Techniques

In slow freezing—the main technique for freezing cells in the past—cells immersed in cryoprotectants are slowly cooled to a specific temperature to dehydrate and freeze by a difference in osmotic pressure caused by ice crystals occurring outside cells. Not only does this procedure require a dedicated device called a program freezer, it also requires more time to complete. Vitrification, on the other hand, does not require any specific devices and can be completed in a much shorter time frame. As a result, the technique enjoys widespread use.
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Freezing
Thawing
Others

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.

Freezing

Can an immature oocyte be frozen?
Yes, an immature oocyte at the GV or MI stage can be frozen by the same procedure.
How is an expanded blastocyst frozen?
In the case of an expanded blastocyst (≥220µm of diameter), a Pre-shrink procedure should be conducted before freezing. Diluent Solution (DS) and Washing Solution (WS) are mixed at a 2：3 ratio to prepare hypertonic solution. By immersing a blastocyst into the mixed solution, the blastocyst is shrunken by osmotic pressure difference. Once the diameter of the blastocyst becomes less than 220 µm, then you can proceed to the ES step. The subsequent process is the same as that for oocytes/embryos. The blastocyst that is shrunken by the Pre-shrink procedure should be transferred to VS after waiting for up to 15 minutes.
Can a blastocyst after being used for biopsy also be frozen?
Such a blastocyst can be also cryopreserved by the identical process as usual. If the recovery of volume of the blastocyst in ES is difficult to determine because it was frozen from a shrunken state, the blastocyst should proceed to the VS step after the maximum immersing duration of 15 minutes.
Is there any time restriction for the ES step?
Only the maximum duration of submersion is specified: 15 minutes for oocytes and blastocysts, and 12 minutes for cleavage stage embryos. Since no minimum duration is specified, you can proceed to VS at the time when volumes of oocytes/embryos are recovered to the same size as that before freezing. In case that volumes of these cells have not been completely recovered, equilibration would be completed and you should proceed to VS treatment.
Are there any time restrictions on the VS1/VS2 steps?
No. At the VS1 step, you can proceed to the VS2 step at the time of confirmation that floating of oocytes/embryos stops. At the VS2 step, you can proceed to the loading step after confirming re-shrink of oocytes/embryos. At any step, you can focus on observation of oocytes/embryos because there is no need to check the time.
When multiple oocytes are frozen, recovery of these oocytes would be varied during the ES and/or VS steps. Should I proceed to the next step by tailoring to individual oocytes?
It is desired to proceed to the next step by tailoring to each oocyte, if you can freeze individual oocytes on separate Cryotec. However, if you put multiple oocytes on the same Cryotec for freezing, or you must significantly delay manipulation of oocytes/embryos that are late to recover from shrinkage, you should start manipulation on all the oocytes/embryos together with those recovered later.
At loading, how much size is appropriate for a droplet?
The diameter of a droplet should be 300 to 500 µm. The width of the Cryotec sheet is 1400 µm, so the droplet should be approximately one third of the width.
What should I do to load multiple oocytes/embryos on the same sheet?
Please ensure one oocyte/embryo per droplet. In case multiple oocytes/embryos are incidentally entered into the same droplet, you must retake another oocyte/embryo and make another droplet on a different site of the sheet.
What is wrong with too small/big droplet?
If the size of a droplet is too small, oocyte/embryo may be damaged by surface tension. On the other hand, an excessive size of droplet may cause ice crystal formation and/or crack of droplet because of a reduced speed for cooling and/or warming. Please comply with the appropriate droplet size to accomplish safer vitrification.
Are there any tips to tightly close the straw cap?
When you put the straw cap on Cryotec, please make sure that the cap is adequately cooled, and no air bubbles come from the inside of it. Cool the cap at least 15 seconds before use. If any air bubbles remain inside the cap, it can easily come off by pressure of the bubbles. Or, after putting the cap on using tweezers, you can close it more tightly with your fingers.

