Techniques
In slow freezing, which was the main technique for cell freezing in the past, cells immersed into cryoprotectants are slowly cooled to a specific temperature to dehydrate and freeze cells by a difference in osmotic pressure due to ice crystals that occurred extracellularly. This procedure requires a dedicated device called a “program freezer” and a prolonged time of working. Compared to the slow freezing, vitrification requires no specific devices and much shorter time for the procedure, resulting in widespread use of this technique.
However, it also requires accurate and minute skills for embryologists and each step for vitrification to be previously conducted according to rigorous procedures specified on a second-by-second basis. Cryotec Method has been developed by continuous improvement to provide a technique by which “Anyone can obtain the same results” because we are concerned that the vitrification method, which requires experience and skills, can cause slight, artificial differences of results, if any, and psychological stress to embryologists.
Modification of solutions
Cryoprotectants used for vitrification are known to have not only protective effects for cells from ice crystals, but also cytotoxicity. Therefore, embryologists have worked while being pushed by a timer to protect cells from this toxicity even if only slightly. In the Cryotec Method, modified solutions enable a decrease in cytotoxicity from cryoprotectants and ease time restrictions for individual steps.
Improvement of device
For the Cryotec Method, we also stick to the design of the device (containers) in addition to the solutions in order to pose no rigorous time restriction for each step and provide a reproducible technique. First, we developed a dedicated plate for vitrification and thawing, which was not present in previous methods, resulting in accomplishment of improved simplicity and cell protective performance by slowing of osmotic changes. In addition, a design focused on ease of handling and safety was pursued for Cryotec (Straw), creating a square-pole shape that is easy to write names on and hold.
Visualization of VS steps
Conventionally, operation with Vitrification Solution (VS) was one of the steps where inter-operator differences frequently occurred. It is very important to confirm substitution of ES (Equilibrium Solution) to VS around cells and proceed to the cooling step with liquid nitrogen, but the criteria for judgement is ambiguous and there were rigorous time restrictions as mentioned above. Because of the design of the Cryotec Method that individual solutions have appropriate specific gravity, the substitution from ES to VS can be clearly identified by “anyone.”
・Elimination of minimization measure against drop formation at freezing
Oocytes or embryo was previously submerged into the minimum volume of VS to accelerate the cooling rate at the time of inserting into liquid nitrogen in order to reduce the risk of ice crystal formation. This is accomplished by aspirating VS around the sample placed on a Cryotec sheet, which could increase the cooling rate, but also causes physical damage by giving excessive surface tension to the cell. The Cryotec Method has successfully eliminated this step by improving the solutions and devices, enabling safer vitrification.
