Multiscale and cell-preserving 3D bioprinting of human cells by nozzle-free acoustic droplet ejection (AcousticBioprinting)

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In established bioprinting processes, the print nozzles used are the limiting factor, because they limit the print resolution. If the nozzle diameter is too small, the nozzles become clogged. In addition, the shear stress on the cells increases significantly as the nozzle diameter is reduced. Above a critical shear stress, the printed cells are mecha¬nically irreversibly damaged. These limitations and problems could be successfully addressed by the Acoustic Droplet Ejection (ADE) method. In the first phase of the project, we could show that cell-laden hydrogel structures can be built up three-dimensionally on a millimeter scale using the ADE technique. Human mesenchymal stem cells embedded in the single droplets showed a very high viability after the printing process. Using fluid mechanics finite element simulations, we were able to show that the shear stress when printing cells using ADE technology is almost three times lower compared to bioprinting using a microvalve-based inkjet process. In a second project phase, we want to find answers to a number of fundamental scientific questions that have not yet been conclusively answered in the first research phase. In line with these scientific questions, we have stated four working hypotheses for the second phase of the project: 1.) The fact that the ADE process does not require a nozzle means that, in contrast to established bioprinting techniques, it is also possible to print droplets with extremely high cell concentrations close to the physiological cell density. 2) By combining ADE and microfluidics, single cells are seperated, transferred to the ejection spot and can be precisely 3D printed. 3) Gels with complex gelation techniques can also be processed using ADE. 4) The analysis and process control of the behavior of the cell-laden droplets upon impact on the build platform is a basic prerequisite for understanding how three-dimensional cell-laden hydrogel structures can be reproducibly built using ADE technology. An essential key to test the working hypotheses in the second phase of the research project is the additional implementation of high-precision 3D measurement technology to the acoustic bioprinting system. This will make it possible to measure the printed objects even during the printing process and to investigate in detail the effects of density, velocity, droplet diameter, and surface tension on the impact of the cell-laden droplets on the building platform and the fusion of the droplets with each other, as well as the process-related response of the bioprinted cells. Based on the expected results and new findings from the second project phase, the acoustic bioprinting method can finally be comprehensively scientifically evaluated in direct comparison with established nozzle-based printing processes.