VitriFreeze – VitriThaw
Medium for vitrifying human embryos
VitriFreeze and VitriThaw media are sets of cell culture media designed for the vitrification and thawing of morula and blastocyst stage human embryos.
We also have a set of vitrification media for 2PN to 8-cell stage embryos: VitriFreeze ES and VitriThaw ES.
Our vitrification media can be used with the aseptic HSV® vitrification device (Cryo Bio Systems), amongst other vitrification devices.
VitriFreeze and VitriThaw media have a 12 month shelf life. Information about the composition of the products van be found in the material safety data sheet.
Regulatory
Europe: CE-marked – USA: US FDA Cleared
Product order codes
VF_KIT1 : VitriFreeze kit – 4 procedures
VT_KIT1 : VitriThaw kit – 4 procedures
| pH | 7.2-7.4 |
| Osmolality | VitriFreeze Pre-incubation medium: 270-290mOsm/kg VitriThaw Thawing medium 1: 805-850mOsm/kg VitriThaw Thawing medium 2: 535-565 mOsm/kg VitriThaw Thawing medium 3: 405-435mOsm/kg VitriThaw Thawing medium 4: 270-290mOsm/kg |
| Sterility | Sterile |
| Endotoxine | < 0.25 EU/mL |
| Mouse embryo assay | ≥ 80% blastocysts after 96h incubation |
| Shelf life | 12 months from date of produce |
| Albumin | FDA (USA) and EMA (Europe) compliant |
| 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). |
| Component | Benefit |
| 18-20 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 (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). |
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Vanneste E., Melotte C., Voet T., et al. For a complex chromosomal rearrangement by array comparative genomic hybridization, Human Reproduction (2011),Vol.26,No.4,pp.941-49
Zech N., Vitrification of hatching and hatched human blastocysts: effect of an opening in the zona pellucida before vitrification, RBM Online (2005),Vol.11,No.3,pp.355-361
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
