Micromixer driven by bubble-induced acoustic microstreaming for multi-ink 3D bioprinting

Recently, the 3D printing of cell-laden hydrogel structures, known as bioprinting, has received increasing attention owing to advances in tissue engineering and drug screening. However, a micromixing technology that efficiently mixes viscous bioinks under mild conditions is needed. Therefore, this study presents a novel method for achieving homogeneous mixing of multiple inks in 3D bioprinting through acoustic stimulation. This technique involves generating an acoustic microstream through bubble oscillations inside a 3D bioprinting nozzle. We determined the optimal hole design for trapping a bubble, hole arrangement, and voltage for efficient mixing, resulting in a four-fold increase in mixing efficiency compared to a single bubble arrangement. Subsequently, we propose a nozzle design for efficient mixing during bioprinting. The proposed nozzle design enabled the successful printing of line structures with a uniform mixture of different viscous bioinks, achieving a mixing efficiency of over 80% for mixing 0.5-1.0 wt% sodium alginate aqueous solutions. Additionally, acoustic stimulation had no adverse effects on cell viability, maintaining a high cell viability of 88% after extrusion. This study presents the first use of a bubble micromixer in 3D bioprinting, demonstrating gentle yet effective multi-ink mixing. We believe this approach will broaden 3D printing applications, particularly for constructing functional structures in 3D bioprinting.