Advancing Intra‐Diffusion of Peptide Colloids in Cell‐Clustered Spheroids for Enhanced Cryo‐Recovery

ABSTRACT 3D cellular models have been increasingly integrated into the drug development process, but maintaining their functional integrity over extended periods remains a major barrier to clinical translation and commercialization. Cryopreservation represents a key strategy for enabling long‐term storage of intact 3D cellular systems; however, the limited diffusion of conventional agents into densely organized structures often compromises post‐thaw recovery. We introduce a colloidal solution containing nanoscale peptide‐colloid hybrids designed to preferentially interact with the extracellular matrix. This solution infiltrated spheroids and distributed homogenously through 3D spheroidal space, which outperformed conventional cryoprotectants in preserving post‐thaw structural integrity and functional activity. At sub‐picomolar colloidal concentration, the solution leverages a synergetic Gibbs effect to generate an ice‐melting thermodynamic efficiency comparable to high‐concentration colligative dimethyl sulfoxide (DMSO). Remarkably, the colloid nanoparticles can be readily removed from the spheroids with a single step of post‐thaw rinsing, mitigating the risks associated with residual cytotoxicity.