6A, B). Affected colonies completely lost adherence to the cultivation surface during the first 48 h of post-thawing cultivation. This effect could be avoided by careful manual aspiration of the CPA medium prior to vitrification. A few experiments resulted in fissures running through the complete cultivation area in a circular fashion (Fig. 6C–G). Affected areas of the hESC colonies and feeder cell layer showed dead cells and cell loss immediately after the thawing process (Fig. 6E–G). Due to the very low number of devices containing these fissures and because this kind of damage is probably caused by limitations of the materials
rather than by weakness in the “twisted vitrification” technique itself, Belinostat datasheet those samples were not integrated into the final evaluation and discarded. The protocol allows cultivation, bulk vitrification, storage and post-thaw cultivation of hESCs in the same device without detachment of the colonies from the surface (Fig. 2) without the use of serum in the cryopreservation media. The prototype (Fig. 3 and Fig. 4) showed very high survival rates and immunological FACS analysis confirmed an undifferentiated state after thawing and further passage (Fig. 5). Vitrification currently seems to be the best choice for hESC cryopreservation,
showing much higher survival and lower differentiation rates after thawing than slow rate freezing approaches  and . Optimal cell dehydration and ice crystal GDC-0199 cost formation, both very important in slow-rate freezing, might be affected by the tight colony morphology of hESCs and result in low success rates ,  and . Cell-to-cell contact is very important for hESCs, which have high numbers of tight junctions, gap junctions and cell adhesion molecules. Its disruption through intra- or extracellular ice crystallization reduces cryopreservation tuclazepam success . Hence, complete prevention of ice crystallization through vitrification can greatly improve cryopreservation success for hESCs , ,  and . Many different vitrification procedures for hESCs have been developed, showing high
survival and low differentiation rates after thawing , ,  and . However, due to heat transfer issues, the number of cells that can be vitrified simultaneously is limited. Successful vitrification requires very high cooling rates, surface-to-volume ratio of the samples therefore is of great importance for a prevention of ice crystallization . However, protocols are usually difficult and awkward, leading to imprecise incubation times in the high concentrated toxic media and a high dependency of cryopreservation success on the skills of the operator. Previous surface-based vitrification techniques using Thermanox© discs gave high survival and low post-thaw differentiation and could handle bulk quantities of hESCs .