A two-step directional freezing method was utilized to assess the membrane integrity in frozen/thawed adult stem cells (ASCs). Using a custom built directional solidification stage (DSS) we determined that cells cooled at two different rates (i.e., a slower rate between room temperature, 27 °C and −10 °C and a higher rate between −10 °C and −60 °C) experienced significantly higher loss of cell viability when compared with those cooled at a single and uniform slower cooling rate between 27 °C and −60 °C. Specifically, the cells cooled using the two-step freezing method exhibited a post-thaw cell viability of ∼17% while those that were cooled using the traditional method exhibited a cell viability of 49%. We also found that varying the cooling rate between 27 °C and −10 °C did not significantly affect the post-thaw viability of ASCs, suggesting that they are not susceptible to supra-zero cooling injury.

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