities in different physiological ranges. To demonstrate the advantages of ADE over other drop-ondemand (DoD) bioprinting methods, cell suspensions at a concentration of at least 10 8 cells per milliliter are transferred acoustically and using a commercial piezoelectric DoD printhead as control. In addition, the impact of ADE on the post-printing behavior of induced Pluripotent Stem Cells (iPSC) is analyzed. While the acoustic droplet transfer of the cell suspensions is reliably performed until the depletion of the reservoir, the commercial printhead fails due to nozzle clogging. Live/dead staining measurements show no significant differences between acoustically transferred and pipetted iPSC. Immunofluorescence micrographs and flow cytometry measurements confirm the preservation of the cells' biological functionality, showing that acoustically transferred iPSC remain pluripotent seven days post-printing. In conclusion, ADE allows for the transfer of cell densities in physiological ranges, while even preserving the biological functionality of delicate iPSC.