Cryobioprinted human tumor models with shelf-stable programmability

Cryobioprinting enables simultaneous fabrication and cryopreservation of tissue analogs, surpassing the limitations of print-to-use biofabrication approaches. However, cryopreservation of living constructs remains challenging, requiring optimal cryopreservation conditions tailored to specific cell types and hydrogel bioinks. To address this, we explored the formulation of a decellularized extracellular matrix (dECM)-based hydrogel bioink containing cryoprotective agents (CPAs) to generate shelf-ready tumor-stroma pancreatic cancer models. Combinatorial screening of CPAs led to the discovery of a novel melezitose-glycerol-dECM formulation that exhibited superior cryoprotective properties in both tumor and stroma compartments. Exometabolomics analysis revealed that cryopreserved constructs exhibited similar metabolic activity to nonfrozen counterparts 14 days post thawing. Cryobioprinted tumor-stroma models in dECM-CPA bioinks also exhibited increased cell viability post thawing and suitable features for in vitro drug screening. Thus, our optimized cryoprotective strategy opens new opportunities to potentially explore any type of tissue decellularized bioinks for cryobioprinting off-the-shelf living constructs for widespread drug screening and beyond.