DeepFreeze 3D‐biofabrication for Bioengineering and Storage of Stem Cells in Thick and Large‐Scale Human Tissue Analogs

Abstract 3D bioprinting holds great promise for meeting the increasing need for transplantable tissues and organs. However, slow printing, interlayer mixing, and the extended exposure of cells to non‐physiological conditions in thick structures still hinder clinical applications. Here the DeepFreeze‐3D (DF‐3D) procedure and bioink for creating multilayered human‐scale tissue mimetics is presented for the first time. The bioink is tailored to support stem cell viability, throughout the rapid freeform DF‐3D biofabrication process. While the printer nozzle is warmed to room temperature, each layer solidifies at contact with the stage (‐80 °C), or the subsequent layers, ensuring precise separation. After thawing, the encapsulated stem cells remain viable without interlayer mixing or delamination. The composed cell‐laden constructs can be cryogenically stored and thawed when needed. Moreover, it is shown that under inductive conditions the stem cells differentiate into bone‐like cells and grow for months after thawing, to form large tissue‐mimetics in the scale of centimeters. This is important, as this approach allows the generation and storage of tissue mimetics in the size and thickness of human tissues. Therefore, DF‐3D biofabrication opens new avenues for generating off‐the‐shelf human tissue analogs. It further holds the potential for regenerative treatments and for studying tissue pathologies caused by disease, tumor, or trauma.