Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

Abstract

The aim of this study was to determine the most efficient vitrification protocol for the cryopreservation of day 7 in vitro produced (IVP) porcine blastocysts. The post-warm survival rate of blastocysts vitrified in control (17% dimethyl sulfoxide + 17% ethylene glycol [EG] + 0.4 mol/L sucrose) and commercial media did not differ, nor did the post-warm survival rate of blastocysts vitrified in medium containing 1,2-propandiol in place of EG. However, vitrifying embryos in EG alone decreased the cryosurvival rate (55.6% and 33.6%, respectively, p < .05). Furthermore, the post-warm survival rates of blastocysts vitrified with either trehalose or sucrose as the non-penetrating cryoprotectant did not differ. There was also no significant difference in post-warm survival of blastocysts vitrified in control (38°C) media and room temperature (22°C) media with extended equilibration times, although when blastocysts were vitrified using control media at room temperature, the post-warm survival rate increased (56.8%, 57.3%, 72.5%, respectively, p < .05). The findings show that most cryoprotectant combinations examined proved equally effective at supporting the post-warm survival of IVP porcine blastocysts. The improved post-warm survival rate of blastocysts vitrified using media held at room temperature suggests that the cryoprotectant toxicity exerted in 22°C media was reduced.

Cryopreservation: An Overview of Principles and Cell-Specific Considerations

“…Because permeating cryoprotectants interact strongly with water through hydrogen bonding, the freezing point of water is depressed, and less water molecules are available to interact with themselves to form critical nucleation sites required for crystal formation 12 . Formation of solid water with an irregular, amorphous structure is known as vitrification, and is achieved by utilizing a cryoprotectant accompanied by an appropriate cooling rate 15 . To minimize toxicity, vitrification mixtures are often added in a stepwise fashion at temperatures near 0ºC 14 .…”

“…They are typically larger, and covalently linked as either polymers, dimers, or trimers. Some commonly-used agents in this class are: Polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), raffinose, sucrose, and trehalose 14,15,18 . Non-permeating agents induce vitrification by the same mechanism as permeating agents but extracellularly and to a lesser extent.…”

Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models

“…They cannot pass through the cell membrane and exert their protective effect extracellularly. Most commonly used non-penetrating CPAs are polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), raffinose, sucrose, and trehalose [42,43]. The mode of action of non-permeating agents is similar to permeating agents by controlling osmolarity but works extracellularly and at a lower degree.…”

Development of Multilayer Mesenchymal Stem Cell Cell Sheets

“…Modification of the DNA methylation pattern of cells and cell sheets, during the freezing/thawing steps, could affect any gene controlling tumorigenicity [197]. In porcine blastocysts, the expression of IGF2 and IGF2R were downregulated by the vitrification process, but their expression was like that of nonvitrified cells, in the presence of ethylene glycol [198]. The DNA methylation pattern of those two imprinted genes was not studied in the vitrified cell sheets, but the alteration of the gene imprinting, during the cryopreservation/thawing steps, could affect their expression and causes diseases in a long term [199][200][201][202][203].…”