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

Abstract

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...