Analyses of human granulosa cell vitality by fluorescence activated cell sorting after rapid cooling

In reproductive medicine, the technique of rapid cooling becomes increasingly important for the preservation of tissue and cells. In order to protect the cells, incubation in different cryopreservation solutions is essential. The speed of the cooling process also makes a pivotal contribution to the success of this method. Using Flourescence Activated Cell Sorting (FACS), we investigated the impact of an open rapid and a closed rapid cooling technique on the vitality of human granulosa cells. Furthermore, we examined effects of the different solutions used for rapid cooling and warming before and after rapid cooling. We found a significant lower proportion of vital cells after rapid cooling compared to untreated controls independently of the technique and the tube size. However, we did not find any significant differences between open and closed rapid cooling. In both, a lower proportion of vital granulosa cells were found after incubation in rapid cooling solution only compared to warming solution only. Our results lend support to the conclusion that the difference of cooling-speed between open and closed rapid cooling is, in our settings, not crucial for the success of the procedure and that cryoprotective agents in the rapid cooling solutions have a higher potential to cause severe cell damage than agents used for warming.