Cryopreservation of mammalian oocytes by using sugars: Intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates

Abstract

Accumulation of intra- and extracellular sugars such as trehalose, glucose, and raffinose is central to survival strategies of a variety of organisms coping with extreme conditions including freezing and almost complete drying. The objective of the present study was to investigate the potential application of intra- and extracellular raffinose in combination with low concentrations of dimethylsulfoxide (Me2SO) to mammalian oocyte cryopreservation. To this end, the fertilization and embryonic development of cryopreserved metaphase II (M II) mouse oocytes were studied in comparison to unfrozen controls. For cryopreservation, M II oocytes were microinjected with 0.1 M raffinose, and then cooled to -196°C in the presence of either 0.3 M raffinose and 0.5 M Me2SO (cryopreservation group 1) or 0.3 M raffinose and 1.0 M Me2SO (cryopreservation group 2). The control groups included untreated oocytes (untreated control) and oocytes microinjected with raffinose, but not frozen (injection control). The post-thaw survival rates were 83.9% and 80.6% for the cryopreservation group 1 and 2, respectively. The fertilization and blastocyst rates in the cryopreservation group 1 (90.0% and 77.8%, respectively) and 2 (94.6% and 72.5%, respectively) were also high and similar to the ones of the injection controls (97.8% and 78.5%, respectively) and untreated controls (98.8% and 83.6%, respectively). These results are consistent with the findings of our earlier studies and support the use of sugars as intra- and extracellular cryoprotectants. Furthermore, the results of the present study indicate that the presence of intra- and extracellular sugars alleviates high concentrations of conventional penetrating cryoprotectants, and thus minimizes their toxicity.

Inulin Can Improve Red Blood Cell Cryopreservation by Promoting Vitrification, Stabilizing Cell Membranes, and Inhibiting Ice Recrystallization

“…The ultimate aim of vitrification is to bring cells safely below the Tg′, where the cells and surrounding medium vitrify. Therefore, a high Tg′ could greatly facilitate the cryopreservation process. , As shown in Figure C, for inulin solutions with different concentrations, they are highly effective glass formers with a high Tg′ of around −21 °C …”

Inulin Can Improve Red Blood Cell Cryopreservation by Promoting Vitrification, Stabilizing Cell Membranes, and Inhibiting Ice Recrystallization

“…The ultimate aim of vitrification is to bring cells safely below the Tg′, where the cells and surrounding medium vitrify. Therefore, a high Tg′ could greatly facilitate the cryopreservation process. , As shown in Figure C, for inulin solutions with different concentrations, they are highly effective glass formers with a high Tg′ of around −21 °C …”

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds

“…Their high glass transition temperatures ( T g ) in comparison to conventional CPAs, such as ethylene glycol (EG), DMSO, and 1,2-propanediol (PROH), are considered a significant advantage. Carbohydrates also stabilize lipid membranes through hydrogen bonding, thus avoiding intracellular protein aggregations, and by behaving as osmolytes to prevent osmotic stresses . In addition to its non-toxic nature, sugar-based CPAs can be injected directly into patients as compared to traditional CPAs, which need to the removed prior to injection .…”

“…Carbohydrates also stabilize lipid membranes through hydrogen bonding, thus avoiding intracellular protein aggregations, and by behaving as osmolytes to prevent osmotic stresses. 44 In addition to its non-toxic nature, sugar-based CPAs can be injected directly into patients as compared to traditional CPAs, which need to the removed prior to injection. 23 Due to the impermeability of their cell membranes, sugars have been added as extracellular components, but recently, different approaches have been used such as thermotropic lipid phase transition, 7 reversible poration, 21 transfection, 45 and microinjection 46 to overcome this barrier.…”

Freeze-Dried Extracellular Vesicles From Adipose-Derived Stem Cells Prevent Hypoxia-Induced Muscle Cell Injury

“…The protective effect of trehalose during drying and rehydration has also been demonstrated in different model systems (e.g., liposomes, membranes, and proteins) and mammalian cells (Crowe et al, 1993a(Crowe et al, ,b, 1987(Crowe et al, , 1994Remmele et al, 1997;Guo et al, 2000;Chen et al, 2001). As a non-reducing disaccharide, trehalose has a remarkably high glass transition temperatures (Tg) (Levine and Slade, 1988;Eroglu et al, 2009;Eroglu, 2010), which would support the resulting vitrified state at high temperatures. Therefore, trehalose has been extensively used for preservation of biological materials (Bissoyi et al, 2016).…”

Inulin Can Improve Red Blood Cell Cryopreservation by Promoting Vitrification, Stabilizing Cell Membranes, and Inhibiting Ice Recrystallization

