The effects of vitrification on oocyte quality

Vitrification, is an ultra-rapid, manual cooling process that produces glass-like (ice crystal free) solidification. Water is prevented from forming intercellular and intracellular ice crystals during cooling as a result of oocyte dehydration and the use of highly concentrated cryoprotectant. Though oocytes can be cryopreserved without ice crystal formation through vitrification, it is still not clear whether the process of vitrification causes any negative impact (temperature change/chilling effect, osmotic stress, cryoprotectant toxicity, and/or phase transitions) on oocyte quality that translate to diminished embryo developmental potential or subsequent clinical outcomes. In this review, we attempt to assess the technique’s potential effects and the consequence of these effects on outcomes.

A Non-Parametric Method for Estimating Rates of Intracellular Ice Formation Due to Independent Competing Mechanisms

The probability of intracellular ice formation (IIF) has conventionally been analyzed by counting the cumulative number of IIF events observed in a cell population, and normalizing to the total cell count to estimate the cumulative IIF probability. However, this method is invalid when attempting to distinguish among multiple, independent IIF mechanisms, because of confounding effects due to competition for a finite pool of unfrozen cells. Therefore, an alternative approach for analyzing IIF data is proposed, based on treating IIF as a marked point process, in which the points represent IIF events and the marks represent different mechanisms of IIF. Using the new method, it is possible to quantify the kinetics associated with any IIF mechanism for which corresponding events can be marked (experimentally distinguished from competing IIF mechanisms). The proposed approach is non-parametric, making possible characterization of IIF mechanisms that have not yet been fully elucidated. The new analytical approach was compared to the conventional method of IIF analysis using data from a simulated experiment, demonstrating that the new method yielded superior estimates of the cumulative distribution function of IIF times when two competing mechanisms of IIF were active. The proposed algorithm was also applied to cryomicroscopic IIF observations in adherent endothelial cells, yielding rate estimates for two concurrent IIF processes. Furthermore, a proof is presented to demonstrate that when the proposed data analysis algorithm is applied to IIF data from a single mechanism of IIF, the results are equivalent to those obtained by the conventional method of analysis.

The transfer temperature from slow cooling to cryogenic storage is critical for optimal recovery of cryopreserved mammalian cells

Cryopreservation is a key step for the effective delivery of many cell therapies and for the maintenance of biological materials for research. The preservation process must be carefully controlled to ensure maximum, post-thaw recovery using cooling rates slow enough to allow time for cells to cryodehydrate sufficiently to avoid lethal intracellular ice. This study focuses on determining the temperature necessary at the end of controlled slow cooling before transfer to cryogenic storage which ensures optimal recovery of the processed cell samples. Using nucleated, mammalian cell lines derived from liver (HepG2), ovary (CHO) and bone tissue (MG63) this study has shown that cooling must be controlled to -40°C before transfer to long term storage to ensure optimal cell recovery. No further advantage was seen by controlling cooling to lower temperatures. These results are consistent with collected differential scanning calorimetry data, that indicated the cells underwent an intracellular, colloidal glass transition between -49 and -59°C (Tg’i) in the presence of the cryoprotective agent dimethyl sulfoxide (DMSO). The glass forms at the point of maximum cryodehydration and no further cellular dehydration is possible. At this point the risk of lethal intracellular ice forming on transfer to ultra-low temperature storage is eliminated. In practice it may not be necessary to continue slow cooling to below this temperature as optimal recovery at -40°C indicates that the cells have become sufficiently dehydrated to avoid further, significant damage when transferred into ultra-low temperature storage.

Fast Drying Combined With Rapid Cooling Can Enhance The Survival of The Desiccation Sensitive Wheat Pollen After Ultra-Low Temperature Storage

Long-term storage of pollen is important for the fertilization of spatially or temporally isolated female parents, especially during hybrid breeding. Wheat pollen is dehydration-sensitive and rapidly loses viability after shedding. To preserve wheat pollen, we hypothesized that fast-(flash)-drying and fast cooling (150°C min-1) compared to slow-(air)-drying and slow cooling (1°C min-1) would increase the rate of intracellular water content (WC) removal, decrease intracellular ice crystal formation, and increase viability after exposure to ultra-low temperatures. High correlations were found between pollen WC and viability analyzed by impedance flow cytometry (IFC viability: r=0.92, P<0.001) and pollen germination (r=0.94, P<0.001). After 10 min of air-drying, 66% WC was lost and pollen germination was at 12.2±12.3%. After 10 min of flash-drying, WC of pollen reduced by 74%. IFC viability decreased from 90.2±6.7 to 39.4±17.9%, and pollen germination dropped from 33.7±16.9 to 1.9±3.9%. After 12 min of flash-drying, WCs decreased to <0.34 mg H2O mg-1 DW, ice crystal formation was completely prevented (ΔH=0 J mg-1 DW), and pollen germination reached 1.2±1.0%. After slow and fast cooling, flash-dried pollen (WC 0.91±0.11 mg H2O mg-1 DW) showed less ice crystal formation during cryomicroscopic-video-recordings and had IFC viability of 4.5±7.0% (slow) and 6.1±8.8% (fast), respectively, compared to air-dried pollen which lost all viability. Generally, fast-(flash)-drying and increased cooling rates may enable the survival of wheat pollen likely due to (1) a fast rate of intracellular WC loss that reduces deleterious biochemical changes associated with the drying process and (2) a delay and reduction in intracellular ice crystal formation.

Strategies for Highly Efficient Rabbit Sperm Cryopreservation

The rabbit is a valuable animal for both the economy and biomedical sciences. Sperm cryopreservation is one of the most efficient ways to preserve rabbit strains because it is easy to collect ejaculate repeatedly from a single male and inseminate artificially into multiple females. During the cooling, freezing and thawing process of sperms, the plasma membrane, cytoplasm and genome structures could be damaged by osmotic stress, cold shock, intracellular ice crystal formation, and excessive production of reactive oxygen species. In this review, we will discuss the progress made during the past years regarding efforts to minimize the cell damage in rabbit sperms, including freezing extender, cryoprotectants, supplements, and procedures.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol, 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2,613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18,467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Molecular mechanisms of cell cryopreservation with polyampholytes studied by solid-state NMR

Polyampholytes are emerging macromolecular membrane non-penetrating cryoprotectants; however, the mechanism behind their cryopreservation remains unclear. Here, we investigated the mechanism using solid-state NMR spectroscopy. The polymer-chain dynamics and the water and ion mobilities in the presence of various membrane penetrating and non-penetrating cryoprotectants were monitored at low temperatures to mimic cryopreservation conditions. NMR experiments revealed that the water, Sodium-ion, and polymer-chain signals in a carboxylated poly-ʟ-lysine (COOH-PLL) solution broadened upon cooling, indicating increasingly restricted mobility and increased solution viscosity. Moreover, strong intermolecular interactions facilitated the COOH-PLL glass transition, trapping water and salt in the gaps of the reversible matrix, preventing intracellular ice formation and osmotic shock during freezing; this reduced cell stress is responsible for cryoprotection. This simple NMR technique enabled the correlation of the cryoprotective properties of polymers that operate through mechanisms different from those of current cryoprotectants, and will facilitate the future molecular design of cryoprotectants.