Cooling-rate dependence of the cryopreservation of aquaporin-overexpressing cells with a non-permeable cryoprotectant

Abstract

Dehydration of intracellular water is an important factor in the cryopreservation of cells, but questions remain as to the appropriate amount and timing of dehydration and the detailed mechanism of the freezing process. Answering these questions will lead to improvements in cryopreservation methods that have remained unchanged for more than half a century and to an increase in the number of cell types that can be cryopreserved. Therefore, we aimed to reveal the time point when cells were dehydrated in their cooling process and how much their viabilities were improved by dehydration. We conducted cryopreservation experiments using cells with enhanced water permeability due to membrane overexpression of the water transport channel protein (AQP4). The AQP4-expressing cells or non-AQP4-expressing cells were cryopreserved under different cooling rates after addition of the membrane-permeable cryoprotectant (CPA) Me₂SO, the non-membrane-permeable CPA trehalose, or no CPA. The results showed that no cryopreservation was successful without CPAs, even in the AQP4-expressing cells with increased water permeability. At slow freezing rates below 35 °C/min, viability with Me₂SO was maintained with decreasing in the cooling rate, but with trehalose, the viability decreased. At cooling rates above 80 °C/min, the viability of AQP4-expressing cells was significantly higher than that of AQP4-non-expressing cells. These results suggest that dehydration due to the osmotic-pressure difference generated after extracellular freezing is fatal to cells.