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Cryopreservation is the only fully established procedure to extend thelifespan of living cells and tissues, a key to activities spanning fromfundamental biology to clinical practice. Despite its prevalence and impact,central aspects of cryopreservation, such as the cell's physico-chemicalenvironment during freezing, remain elusive. Here we address that question bycoupling in situ microscopic directional freezing to visualize cells and theirsurroundings during freezing with the freezing medium phase diagram. We extractthe freezing medium spatial distribution in cryopreservation, providing a toolto describe the cell vicinity at any point during freezing. We show that twomajor events define the cells' local environment over time: the interactionwith the moving ice front and with the vitreous moving front - a term weintroduce here. Our correlative strategy may be applied to cells relevant inclinical research and practice, and help designing new cryoprotective mediabased on local physico-chemical cues.

Unveiling cells' local environment during cryopreservation by correlative in situ spatial and thermal analyses