Research articleImplications of storage and handling conditions on glass transition and potential devitrification of oocytes and embryos
Introduction
Low temperature preservation of oocytes and embryos is a fundamental cornerstone of assisted reproductive technologies. Cryopreservation of reproductive cells has been traditionally achieved by slow cooling the samples at specific rates to allow cell dehydration [1], [2]. However, the preservation outcome of oocytes and embryos by slow freezing equilibrium protocols is negatively affected by cryoinjury due to formation of intracellular and extracellular ice crystals, concentration of solutes during the freezing process, and prolonged cell exposures to toxic cryoprotectant and chilling temperatures [2], [3], [4].
Storage of cryopreserved reproductive cells is conducted in specialized cryogenic, thermally insulated vacuum flask dewars that hold cryogenic fluids such as liquid N2 below their boiling point [5]. Smaller to medium-sized tanks (20–50 L) used by veterinary practitioners and in some laboratories are routinely filled with cryogenic fluid to maintain adequate chamber temperatures [6]. Although the recommendation is to maintain the dewars full at all times [5], [7], manufacturers provide guideline static evaporation rates for individual models and suggest close monitoring of liquid N2 levels based on specific usage conditions [7].
Cells stored in these containers are kept either immersed in liquid N2 or in the immediate vapor phase [5]. Because the temperature of the vapor phase is not a constant (as opposed to liquid N2, −196 °C), a lack of temperature homogeneity within the chamber is observed [8]. Noteworthy, storage recommendations for oocytes and embryos in vapor phase of liquid N2 dewars were originally formulated for cells that had been cryopreserved using equilibrium, slow freezing protocols [5], [6]. Cells cryopreserved under those conditions have been reported to undergo sufficient dehydration and minimal cytoplasm supercooling and thus are less likely to be damaged during warming. However, these recommendations may not be applicable to vitrified material, which has a higher risk of devitrification and can suffer irreversible cryoinjury.
Vitrification, the process of solidification of a sample into an amorphous, glassy state in absence of intracellular and extracellular ice crystals, requires high concentrations of cryoprotectants, extremely rapid cooling rates, and reduced volume handling. In the last decade, vitrification has progressively become the method of choice for the cryopreservation of human oocytes and embryos [9], [10], [11], and this trend is now being followed by veterinary and animal science practitioners for domestic and exotic animal species [12], [13].
Devitrification is defined as the process of crystallization in a formerly crystal-free, amorphous glass solution [12], [14], [15], [16]. Early experiments to study the warming behavior of vitrified aqueous solutions were conducted by Luyet [14], [15], [16] and Luyet and Rasmusen [17], [18] using differential thermal analyses to detect enthalpy changes associated with transition events [17], [18]. Unlike melting point, devitrification phenomenon has been described not as an individual point but as a temperature range determined by the composition of solution, presence of nucleating particles, among other factors [14], [15], [18], [19], [20], [21].
The devitrification of the intracellular solution and the surrounding extracellular medium can lead to significant damage during the warming of cells [20], [21]. Several authors have indicated that above the glass transition temperature (Tg) of the cytoplasm (approximately −120 °C to −130 °C), the vitrified cytoplasm of oocytes and embryos could enter a liquid transition, promoting devitrification and subsequent ice nucleation and crystallization [22], [23], [24]. There are limited reports on Tg of cryopreservation solutions [20], [21], [22], and those available are mostly water–sugar solutions and not the complex mixtures of balanced salt solutions, permeating and nonpermeating cryoprotectants used in current oocyte and embryo vitrification protocols [25], [26], [27].
To date, there are no reports on Tg of vitrification solutions used in the storage of oocytes and embryos. This information would be of value to calculate critical devitrification thresholds and to update recommendations for the storage of vitrified oocytes and embryos. Therefore, the objective of this study was to determine the Tg of a cryopreservation solution typically used in the vitrification, storage, and warming of mammalian oocytes and embryos. To analyze devitrification thresholds, a numerical modeling of heat transfer for a common vitrification carrier (open-pulled straw, OPS) was conducted. Finally, the implications of these results on specimen storage and handling conditions in N2 vapor phase were discussed.
Section snippets
Measurement of the Tg of the vitrification solution by differential scanning calorimetry
Current vitrification protocols require that cells be successively moved through increasing cryoprotectant concentrations (permeable and nonpermeable) before their vitrification by direct plunging into liquid N2 and long-term storage. Therefore, the Tg of the final vitrification solution routinely used in our laboratory was determined by differential scanning calorimetry (DSC).
The Tg of the vitrification solution consisting of 2.8 M Me2SO (Sigma D2650) + 3.6 M EG (Sigma 102466), and 0.65 M
Measurement of glass transition and devitrification temperatures of a vitrification solution by DSC
The Tg of a vitrification solution commonly used in which oocytes and embryos are later stored was determined by DSC. Because most of the oocytes and embryos are vitrified and stored in vitrification supports individually (one oocyte/embryo is loaded, vitrified, and stored per support device), the Tg of the system is dominated by the glass transition of the surrounding medium, and the contribution of the cytoplasm assumed to be negligible. A schematic representation of heat flow process is
Discussion
Correct storage management of cryopreserved material is a fundamental aspect of cell survival and viability after thawing or warming. Rapid cooling, vitrification protocols have now mostly replaced slow-cooling equilibrium protocols in the cryopreservation of human oocytes and embryos because of improved viability and development after warming [31]. This improved cell survival determines that vitrification is also progressively becoming the method of choice for cryopreservation of domestic and
Acknowledgments
Authors have no financial relationships to disclose. This research was funded by Facultad de Ciencias Agrarias, Universidad Católica Argentina, Ciudad Autónoma de Buenos Aires, Argentina y Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA-CONICET), Universidad Nacional de La Plata, Buenos Aires, Argentina.
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