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

在现有的生物打印工艺中，使用的打印喷嘴是限制因素，因为它们限制了打印分辨率。如果喷嘴直径太小，喷嘴就会堵塞。此外，随着喷嘴直径的减小，细胞上的剪切应力显著增加。在临界剪切应力以上，打印细胞在机械上不可逆转地损坏。声学液滴喷射（ADE）方法可以成功地解决这些局限性和问题。在项目的第一阶段，我们可以证明，利用ADE技术，可以在毫米尺度上三维构建细胞负载的水凝胶结构。在打印过程中，嵌入的人间充质干细胞显示出非常高的活力。通过流体力学有限元模拟，我们能够证明，与使用基于微阀的喷墨工艺进行生物打印相比，使用ADE技术打印细胞时的剪切应力几乎降低了三倍。在第二个项目阶段，我们希望找到一些基础科学问题的答案，这些问题在第一个研究阶段尚未得到最终的回答。根据这些科学问题，我们为项目的第二阶段提出了四个工作假设：事实上，ADE工艺不需要喷嘴，这意味着，与现有的生物打印技术相比，它也可以打印具有极高细胞浓度的液滴，接近生理细胞密度。2)结合ADE和微流体，将单个细胞分离，转移到弹射点，可以进行精确的3D打印。3)复杂凝胶技术的凝胶也可以用ADE进行加工。4)承载细胞的液滴在撞击构建平台时的行为分析和过程控制是理解如何使用ADE技术可重复构建承载细胞的三维水凝胶结构的基本前提。在研究项目的第二阶段，测试工作假设的关键是在声学生物打印系统中额外实施高精度3D测量技术。这将使在打印过程中测量打印对象成为可能，并详细研究密度、速度、液滴直径和表面张力对承载细胞的液滴在建筑平台上的冲击和液滴彼此融合的影响，以及生物打印细胞的过程相关响应。基于预期结果和第二阶段项目的新发现，声波生物打印方法最终可以与现有的基于喷嘴的打印工艺进行直接比较，从而全面科学地评估。

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.