Cryopreservation and post-thaw differentiation of monocytes enabled by macromolecular cryoprotectants which restrict intracellular ice formation.

THP-1 is a monocytic cell line which can differentiate into macrophage and dendritic cells, widely used in immunology. Immune cells are particularly sensitive to cryopreservation, leading to low recovery and/or reduced differentiation capacity compared to non-frozen cells. Current cryopreservation protocols are unsuitable to cryopreserve THP-1 cells in 'assay-ready' format, due to the time and resource intensive culturing steps required post-thaw to recover functional cells. We report the cryopreservation of THP-1 cells in vial and multi-well plate format, with significantly enhanced recovery compared to commercial cryoprotectants. This was achieved using macromolecular cryoprotectants (polyampholytes and ice nucleators) which doubled post-thaw recovery relative to DMSO-alone and improved macrophage phenotype post-differentiation comparable to non-frozen controls. Cryo-Raman microscopy demonstrated that the polyampholytes reduced intracellular ice formation compared to DMSO-alone. These results will enable routine banking and 'assay-ready' THP-1 cells direct from the freezer, accelerating immunological research.