Cryopreservative Bioink Enables Direct Bioprinting of Adherent Cells

Cryopreservation-integrated bioprinting represents a promising approach for tissue regeneration by combining cell-laden bioink freezing with direct post-thaw printing, bypassing traditional culturing steps. However, key challenges remain: ice crystallization compromises cellular viability, while hydrogel structural integrity deteriorates, impairing printability. We present a biphasic bioink platform for cryopreservation-enabled three-dimensional (3D) bioprinting-CAMP (Cryopreservation for Adhesion and Maintenance Printing), which enables direct 3D printing at 4-8°C post liquid nitrogen storage (-196°C). CAMP inhibits ice recrystallization through hydrogen bond-mediated water immobilization, achieving approximately 80% cell viability without the use of toxic cryoprotectants. Cryopreserved cells in the bioink retained focal adhesions and increased phosphorylated FAK expression, and the bioink exhibited approximately ten fold higher ice recrystallization inhibition than phosphate-buffered saline. Mechanistically, CAMP suppressed cell death via phospho-FAK signaling. In vivo evaluation using a rat femoral defect model demonstrated the therapeutic efficacy of CAMP, with cryopreserved constructs promoting complete bone regeneration within three months. CAMP overcomes the key limitations of conventional biofabrication by combining cell cryopreservation, bioprinting, and functional tissue formation into a single workflow. By bridging cryopreservation and bioprinting, CAMP represents a significant advance toward clinically viable, ready-to-implant engineered tissues.