Effect of Pectins on Water Crystallization Pattern and Integrity of Cells During Freezing.

The ability of various pectin polysaccharides to modify the morphological structure of ice during the phase transitions from water to ice was studied. Pectins were isolated from Sosnowsky's hogweed Heracleum sosnowskyi Manden (heracleuman-N6HS), tansy Tanacetum vulgare L. (tanacetan-N7TVF), and Rauwolfia serpentina Benth callus (rauwolfian-N8RS). Pectins were isolated by multistep extraction. The effect of pectins was assessed using osmometry, thermographic analysis, and cryomicroscopy. A concentrate of leukocytes was used as the sample for the subsequent freezing step. The condition of the leukocyte membrane, and lysosomal and phagocytic activity after a freezing-warming process were assessed. Osmotic concentrations of the pectin polysaccharide solutions were found to be very low. The 0.4 wt % N7TVF solution had the highest osmotic concentration as well as freezing point; however, the duration of its crystallization plateau was lower than that of the 0.4 wt % and 0.2 wt % N6HS solutions. All studied polysaccharide solutions demonstrated a high linear rate of ice crystal growth. There were statistically significant differences between the melting rates for the 0.2% solutions of the pectins, N6HS and N7TVF, N6HS and N8RS, as well as between concentrations for the pectin N7TVF and between concentrations for the pectin N8RS. The data on the integrity of cells that are frozen in a medium containing polysaccharides may indicate a cryoprotective effect of the N7TVF and N8RS pectins, that is, tanacetan from tansy and rauwolfian from rauwolfia. The most effective modifier among the substances, which were studied by us, was the N7TVF pectin polysaccharide (tanacetan from tansy).

Towards biobanking technologies for natural and bioengineered multicellular placental constructs.

Clinical application of a large variety of biomaterials is limited by the imperfections in storage technology. Perspective approaches utilizing low-temperature storage are especially challenging for multicellular structures, such as tissues, organs, and bioengineered constructs. Placenta, as a temporary organ, is a widely available unique biological material, being among the most promising sources of various cells and tissues for clinical and experimental use in regenerative medicine and tissue engineering. The aim of this study was to analyse the mechanisms of cryoinjuries in different placental tissues and bioengineered constructs as well as to support the viability after low temperature storage, which would contribute to development of efficient biobanking technologies. This study shows that specificity of cryodamage depends on the structure of the studied object, intercellular bonds, as well as interaction of its components with cryoprotective agents. Remarkably, it was possible to efficiently isolate cells after thawing from all of the studied tissues. While the outcome was lower in comparison to the native non-frozen samples, the phenotype and expression levels of pluripotency genes remained unaffected. Further progress in eliminating of recrystallization processes during thawing would significantly improve biobanking technologies for multicellular constructs and tissues.

Influence of temperature fluctuations during cryopreservation on vital parameters, differentiation potential, and transgene expression of placental multipotent stromal cells.

BACKGROUND: Successful 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. METHODS: In 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. RESULTS: It 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. CONCLUSIONS: Alterations 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.

Physical states of aqueous solutions of oxyethylated glycerol with polymerization degree of n=30 at temperatures lower than 283 K.

A binary system water-oxyethylated glycerol with polymerization degree of n=30 (OEGn=30) is studied over the concentration range of 0-100% (w/w) and the temperature range of 123-283 K by differential scanning calorimetry (DSC). A phase diagram of this system is constructed at the average cooling rate of 200 K-1 per min and the warming rate of 0.5 K-1 per min. Ice crystallization occurs and 70% (w/w) unfrozen residues remain during cooling in the OEGn=30 concentration range of 0-45% (w/w). The system hardens in the amorphous state during cooling and ice crystallization occurs during warming in the range of 46-62% (w/w). Crystallization is observed neither during the cooling nor the subsequent warming in the range of 63-74% (w/w). OEGn=30 crystallization occurs in the range of 75-100% (w/w). Each OEGn=30 molecule can strongly bind to 48 water molecules. In parallel with dsc study, the water-OEGn=30 system is investigated by cryomicroscopy in the temperature range of 173-283 K. Cryomicrographs of the solidified solutions representing each of the four concentration bands mentioned above are given.