Coaxial temperature controlled cryoprinting: A biomimetic technology inspired by the freezing survival mechanisms of the frog Rana sylvatica

Authors: LOU, LEO - University Of California Berkeley; WARBURTON, LINNEA - University Of California Berkeley; Bilbao-Sainz, Cristina; JEAN, NICHOLAS - University Of California Berkeley; RUBINSKY, BORIS - University Of California Berkeley

Submitted to: Journal of Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2025
Publication Date: 12/13/2025
Citation: Lou, L., Warburton, L., Bilbao-Sainz, C., Jean, N., Rubinsky, B. 2025. Coaxial temperature controlled cryoprinting: A biomimetic technology inspired by the freezing survival mechanisms of the frog Rana sylvatica. Journal of Cryobiology. 122. Article 105569. https://doi.org/10.1016/j.cryobiol.2025.105569.
DOI: https://doi.org/10.1016/j.cryobiol.2025.105569

Interpretive Summary: We developed Temperature Controlled Cryoprinting (TCC) to enable the immediate freezing of each 3D printed voxel. This instant freezing provides immediate structural rigidity, facilitating the fabrication of complex and large-scale structures that might otherwise collapse or deform. However, to keep the rigidity of the material after thawing, crosslinking is a requirement. Sodium alginate, a polymer derived from seaweed, is widely used as a hydrogel in both 3D printing and Temperature Controlled Cryoprinting (TCC) due to its low cost and excellent biocompatibility. Crosslinking sodium alginate with CaCl2 is a simple and effective method for imparting rigidity to printed structures. However, printing with sodium alginate presents a dilemma. Crosslinked sodium alginate is far too viscous to extrude through the printing nozzle, but uncrosslinked sodium alginate lacks sufficient rigidity to maintain structural fidelity after deposition. Therefore, we have developed a process in which melting progresses outward from the object's surface, synchronized with the diffusion of CaCl2 into the thawed region. This synchronization ensures that the rigidity initially provided by freezing is seamlessly replaced by rigidity resulting from crosslinking.

Technical Abstract: Inspired by the freeze-survival mechanism of the frog Rana sylvatica, we developed a co-axial, self-crosslinking approach to be used when fabricating frozen scaffolds. Soft biomaterial can be fabricated into scaffolds using methods such as Temperature Controlled Cryoprinting(TCC), which uses a freezing plate to freeze the biomaterial as it is extruded. However, it remains a challenge to uniformly crosslink the scaffolds during thawing without them losing their shape. In this paper, we use coaxial printing to generate locally variable phase-transition compositions, mimicking the frog's glucose distribution during freezing, and electrical Joule heating to simulate metabolic heat production. Unlike previous methods, in our novel approach the crosslinker is located already within the frozen scaffold, and there is no need to submerge the scaffold in a bath. Both mathematical modeling and experimental validation confirmed the concept. A finite thawing-diffusion-crosslinking model was developed to analyze parameters such as temperature, thawing interface, concentration, and crosslinking interface. Experimental evaluations, including mechanical testing, swelling performance, and electron microscopy, demonstrated that coaxial self-crosslinking enables controlled thawing and crosslinking of frozen scaffolds, which improved scaffold fabrication. Results demonstrated that Coaxial TCC self-crosslinking scaffolds is feasible, allowing for a more controlled thawing and crosslinking process in cryoprinted scaffolds.