Thawing

Why is TS warmed at 37 °C?
The temperature of Warming Solution (TS) should be 37 °C which is the maximum acceptable temperature to be used for oocyte/embryo and it maximizes the warming speed to pass the temperature that may cause ice crystal formation. Please maintain the temperature of TS at 37 °C until just before use.
Can TS be warmed in a CO2 incubator?
We recommend warming TS in an incubator or keep-warm chamber without CO2 because HEPES is added in TS. If you use a CO2 incubator, the TS vial should be re-tightened and wrapped with parafilm. If you are still concerned, it is safer to prepare the vial by putting it into a larger container, such as a conical tube, then tightly close the cap of the container.
Planned embryo transfer was suddenly canceled. Can warmed TS be used anymore?
Unused TS can be used without a problem. Stop warming and store the TS in the refrigerator again.
When an oocyte was submerged into TS, it immediately left from the sheet. Before one minute has passed, can only Cryotec be removed from TS?
In addition, why should Cryotec be kept in TS for 1 minute?
If you move Cryotec before 1 minute, a flow occurs in TS to decrease the temperature of TS more quickly than intended. In addition, the moment when an oocyte/embryo is submerged into TS is the most stressful for the cell. Therefore, it is necessary to wait without moving a muscle in order not to give the cell further stress. For this reason, you should not move Cryotec and keep it in TS for 1 minute, even if the oocyte/embryo floats.
At thawing, what should I do if an oocyte/embryo is not removed from the Cryotec sheet?
If an oocyte/embryo is adhered to the sheet after more than 1 minute has passed since it was submerged into TS, gently shake Cryotec in a horizontal fashion and wait until the oocyte/embryo spontaneously falls down. In case that the oocyte/embryo is not removed, squirt TS slowly using a pipette to take off. Picking the oocyte/embryo directly by a pipette may cause damage to the cell, so it is recommended to try the method above if possible.
At thawing, can I shorten the time with each solution if the embryo seems to be recovered?
The times required for thawing steps are 1 minute for TS, 3 minutes for DS, and 5 minutes for WS1, which are specified as the shortest limits of time. Even if an embryo already shows recovery, ensure that it is kept in each solution for the specified time.
What is the appropriate culture duration for oocyte from thawing to intracytoplasmic sperm injection (ICSI)?
It is recommended to incubate an oocyte for 2 hours after thawing, except the case that the mitotic spindle can be seen after thawing.
How long is optimal for embryo transfer to wait after thawing?
It is recommended to conduct embryo transfer as quickly as possible after viability of the embryo is confirmed, because we believe that an in vivo environment is more appropriate for embryos to survive in than in vitro. Usually, we recommend at approximately 1 hour of culture after thawing for embryo transfer, but in the end follow the clinic’s policy for the timing.
Air bubbles often occur when Cryotec touches TS. What should I do to prevent it?
Residue of liquid nitrogen on the Cryotec sheet would become air bubbles in TS solution. Therefore, Cryotec should be pulled out from the solution as vertically as possible. In addition, when you land the Cryotec sheet in TS, inserting the sheet at 20 to 30 degrees of incident angle along the curve of the TS well enables reduction of air bubbles.
I lost sight of an oocyte/embryo in TS. What should I do?
If no minimization of droplet is conducted at loading, an oocyte/embryo may immediately leave from the Cryotec sheet and float when Cryotec is inserted into TS. You may also lose sight of oocyte/embryo because of not coming into focus on the tip of the sheet. However, if the oocyte/embryo was certainly loaded on the sheet, it must be present in the TS solution. After 1 minute has passed after inserting the sheet into TS, you should confirm that there is no oocyte/embryo on the sheet, then carefully search for it on the surface and the side of the TS well.

Others

What are cryoprotectants used in this system?
Ethylene glycol and DMSO as permeable cryoprotectant agents, and trehalose and hydroxypropylcellulose (HPC) as impermeable cryoprotectant agents are added.
Should a heating plate be used on a microscope or a clean bench?
Do not use a heating plate. Increased temperature of solutions may accelerate chemical reactions. It may induce toxicity of the cyroprotectants. Therefore, you should carefully manage the temperature of solutions.
How long is the shelf life?
For both freezing and thawing solutions, the shelf life is 1 year from manufacturing, but 3 months at room temperature. The shelf life for plastic products (plates and Cryotec) is 2 years from manufacturing.
Can oocytes/embryos frozen by other manufacturer’s products be thawed by Cryotec products?
Yes, but we cannot guarantee similar results to those obtained by using Cryotec products for the entire process.
Can oocytes/embryos frozen by the closed system be thawed by the Cryotec method?
You can use the Cryotec method to thaw oocytes/embryos frozen by the closed system, since the thawing process of the closed system is basically similar to the open method. However, we cannot guarantee similar results to those obtained by using Cryotec products for the entire process.
Should I remove air bubbles that were formed on the surface when solutions were added to the well?  
Air bubbles, if formed, spontaneously disappear. You can leave such bubbles as they are because removing air bubbles may change the solution volume.
How much magnification of a microscope should I use for this vitrification process?
We recommend a magnification so that the circle of the well overlaps with the circle of the microscopic visual field (12 to 15x) for general operations such as embryo transfer, and the highest magnification of the microscope (approximately 45 to 55x) for steps that require observation of oocyte/embryo.
The aspiration volume of a pre-solution is specified for each step (for example, volume consistent with 3 mm-length of the pipette tip after aspirating oocyte/embryo). However, what should I do in case the inner diameter of a pipette is not constant?
You can aspirate the volume as indicated despite differences in the inner diameter of the pipette used because we specify the minimum volume required as a rough indication of solutions used. We have determined the rough indication by assuming appropriate inner diameters (140 to 150 µm for oocyte/cleavage stage embryo, and 200 to 250µm for blastocyst), so please refrain from using a pipette with an excessively large inner diameter.
Should I close the lid of the plate during operation?
We recommend operation while opening the lid because the volume of solutions required is at least 300 µL, which causes no problems by continuous use without the lid. In particular at thawing, slow dilution by mixing pre-solution and current-solution over time is important. Vibration by opening/closing the lid may cause adverse impacts on the result of thawing.
Can I purchase individual products of the Cryotec, for example, only Cryotec or plates?
Yes, all the Cryotec products can be separately purchased.

To reach desirable result