Vitrification has become a method of choice for hESCs and other delicate cell types because of high survival rates and good functionality after thawing [21], [29], [41], [50] and [51]. However, disadvantages of current vitrification-protocols are the small numbers of colonies that can be vitrified at the same time (about 5–10 hESC-clumps) and enzymatic stress due to detachment of cell clumps. Additionally, success is highly dependent on the expertise of the researcher. Handling is difficult and there can be significant cell loss caused by inaccurate incubation times in the highly toxic cryopreservation media [25], [26], [27], [42] and [47]. Vitrification in straws for example

is quite challenging when it comes to transferring the cell ICG-001 clumps into different CPA solutions manually, while maintaining exact incubation times or recovering the few cell clumps from the warming solutions after thawing. Other vitrification principles, e.g. cryoloop vitrification, are only designed to vitrify very few samples at a time, while those developed for more colonies suffer from sterility issues or complicated workflows including manual colony detachment which make them unsuited for automated throughput systems. Although the recently introduced surface-based cryopreservation technique resulted in very high learn more post-thawing survival

rates and low differentiation, it still has Cytidine deaminase limitations that need to be overcome. To achieve the very rapid cooling and warming rates that are needed to successfully

vitrify and devitrify the cells, the samples were immersed directly into liquid nitrogen [5] and [49]. However, the non-sterile properties of liquid nitrogen increase the risk of contamination and infection and can lead to a propagation of contamination from one sample to another [7], [10], [15], [32] and [33]. Even though sterile liquid nitrogen sources are available, maintaining sterile working conditions remains expensive and awkward [35] and [36]. Hence, the development of a successful sterile cryopreservation method for bulk quantities of hESCs is highly appreciated. Also, the technique on modified Thermanox© slides is error prone due to difficult handling issues, overlapping of the discs and the need to explicitly cultivate the cell samples on the modified discs prior to cryopreservation and storage. Therefore this technique needs improvements in sterility, handling, and practicability. The aim of this study was to develop a new cryopreservation technique using a combined cultivation and cryopreservation device. The technique should enable the cultivation of hESCs on a cell culture surface in combination with an efficient vitrification of the adherent stem cell colonies, including the feeder layer, without direct contact with liquid nitrogen.