Vitrification is a technology that is used in the embryo and egg freezing process so that they can be stored for later use. It is a technology that has many uses outside of fertility care with egg and embryo freezing, as it allows something with a crystalline structure to be converted into something very smooth. The classic example is the creation of glass using sand (which is crystalline) as the main ingredient. Other examples include the manufacture of china plates and cups (also using sand), cotton candy from sugar (also crystalline) or ice cream which is smooth to the taste and contains no ice crystals despite its frozen state.
When we freeze cells in a lab, the main focus of the process is avoiding ice crystal formation as the fluid in the cell cools to subzero temperatures. Ice crystals pose 2 significant and deadly problems for cells. First, despite its beauty, an ice crystal is razor sharp and will readily shred any cell membrane, killing the cell. Second, as water in the cell turns to ice, it expands in volume, rupturing (killing) the cell. As a result, processes must be developed that allow cells to be frozen while avoiding the formation of ice. This science, called cryobiology, has come up with 2 methods that work well with human embryos, slow freezing and vitrification.
Human embryos were first successfully frozen and used to establish a pregnancy in Australia in 1984. The procedure used to preserve the embryos was aptly called slow freezing since the embryos were cooled at a rate of just 0.3ºC/minute until their temperature had passed below -30ºC. They were then stored in liquid nitrogen (at -196ºC) until the time of thawing. Ice crystal formation was avoided by incubating the embryos in chemicals called cryoprotectants, which draw water out of the cells in the embryo before the embryos freeze. By removing much of the water and replacing it with cryoprotectants, ice crystal formation is limited or avoided inside the cells.
Cryoprotectants work by drawing water out of a cell by simple osmosis, and then crossing the cell membrane to replace that lost water. When an embryo is placed in a mild solution of cryoprotectant, it initially shrivels slightly as it loses water, and then returns to normal size as the cryoprotectant works its way into the cells. The water molecule is small and leaves the cell very quickly, compared to the larger cryoprotectant molecule that takes longer to cross the cell membrane. Cryobiologists have successfully used many cryoprotectants to preserve embryos, but the most commonly used during slow freezing are ethylene glycol, glycerol and propylene glycol.
When slow frozen embryos are thawed, the water is allowed back into the cells and the cryoprotectant is drawn out. During thawing, water again crosses into the cell before the cryoprotectant can leave so there is a risk that cells can over expand and rupture. Thawing is therefore performed slowly and in small steps to try to prevent cells rupturing and dying during the process.
Slow freezing has been used successfully as a method for preserving human embryos for just about 30 years now and many hundreds of thousands of babies have been born after spending time in the freezer as an embryo. The process is considered extremely safe and it works reasonably well for embryos at all stages of development.
One limitation of the slow freezing process was that it never worked very reliably for freezing unfertilized eggs. Cryobiologists worked on trying to develop a method for egg freezing for 25 years, and despite some individual successes, a repeatable and reliable process was never worked out. The human egg is a huge cell, the largest cell in the human body by some distance, and it has stubbornly resisted most attempts at slow freezing. Because of its size, it has an unusually large surface area to volume ratio, and this significantly affects the rate at which it can be dehydrated or re-hydrated during freezing and thawing. The egg is also in a very delicate state at ovulation as it is in the process of reducing its own DNA content in preparation for the arrival of the sperm which brings some new DNA. The egg has been found to be ultra-sensitive to temperature changes and to chemical (cryoprotectant) exposure, and because it is such a large cell, these exposures needed to be longer and cooling also took more time than for embryos. Unfertilized eggs did not tolerate these insults well and worldwide results with egg freezing were poor.
Cryobiologists eventually gave up on slow freezing eggs and looked at another technology, vitrification as a possible method of preservation. Vitrification of mouse embryos was first demonstrated in 1985, but the procedure never became popular, perhaps because it used much higher concentrations of cryoprotectants than slow freezing. However, 20 years later, in the mid 2000's the technique was revitalized and shown to work well for freezing unfertilized eggs. The technique worked so well in fact, that it has now become the preferred technique for freezing embryos too.
Tips for Embryo Vitrification
- All procedures are to be done at ROOM TEMPERATURE (22-27°C). DO NOT USE HEATED STAGE.
- Have all necessary material, tools and equipment ready and easily accessible before starting procedure.
- CryoTips should be pre-labeled with patient information, and assembled with connector and syringe or pipette (for loading), prior to starting procedure. To protect the finely pulled tip from damage, keep it covered with metal cover sleeve until ready to load specimen(s).
- HSV device should be pre-labeled with patient information, and the capillary tube should be connected with the longer end of the blue plastic insertion device, prior to starting procedure.
- Where possible, select only the best quality embryos (2PN to Blastocyst) for vitrification.
- The recommended CryoTip or HSV Device capacity is a MAXIMUM of 2 specimens.
- Process only as many specimen(s) as will be loaded per CryoTip or HSV device at one time.
- Minimize exposure of specimens to light during equilibration in ES and VS solutions.
- Transfer specimens between drops using a minimal volume of medium.