Development and Application of an Additively Manufactured Calcium Chloride Nebulizer for Alginate 3D-Bioprinting Purposes

Three-dimensional (3D)-bioprinting enables scientists to mimic in vivo micro-environments and to perform in vitro cell experiments under more physiological conditions than is possible with conventional two-dimensional (2D) cell culture. Cell-laden biomaterials (bioinks) are precisely processed to bioengineer tissue three-dimensionally. One primarily used matrix material is sodium alginate. This natural biopolymer provides both fine mechanical properties when gelated and high biocompatibility. Commonly, alginate is 3D bioprinted using extrusion based devices. The gelation reaction is hereby induced by a CaCl₂ solution in the building chamber after material extrusion. This established technique has two main disadvantages: (1) CaCl₂ can have toxic effects on the cell-laden hydrogels by oxygen diffusion limitation and (2) good printing resolution in the CaCl₂ solution is hard to achieve, since the solution needs to be removed afterwards and substituted by cell culture media. Here, we show an innovative approach of alginate bioprinting based on a CaCl₂ nebulizer. The device provides CaCl₂ mist to the building platform inducing the gelation. The necessary amount of CaCl₂ could be decreased as compared to previous gelation strategies and limitation of oxygen transfer during bioprinting can be reduced. The device was manufactured using the MJP-3D printing technique. Subsequently, its digital blueprint (CAD file) can be modified and additive manufactured easily and mounted in various extrusion bioprinters. With our approach, a concept for a more gentle 3D Bioprinting method could be shown. We demonstrated that the concept of an ultrasound-based nebulizer for CaCl₂ mist generation can be used for 3D bioprinting and that the mist-induced polymerization of alginate hydrogels of different concentrations is feasible. Furthermore, different cell-laden alginate concentrations could be used: Cell spheroids (mesenchymal stem cells) and single cells (mouse fibroblasts) were successfully 3D printed yielding viable cells and stable hydrogels after 24 h cultivation. We suggest our work to show a different and novel approach on alginate bioprinting, which could be useful in generating cell-laden hydrogel constructs for e.g., drug screening or (soft) tissue engineering applications.