Viability of the Human Adenocarcinoma Cell Line Caco-2: Influence of Cryoprotectant, Freezing Rate, and Storage Temperature

The freezing process as well as cryostorage may induce molecular and cellular changes due to osmotic stress. Currently, standard protocols for cryopreservation of mammalian cells recommend slow freezing and rapid thawing to avoid intracellular ice crystal formation and osmotic damage. As an appropriate freezing process should guarantee adequate cell viability, we evaluated the impact of two cryoprotectants, a commercially available cryopreservation medium, two freezing rates, as well as two storage temperatures on the viability of Caco-2 cells. The freezing parameters were optimized by carefully determining vitality, cell count, proliferation, and functional differentiation of the cells. Though at least 90% of the cells were viable after one freezing/thawing cycle, adequate recovery of proliferation and differentiation is obtained not until 10 days post thawing. Interestingly, addition of 10% PEG 200 as cryoprotectant yielded best results. All in all, our results should give valuable advice for the optimization of the cryopreservation protocol of individual cell lines.