“…The ultimate aim of vitrification is to bring cells safely below the Tg′, where the cells and surrounding medium vitrify. Therefore, a high Tg′ could greatly facilitate the cryopreservation process. , As shown in Figure C, for inulin solutions with different concentrations, they are highly effective glass formers with a high Tg′ of around −21 °C …”

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds

“…Their high glass transition temperatures ( T g ) in comparison to conventional CPAs, such as ethylene glycol (EG), DMSO, and 1,2-propanediol (PROH), are considered a significant advantage. Carbohydrates also stabilize lipid membranes through hydrogen bonding, thus avoiding intracellular protein aggregations, and by behaving as osmolytes to prevent osmotic stresses . In addition to its non-toxic nature, sugar-based CPAs can be injected directly into patients as compared to traditional CPAs, which need to the removed prior to injection .…”

“…Carbohydrates also stabilize lipid membranes through hydrogen bonding, thus avoiding intracellular protein aggregations, and by behaving as osmolytes to prevent osmotic stresses. 44 In addition to its non-toxic nature, sugar-based CPAs can be injected directly into patients as compared to traditional CPAs, which need to the removed prior to injection. 23 Due to the impermeability of their cell membranes, sugars have been added as extracellular components, but recently, different approaches have been used such as thermotropic lipid phase transition, 7 reversible poration, 21 transfection, 45 and microinjection 46 to overcome this barrier.…”

Freeze-Dried Extracellular Vesicles From Adipose-Derived Stem Cells Prevent Hypoxia-Induced Muscle Cell Injury

“…The protective effect of trehalose during drying and rehydration has also been demonstrated in different model systems (e.g., liposomes, membranes, and proteins) and mammalian cells (Crowe et al, 1993a(Crowe et al, ,b, 1987(Crowe et al, , 1994Remmele et al, 1997;Guo et al, 2000;Chen et al, 2001). As a non-reducing disaccharide, trehalose has a remarkably high glass transition temperatures (Tg) (Levine and Slade, 1988;Eroglu et al, 2009;Eroglu, 2010), which would support the resulting vitrified state at high temperatures. Therefore, trehalose has been extensively used for preservation of biological materials (Bissoyi et al, 2016).…”

Inulin Can Improve Red Blood Cell Cryopreservation by Promoting Vitrification, Stabilizing Cell Membranes, and Inhibiting Ice Recrystallization

“…The ultimate aim of vitrification is to bring cells safely below the Tg′, where the cells and surrounding medium vitrify. Therefore, a high Tg′ could greatly facilitate the cryopreservation process. , As shown in Figure C, for inulin solutions with different concentrations, they are highly effective glass formers with a high Tg′ of around −21 °C …”

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds

“…Their high glass transition temperatures ( T g ) in comparison to conventional CPAs, such as ethylene glycol (EG), DMSO, and 1,2-propanediol (PROH), are considered a significant advantage. Carbohydrates also stabilize lipid membranes through hydrogen bonding, thus avoiding intracellular protein aggregations, and by behaving as osmolytes to prevent osmotic stresses . In addition to its non-toxic nature, sugar-based CPAs can be injected directly into patients as compared to traditional CPAs, which need to the removed prior to injection .…”

“…Carbohydrates also stabilize lipid membranes through hydrogen bonding, thus avoiding intracellular protein aggregations, and by behaving as osmolytes to prevent osmotic stresses. 44 In addition to its non-toxic nature, sugar-based CPAs can be injected directly into patients as compared to traditional CPAs, which need to the removed prior to injection. 23 Due to the impermeability of their cell membranes, sugars have been added as extracellular components, but recently, different approaches have been used such as thermotropic lipid phase transition, 7 reversible poration, 21 transfection, 45 and microinjection 46 to overcome this barrier.…”

Freeze-Dried Extracellular Vesicles From Adipose-Derived Stem Cells Prevent Hypoxia-Induced Muscle Cell Injury

“…The protective effect of trehalose during drying and rehydration has also been demonstrated in different model systems (e.g., liposomes, membranes, and proteins) and mammalian cells (Crowe et al, 1993a(Crowe et al, ,b, 1987(Crowe et al, , 1994Remmele et al, 1997;Guo et al, 2000;Chen et al, 2001). As a non-reducing disaccharide, trehalose has a remarkably high glass transition temperatures (Tg) (Levine and Slade, 1988;Eroglu et al, 2009;Eroglu, 2010), which would support the resulting vitrified state at high temperatures. Therefore, trehalose has been extensively used for preservation of biological materials (Bissoyi et al, 2016).…”