For a long time, slow freezing has been the method of choice for freezing sperm, oocytes, embryos, or blastocysts. Most typically, embryos are frozen 1, 3 or 5 days after the sperm and egg were put together. Freezing is a stressful process for an embryo, and only embryos that are growing well in the laboratory will tolerate the freezing procedure.
Step 1. Before an embryo can be frozen, all the water that it contains must be removed, otherwise ice crystals will form inside the cells, which is deadly for the cells. To prevent the embryo from shriveling as the water is extracted, we replace the water with “antifreeze,” or a solution of cryoprotective agents such as glycerol, ethylene glycol etc.
Step 2. When most of the water has been removed the embryo, it is inserted into a carefully labeled vial and placed in the cooling chamber of a controlled rate freezer.
Step 3. The embryo is then cooled very slowly at -0.30C per minute, hence the freezing process is termed slow freezing. This allows the embryologist to have precise control over the freezing process to maximize water extraction from the embryo and to prevent formation of large ice shards that could pierce the embryo.
Step 4. The cooled vial is placed into carefully labeled metal canes and lowered into the tank with other frozen embryos. The entire process takes several hours and the embryos are stored frozen at –196 degree C in liquid nitrogen.
Sperm freezing is much easier and more reliable. It needs glycerol as CPA with the addition of sucrose. Egg freezing has for a long time been considered to be more difficult. It is now becoming more reliable and successful with the advent of vitrification. The CPAs most commonly used for blastocyst and egg freezing are EG and DMSO.
Recently, a new method called vitrification has been employed in freezing and is gaining popularity due to its safe and simple technique. Embryos can be frozen at different times after fertilization.
Vitrification is a newer technique that incorporates a higher concentration of cryoprotective agents in combination with ultra-rapid cooling or flash freezing. This method requires addition of cryoprotective agents prior to cooling.
Again, the cryoprotective agents act as antifreeze, which lowers the freezing temperature and increase viscosity. The solution turns into amorphous solid or vitrifies (meaning turning it into a glass-like substance) when submerged into liquid nitrogen for flash freezing.
An egg is a single free-living cell. They have always been more difficult to freeze than one-cell (zygote) or early 2 to 8-cell embryos indicating the problem was more than the fact of being a single cell.
To survive freezing, a cell needs to be dehydrated. Since water expands as it turns to ice, it could burst the cell. Therefore, the water in the cell is replaced with an “antifreeze” or cryoprotectant.
Since an egg is a single free-living cell, it can be dehydrated quickly. However, when the egg is released from the ovary, it is in a very critical phase of development that is very vulnerable to the freezing process.
Since the egg is preparing for a sperm, the DNA within the egg is in a very delicate phase of reorganization. The egg is in the process of getting rid of half of its DNA, a process that is not completed until after the sperm has entered the egg. Freezing the egg may fatally disrupt the DNA reduction process.
Freezing of oocytes by vitrification is now very successful at the very specific metaphase II stage.
Embryos that have grown successfully in the laboratory for 5 or 6 days are called blastocysts.
Blastocysts are typically composed of approximately 100 to 230 cells. They have gone beyond the stages where it was possible to count the number of cells that they contain (e.g. 4-cell or 8-cell stage), and have begun to differentiate into 2 different cell types.
Our lab prefers to freeze blastocysts rather than 1, 2, 4, 6 or 8-cell stage embryos. Some of the advantages of freezing blastocysts include:
It limits the number of embryos stored in tanks of liquid nitrogen in the laboratory.
Cryoprotective Agents (CPAs) are often biological molecules that are byproducts of cellular metabolism that can be used to protect cells from damage during the freezing process. CPAs are low molecular weight organic compounds.
The concept of using CPAs was originally derived from studies of animals that live in cold temperatures. For example, arctic frogs use glucose and arctic salamanders produce glycerol in their livers, which act as “natural antifreeze” and prevent ice formation within the cells, which would result in death. For CPAs to be effective, they must meet two important criteria: first, they must be able to penetrate the cell and secondly, they cannot be toxic to the cell.
Glycerol, ethylene glycol (EG), propylene glycol (PG), propenediol (PROH) and dimethyl sulfoxide (DMSO) are the most commonly used CPAs.
The cooling agent of choice is liquid nitrogen. It is used because it is cheap, abundant, and easy to work with.