Jentsch, Stefan (Department of Dental Materials and Biomaterials Research, RWTH Aachen University Hospital )


"During the last decade, bioprinting technology has gradually made tremendous progress in manufacturing complex cell-laden hydrogel-based constructs especially for advanced 3D in vitro models. However, most models still lack full complexity. The combination of multiple state-of-the-art printing processes is currently expected to make up for this shortcoming and to improve the outcome after cultivation.
Most state-of-the-art bioprinting processes typically use a nozzle to print droplets or extrude material. The nozzles are a critical component because they must be selected with a tiny diameter for high-resolution printing and quickly become clogged during the printing process. As the nozzle diameter is reduced and the speed in the nozzle is increased, the shear stress increases. Critical shear stresses lead to sustained damage of the printed cells. The acoustic droplet ejection process presented here does not require a nozzle at all. Therefore, nozzle-related wall shear stress is eliminated. The technique, which has so far been associated mainly with single-cell printing1, has recently been made usable for macroscopic 3D bioprinting2. However, this printing method is new to the field of biofabrication and has not yet been fully characterized.
Individual droplets were generated by an ultrasonic burst that applied sufficient energy below the liquid surface to overcome the surface tension and inertial forces of the bioink against gravity. Droplets ranging from picoliters to nanoliters were generated. By simple adjustment of the ultrasound, droplets of the order of a single cell can be printed on the one hand, or large droplets of the order of cell agglomerates on the other. The generated cell-laden hydrogel droplets attach gently to a building platform. This way, 3D constructs of several millimeters in size were successfully realized by moving the building platform. The ejected droplets were captured in-flight with a 5-megapixel camera (VCXU-51M, Baumer) and microscope lens. A controlled LED flash captured the droplets during the exposure time. Contour analysis was performed using TwinCAT Vision (Beckhoff). For the evaluation of printable bioinks, Pluronic F-127 and Matrigel were characterized with the camera system. The fluorescent dye CellTracker (Invitrogen, Thermo Fisher Scientific) was used to monitor the location of cells within the 3D-printed construct.
Using the camera approach, it was possible to reveal optimal ultrasound parameters. A characteristic phase map was created for each tested bioink. From these phase maps with their different regimes, the best parameter combinations can be derived for each bioink. 3D structures were printed at best parameter combinations and inspected using confocal microscopy, respectively.
These results are of importance for material-specific optimal bioprinting using acoustic droplet ejection principle. Using the described phase map method, novel hydrogel materials can be quickly evaluated with regard to optimal ejection parameters. This enables optimal acoustic printing and helps to increase the complexity of 3D printed structures. Thus, more precise spacial placement of cell-laden hydrogel droplets at higher printing resolution will be feasible in the future using the acoustic bioprinting technique.
1. Demirci, U., Montesano G., Lab Chip,7, 1139-1145 (2007).
2. Jentsch, S. et al., Small Methods, 5, 2000971 (2021)."


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