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VitriFreeze ES™ - VitriThaw ES™

Medium for vitrifying human embryos
Cryopreservation3531

VitriFreeze ES™ and VitriThaw ES™ media are sets of cell culture media designed for the vitrification of 2PN up to blastocyst stage human embryos.

Our vitrification media have been designed for use with the aseptic HSV® vitrification device (Cryo Bio Systems) and for use with the new aseptic VitriSafe device (VitriSafe website).

VitriFreeze ES™ and VitriThaw ES™ media have a 12 month shelf life.

VitriFreeze ES™ and VitriThaw ES™ media are CE marked as Class III medical devices according to the European Medical Device Directive 93/42/EEC.

VitriFreeze™/VitriThaw™ complies with the following specifications:

pH 7.2-7.4
Osmolality VitriFreeze ES Pre-incubation medium: 270-290mOsm/kg
VitriThaw Thawing medium 2: 805-850mOsm/kg
VitriThaw Thawing medium 3: 535-565 mOsm/kg
VitriThaw Thawing medium 4: 405-435mOsm/kg
VitriThaw Thawing medium 5: 270-290mOsm/kg
Sterility Sterile
Endotoxine < 0.25 EU/mL
 Mouse embryo assay ≥ 80% blastocysts after 96h incubation
Shelflife 12 months from date of produce
Albumin FDA (USA) and EMEA (Europe) compliant


Product order codes

VF_KIT1_ES VitriFreeze ES kit - 4 procedures
VT_KIT1_ES VitriThaw ES kit - 4 procedures


The product composition can be found in the MSDS (see Resources). Additional information on some components is provided below:

VitriFreeze

Component Benefit
Ethylene glycol (5-10-20%)

Compounds such as ethylene glycol (EG) have been shown to penetrate cells rapidly, forming glass during cooling of aqueous solutions (Palasz and Mapletoft 1996). EG and water form clusters of hydrogen bonds in such a way that the water molecules are tightly bound to the cryoprotector, resulting in a lowered mobility and higher viscosity of the solution. This positively contributes to the prevention of ice crystal formation and thus EG became an important component of vitrification solutions (Shaw and Jones 2003).

DMSO (5-10-20%)

Ishimori (1992) described for the first time that the combination of ethylene glycol and DMSO resulted in good survival rates during vitrification. EG has been reported to be a weak glass former (Fahy and Ali 1987). Since vitrification relies on a sufficiently concentrated solution that solidifies as glass, DMSO was added to the medium. Moreover, DMSO is known to increase the permeability of EG (Vicente and Garcia-Ximenez 1994). For these reasons, the combination of EG and DMSO is used as common cryoprotectants.

HSA (10-20 g/L)

Cryoprotective solutions have been supplemented with macromolecules such as sucrose and human serum albumin (HSA). Several studies have shown that such molecules help to reduce physical damage and help to maintain osmotic pressure of the extracellular fluid (Shaw, et al. 2000). Besides its cryoprotective role, HSA facilitates gamete or embryo manipulation by preventing adsorption to the surface of petri dishes and pipettes through saturation of the potential binding sites. Also, the increased viscosity of the media, caused by the addition of HSA, promotes the ease of embryo handling and manipulation (Trounson and Gardner 2000).

Sucrose (only the last step: 0.75 M)

The potential for damage to frozen-thawed embryos by osmotic shock can be minimized by the use of non-permeating compounds, such as sucrose and human serum albumin (HSA), as 'osmotic buffers' to reduce the osmotic gradient between the intracellular and extracellular solutions (Shaw, et al. 2000). More in detail, addition of such non-permeating agents results in a hyperosmotic extracellular solution, promoting cellular dehydration and diminution of ice crystal formation (Shaw and Jones 2003).

Toxicity of the vitrification media is also reduced by sucrose because it probably reduces the necessary amount of intracellular cryoprotectant (Kasai 1996).

Ficoll (only the last step)

It is known that the addition of macromolecules promotes vitrification by increasing the viscosity of the solution (Kasai 1996).

VitriThaw

Component Benefit
17-20 g/L HSA

Cryoprotective solutions have been supplemented with macromolecules such as sucrose and human serum albumin (HSA). Several studies have shown that such molecules help to reduce physical damage and help to maintain osmotic pressure of the extracellular fluid (Shaw, et al. 2000). Besides its cryoprotective role, HSA facilitates gamete or embryo manipulation by preventing adsorption to the surface of petri dishes and pipettes through saturation of the potential binding sites. Also, the increased viscosity of the media, caused by the addition of HSA, promotes the ease of embryo handling and manipulation (Trounson and Gardner 2000).

Sucrose (1 - 0.5 - 0.25 -0.125 - 0 M)

The potential for damage to frozen-thawed embryos by osmotic shock can be minimized by the use of non-permeating compounds, such as sucrose and human serum albumin (HSA), as 'osmotic buffers' to reduce the osmotic gradient between the intracellular and extracellular solutions (Shaw, et al. 2000). More in detail, addition of such non-permeating agents results in a hyperosmotic extracellular solution, promoting cellular dehydration and diminution of ice crystal formation (Shaw and Jones 2003).

Toxicity of the vitrification media is also reduced by sucrose because it probably reduces the necessary amount of intracellular cryoprotectant (Kasai 1996).


Product literature


     Kaartinen, N., et al., The freezing method of cleavage stage embryos has no impact on the weight of the newborns., J Assist Reprod Genet (2016),Vol.33,No.3,pp.393-399


Literature concerning the components


     Fahy, G, Levy, D, & Ali, S., Some emerging principles underlying the physical properties, biological actions and utility of vitrification solutions., Cryobiology (1987),Vol.24,pp.196-213
     Ishimori, H., Tahahashi, Y. , & Kanagawa, H, Viability of vitrified mouse embryos using various cryoprotectant mixtures., Theriogenology (1992),Vol.37,pp.481-487
     Kasai, M., Simple and efficient methods for vitrification of mammalian embryos., Animal Reproduction Science (1996),Vol.42,pp.67-75
     Palasz, A., & Mapletoft, R., Cryopreservation of mammalian embryos and oocytes: Recent advances., Biotechnology Advances (1996),Vol.14,No.2,pp.127-149
     Shaw JM, Oranratnachai A, Trounson AO., Fundamental cryobiology of mammalian oocytes and ovarian tissue., Theriogenology (2000),Vol.53,pp.59-72
     Shaw, JM and Jones, GM., Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos., Human Reproduction Update (2003),Vol.9,No.6,pp.583-605
     Trounson AO, and Gardner, DK., , Handbook of in vitro Fertilization, Boca Raton, Florida: CRC Press (2000),Vol.2
     Vicente, J, Garcia-Ximenez, F., Osmotic and cryoprotective effects of a mixture of DMSO and ehtylene glycol on rabbit morulae., Theriogenology (1994),Vol.42,pp.1204-1215

Resources

Click on the links below for more information.