Cryopreservation of mammalian somatic cells must be carefully controlled to ensure maximum, post-thaw recovery using cooling rates slow enough to allow time for cells to cryodehydrate sufficiently thereby avoiding lethal intracellular ice. In this study, we looked at the effect of different transfer temperatures to cryogenic storage after slow, controlled-rate cooling on the post-thaw recovery of a range of cell lines derived from T lymphocyte (Jurkat), liver (HepG2), ovary (CHO) and bone (MG63). We found that slow, controlled-rate cooling should be conducted down to-50C to ensure optimal cell recovery and metabolic activity post-thaw across all four cell lines tested, as opposed to-80C,-100C or even lower temperatures in common practice. We have also demonstrated that this critical temperature is linked to a physical event that cells undergo between-47 and -59 °C in the presence of the cryoprotective agent dimethyl sulfoxide: an intra cellular, colloidal glass transition (Tg’i), as detected by differential scanning calorimetry (DSC). This study ultimately shows that shorter, more time and cost-efficient cryopreservation protocols can therefore safely be applied, and maybe help expand the adoption of cryopreservation in particular in the cell therapy space.