UC Berkeley

There are over 100,000 patients on the US transplant list alone, and millions more needing transplants globally. Many of these transplant patients have little hope of getting a replacement organ in time to ensure survival. On-demand fabrication of organs and tissues using tissue engineering could provide a solution and save the lives of millions of transplant patients and patients with end-stage organ failure. A major component of fabricating these artificial tissues and organs is the construction of a scaffold, often with the cells incorporated, that simulates the appropriate tissue environment and guides the formation of complex tissue structures. Three-dimensional (3D) bioprinting has emerged as the most promising approach for developing these scaffold constructs, but despite significant advances, the technique faces various challenges. Some factors that have thus far limited the production of clinically relevant constructs include, insufficient mechanical properties for fabrication of full-scale organs, unsustainably long print times, and poor survival of the biological matter during and after printing due to lack of vascularization. Current techniques and emerging methods still fall short of addressing some of these limitations, so in order for continued advancement in this field, alternative approaches are needed.

This work explores in detail a novel technique of doing 3D bioprinting at subfreezing temperatures, called 3D cryoprinting. 3D cryoprinting incorporates the benefits of ice formation at subfreezing temperatures and the flexibility afforded by super soft biomaterials to improve the fabrication of soft tissue scaffolds. This method utilizes controlled freezing to improve mechanical properties of the printed matrix while simultaneously cryopreserving the biological matter. In order to establish 3D cryoprinting as a viable alternative for 3D bioprinting, the challenges and unknown parameters associated with 3D cryoprinting such as the solidification in large objects, cell viability, structural stability during melting, and mass manufacturability are tackled. Several methods and models to control and understand freezing during cryoprinting are developed, including a thermal phase change model for controlling the freezing rate of biological matter during cryoprinting, which substantially improves the process outcome. A method that enables the preservation of the geometric shape after thawing, a crucial element to the viability of 3D cryoprinting, is also developed. And to speed up manufacture and enable mass manufacturing a modified process of cryoprinting is developed, called parallel multilayer cryolithography. The culmination of this work demonstrates that 3D cryoprinting could be a viable alternative for the fabrication of complex 3D tissue and organ scaffolds, especially in cases of super soft tissue scaffolds where other bioprinting methods have proven inadequate.