Although sterile nitrogen sources are available, sterile working conditions are expensive and delicate [33], [35] and [36]. Also, direct handling of Thermanox© substrates is difficult due their small size and overlapping during cultivation. The cultivation surface has to be as thin as possible to achieve the very high heat transfer rates needed Cyclopamine datasheet for vitrification and re-warming. In this work, the consequent advancement of the surface based vitrification technique on modified Thermanox© substrates led to the development of the “twisted vitrification” technique and a respective cultivation and vitrification

device. It is based on a two compartment system with a thin cultivation surface separating the two compartments. It allows the adherent cultivation of hESC colonies and a surrounding feeder layer without constraints to the normal hESC culture. To avoid direct contact with liquid nitrogen Fulvestrant of the samples, vitrification and re-warming of the cells was achieved through the cultivation surface. hESC cell colonies cryopreserved by “twisted vitrification “showed almost no colony- or cell-loss caused by the vitrification and thawing process (Fig. 3A–H). Only small areas in the border regions of the cultivation surface showed partial cell- and colony loss, probably due to inhomogeneities in the thickness of the CPA film covering the cells during vitrification (Fig. 3, asterisks). Too much medium (e.g. a meniscus) reduces

the surface to volume ratio and cooling rates are too low for successful vitrification, resulting in ice crystallization. However, the high survival rates imply that cooling rates achieved through the cultivation surface were high enough to permit successful vitrification although there is no independent confirmation of this. Vital residual areas show an increase in the “twisted vitrification” prototype 99% (±1%) compared to vitrification

on modified Thermanox© discs (89% (±11%). This improvement may be the result of reducing the mechanical stress caused by the constant movement of discs through media and liquid nitrogen. Overall recovery and growth rate of the colonies during the first 24 h post-thaw showed no significant difference Cyclin-dependent kinase 3 from non-frozen control colonies. Apoptosis (seen in slow-rate freezing) can be excluded as a source of cell loss after thawing [17]. Post-thaw functionality is not severely affected by “twisted vitrification”. FACS analysis of Tra-1-81 and Oct-4 was not significantly different from a non-frozen control (Fig. 5) and further passage and cultivation of thawed colonies did not result in morphological differences to control colonies (Fig. 3I–L). Although the overall cryopreservation success and post-thawing functionality are very satisfying, the prototype can be improved. The rim of the nitrogen compartment has to be detached to allow high magnification microscopy inside the device. Otherwise, the working distance is too large, so microscopy is not possible.