Cryogenic bioprinting – that is bioprinting on a cooling plate in a temperature controlled cooling chamber – could open new opportunities in terms of producing soft organ tissue. It has been shown that the stiffness of the majority of human tissues lies within the order of a few kPa. Furthermore, in specific cases, cell differentiation and regeneration are promoted in tissue scaffolds that exhibit mechanical properties similar to those of the real tissue. Therefore, a 3D printing technique that is able to produce geometrically and mechanically accurate scaffolds could hold enormous potential in regenerative medicine and biomimetics. This reinforces the importance of soft 3D printing. As of today there still is a lack of studies focusing on bioprinting very soft materials with stiffness O(1) kPa. One of the causes of this is the inability of extremely soft materials to withstand their own weight: the printed structure is usually too soft to hold its shape or allow further layers to be built on top of it.
Hinton et al. have developed a technique for free-form extrusion-based 3D printing of biological structures (e.g. arterial branches) using alginate, collagen and fibrin gels as printing inks and a gelatine slurry as a support bath. The technique was able to achieve a resolution of ~200 µm demonstrated through the printing of a scaled down human brain using an alginate bioink. However, the stiffness of the alginate ink was reported to be O(10) kPa, and therefore not comparable with that of super soft tissues, such as human brain or lung (O(1) kPa). In another study, Lozano et al. used an RGD modified gellan gum 1 wt% hydrogel bioink with encapsulated cortical neuron cells. The authors were able to demonstrate the ability to print soft 3D cell-laden constructs. However, the printing process was achieved through a hand-held device, hence lacking precision, and the material stiffness was not characterized.
Adamkiewicz et al. introduced a novel cryogenic 3D printing method using liquid nitrogen. The conceptual idea behind the cryogenic method is that it allows inks in a solution state to transform into a solid state, thus allowing stable structures to be built in 3D using a layer-by-layer approach, without the need for a support bath. However, the stiffness of the hydrogel ink was not reported and the precision of the printing method was not discussed. The cryogenic method was also used to create 2D constructs for implants by Wang et al., who utilized a substrate cooled by coolant flow to create the cryogenic stage. Again, mechanical characterization of the printed structure was not reported.
Therefore, this study demonstrates the fabrication of mechanically accurate 3D printed composite hydrogels that mimic the stiffness of super soft tissues through the use of a novel printing setup based on cryogenic theory. Solid carbon dioxide (dry ice) and an isopropanol thermal conductive bath were used to achieve the cryogenic stage, which is a safer alternative to liquid nitrogen. The ink used in this work is a composite hydrogel of poly(vinyl) alcohol (PVA) and Phytagel, which has been pioneered by Leibinger et al. and Forte et al. to mimic soft tissues, such as a brain, with a stiffness of O(1) kPa.
A further advantage of this novel 3D printing technique over traditional cast molding methods resides in the possibility to produce hollow structures of super soft hydrogels. Interconnected holes make soft hollow structures impossible to extract from a mold using traditional cast molding techniques.