Speaker
Description
Introduction
We have already shown that the Acoustic Droplet Ejection (ADE) technology facilitates the precise, nozzle-less transfer of cell-laden bioinks [1]. This technique offers an alternative to traditional bioprinting methods like microextrusion or inkjet, circumventing nozzle-related issues such as clogging and high wall shear stress. By eliminating the nozzle, ADE significantly reduces physical stress on cells and removes limitations on printable cell densities imposed by nozzle dimensions. Now we further hypothesized that the technology could enable the fabrication of more intricate structures and the transfer of physiologically relevant cell densities exceeding 108 cells/ml, which is not possible using established nozzle-based bioprinting methods.
Methods
To enhance the complexity of printed 3D structures, two primary improvements were implemented. First, droplet deposition accuracy was increased using an ultrasonic pulse-receiver system to precisely locate the bioink surface and position the transducer accordingly. Second, a slicing workflow, including a Python script to convert pictures and standard G-codes, was integrated to generate complex geometries readable by the custom acoustic bioprinter. Additionally, the limits of the reachable cell concentrations were tested. For this purpose, different cell types pre-stained with Hoechst solution were centrifuged into a pellet. A negligible amount of cell culture medium was added to increase the moisture content of the pellets, which were then pipetted into the acoustic bioprinter. The cells were then printed onto a six-well well plate in a droplet-wise manner, creating a small pool of cell suspension to increase the ejected volume and keep the cells moist. Further experiments were conducted with a lower cell concentration of at least 108 cells per ml. The survival rate of the printed cells was analyzed under a fluorescence microscope.
Results
The Python scripts were used to transform images or common G-Codes into machine readable commands, allowing for the generation of complex two- and three-dimensional structures. The changes in the workflow also significantly increased the printing speed and allowed for the automatic focusing of the transducer to account for reservoir depletion during printing. Reservoir cell concentrations of more than 108 cells per ml were transferred using the acoustic bioprinter (Fig. 1 a. and b.), showing post-printing viabilities above 95 % (Fig. 1 b.).
Conclusions
The changes implemented into the printing workflow allowed for the generation of significantly more complex two and three-dimensional structures. The results of the cell experiments showed that the printer’s nozzle-less approach allows for ultra-high cell density transfer with excellent post-printing viability.
References
[1] Jentsch S, Nasehi R, Kuckelkorn C, Gundert B, Aveic S, Fischer H (2021). Multiscale 3D bioprinting by nozzle-free acoustic droplet ejection. Small Methods 5:e2000971.
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