The Evolution of the Cryopreservation Techniques in Reproductive Medicine—Exploring the Character of the Vitrified State Intra- and Extracellularly to Better Understand Cell Survival after Cryopreservation

Abstract

Nowadays, cryopreservation of gametes and embryos is a fundamental, integral, and indispensable part of infertility treatment or fertility preservation. Cryopreservation is not only needed for the policy of single embryo transfer and cryopreservation of surplus embryos, but for deferring embryo transfer in the case of ovarian hyperstimulation syndrome, uterine pathologies, and suboptimal endometrium built-up or when preimplantation genetic testing is needed. Several current strategies in assisted reproduction technology (ART) would be inconceivable without highly efficient cryopreservation protocols. Nevertheless, cryopreservation hampered for a long time, especially in terms of low survival rates after freezing and thawing. Only the technical progress during the last decades, namely, in regard to the implementation and advancement of vitrification, leveraged its application, and thus, even allows the cryopreservation of human oocytes—a process that is far from being easy. This review aims to provide a deeper insight into the physical processes of cryopreservation and to explore the character of the vitrified state in the extra and intracellular milieu in order to demonstrate that the common denominator to all cryopreservation procedures is the establishment of an intracellular amorphous condition that hinders the likelihood of crystallization.

Biomaterials for Testicular Bioengineering: How far have we come and where do we have to go?

“…Vitrification is the process of solidifying a liquid substance at extremely high freezing rates, transforming the liquid sample into an amorphous solid state ( 143 ). This freezing process prevents the formation of ice crystals ( 144 ).…”

“…The rapid freezing method is based on the direct contact of samples with liquid nitrogen vapor for at least 10 minutes, in this method the sperm are mixed with cryoprotective agent and placed in cryotubes that will be exposed to nitrogen vapors. After the vapor exposure phase; the samples are immersed in liquid nitrogen (140,141). The addition of CPAs to the samples seeks to minimize osmotic damage and prevent intracellular and extracellular ice crystals from forming, however, CPAs have cytotoxic characteristics (142).…”

Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives

“…64 Furthermore, an increase in viscosity of freezing mixtures led to a delay in nucleation, a decrease in the kinetics of growth of ice crystals, impaired crystal formation, and a reduction in the size of ice crystals. 50,65 2.1.6 Effect on molecular diffusion. The addition of polymeric CPAs in the extracellular compartment induces steric hindrance and restricts microscale diffusion.…”

“…The probability of obtaining a vitrified state is directly proportional to the concentration/viscosity of CPAs used for cryopreservation as well as the cooling and warming rates. 65 In order to demonstrate the effect of polymeric crowders on thermal stability, devitrification temperatures (T d ) of the polymeric macromolecule-based freezing solutions containing high concentrations of solutes (50% w/w) were determined. Different freezing mixtures, each containing sucrose, a polymeric solute (Ficoll s 70, Ficoll s 400, PVP 40, PVP 360), and DMSO in different weight ratios, were prepared and studied using differential scanning calorimetry.…”

“…Hence, in addition to the solute/CPA viscosity and cooling/ warming rates, the probability of achieving a vitrified state in the intracellular compartment is also directly related to the degree of intracellular crowding. 65,78 The cell is expected to be osmotically unresponsive once it is vitrified. The colloidal glass behaves like a molecular sieve and allows the passage of only small molecules.…”