RESUMEN
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.
