BackgroundSuccessful implementation of rapidly advancing regenerative medicine approaches has led to high demand for readily available cellular suspensions. In particular, multipotent stromal cells (MSCs) of placental origin have shown therapeutic efficiency in the treatment of numerous pathologies of varied etiology. Up to now, cryopreservation is the only effective way to preserve the viability and unique properties of such cells in the long term. However, practical biobanking is often associated with repeated temperature fluctuations or interruption of a cold chain due to various technical, transportation, and stocking events. While biochemical processes are expected to be suspended during cryopreservation, such temperature fluctuations may lead to accumulation of stress as well as to periodic release of water fractions in the samples, possibly leading to damage during long-term storage.MethodsIn this study, we performed a comprehensive analysis of changes in cell survival, vital parameters, and differentiation potential, as well as transgene expression of placental MSCs after temperature fluctuations within the liquid nitrogen steam storage, mimicking long-term preservation in practical biobanking, transportation, and temporal storage.ResultsIt was shown that viability and metabolic parameters of placental MSCs did not significantly differ after temperature fluctuations in the range from –196 °C to –100 °C in less than 20 cycles in comparison to constant temperature storage. However, increasing the temperature range to –80 °C as well as increasing the number of cycles leads to significant lowering of these parameters after thawing. The number of apoptotic changes increases depending on the number of cycles of temperature fluctuations. Besides, adhesive properties of the cells after thawing are significantly compromised in the samples subjected to temperature fluctuations during storage. Differentiation potential of placental MSCs was not compromised after cryopreservation with constant end temperatures or with temperature fluctuations. However, regulation of various genes after cryopreservation procedures significantly varies. Interestingly, transgene expression was not compromised in any of the studied samples.ConclusionsAlterations in structural and functional parameters of placental MSCs after long-term preservation should be considered in practical biobanking due to potential temperature fluctuations in samples. At the same time, differentiation potential and transgene expression are not compromised during studied storage conditions, while variation in gene regulation is observed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0512-7) contains supplementary material, which is available to authorized users.

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Two critical factors for obtaining high viable biological samples after cryopreservation by vitrification method are cryoprotectant glass-forming ability while cooling and the stability of its amorphous state during warming. The present work is devoted to the study of the amorphous state stability of plant vitrification solutions (PVS) by differential scanning calorimetry method. The objects of the study were PVS1 modified, PVS2, PVS3 modified, PVS4 and PVSN. The thermograms of PVSs, frozen in liquid nitrogen, were recorded at a warming rate of 0.5 deg/min. The glass transition temperatures of PVS1, PVS2 and PVS3 were –109.0°C, –115.3°C, and –93.9°C, respectively. Neither exo- nor endothermic peaks have been recorded, indicating no crystallization, at the cooling and the warming stages. PVS4 and PVSN thermograms, besides glass transition (–111.5°C and –110.0°C, correspondingly), showed crystallization from the amorphous state (devitrification) and ice melting peaks. Crystallization enthalpy of PVSN was 2.8 times higher compared to PVS4. This fact testifies that the crystalline phase was larger in PVSN compared to PVS4. It should be noted that crystallization in PVS4 and PVSN may be related to the low warming rate used during the experiment. At higher warming rates, this crystallization is likely to be avoided. Thus, PVS1, PVS2 and PVS3 have a high glass-forming ability and a stabile amorphous state. The amorphous state stability of PVS4 and PVSN is significantly lower compared to PVS1, PVS2, and PVS3.