Zwitterionic polymer-mediated intercellular cryoprotection for efficient vitrification of spheroids

Efficient cryopreservation of three-dimensional (3D) cellular assemblies remains challenging because conventional slow-freezing methods using dimethyl sulfoxide are insufficient and vitrification requires highly concentrated cytotoxic solutes. Here, we report a vitrification-based cryopreservation strategy enabled by intracellular delivery of zwitterionic polymers. Sulfobetaine-type zwitterionic polymers and their l (PEG) block copolymer rapidly permeated cells and were efficiently cleared after thawing. Their addition to ethylene glycol-based vitrification solutions (VSs) suppressed ice crystallization during cooling and warming. In mesenchymal stem cell spheroids, intracellular polymer permeation improved post-thaw viability and recovery compared with VS alone or with VS containing nonpermeable polymers. Spheroids vitrified with intracellularly permeable polymers exhibited improved fusion behavior, proliferation, and preserved differentiation capacities after thawing. These protective effects were maintained at reduced ethylene glycol concentrations, indicating reduced vitrification toxicity. These results demonstrate that transient intracellular stabilization using zwitterionic polymers is an effective strategy for vitrification of complex 3D cellular assemblies.