Effect of Slow Freezing Versus Vitrification on the Recovery of Mouse Embryonic Stem Cells

The purpose of this study was to cryopreserve mouse embryonic stem (ES) cells R1 line and determine cell viability, morphology, the number of colonies, and the Alkaline Phosphatase (AP) activity. In addition, the expression of transcription factors such as the stage-specific antigen (SSEA-1) and Octamer-4 (Oct-4) were evaluated before and after cryopreservation. The effects of two methods of cryopreservation, slow freezing and vitrification, were studied. The ES cells were cryopreserved either as single cells or as clumps. The viability of a single cell after slow freezing was 88%, but after vitrification no single cell was recovered. Surviving clumps after slow freezing quickly recovered and exhibited a morphology indistinguishable from noncryopreserved cells. After vitrification, 2 weeks of culture were required for the cells in clumps to proliferate enough for subculturing. Analysis of cloning efficiency and the colonies morphology were based on a mouse colony rating scale and their characteristic. The colonies from the slow-freezing group were compared to colonies from the control group, which were the cells before cryopreservation, and they showed the same cloning efficiency and morphology. The colonies from the vitrified group were compared to the colonies from the control group, and they showed differences in cloning efficiency on the mouse colony rating scale A (<0.05) and C (<0.05) but they did not show differences in their morphology. The biggest clumps from both experimental groups showed a reduction of viability in the center area compared to the fresh ones. The survival rate of the clumps in the slow-freezing and rapid-thawing group was 75% and in the vitrified group 25%. The colonies from the control group and both experimental cryopreservation groups show the same activity of AP, and they were all positive for SSEA-1 and Oct-4. The conventional slow-freezing method of cryopreservation of single cells and clumps is reliable and effective for the cryopreservation of mouse R1 ES cells. Vitrification can be used for cryopreservation of these same cell clumps but with lower recovery using the conditions that we used.