Embryos can be cryopreserved at PN stage, early cleavage stage (2-8 cells) or blastocyst stage. If using Testart’s protocol for early embryos and Menezo’s simplified protocol for blastocysts, the programs are identical except for the finishing temperature (-30ēC for early embryos, -37ēC for blastocysts). It is therefore possible to concurrently freeze blastocysts and early embryos; straws containing early embryos can be removed at –30ēC.

Suggested programs:

START at 22ēC

-2C/min to -7ēC

SEED

HOLD at –7ēC for 10min

-0.3C/min to -30ēC (early embryos) or –37ēC (blastocysts)

END

Refer to the Biotronics manual for details on programming the controlled rate freezer.

Preparation

Prepare dishes or 4-well plates containing 1ml of the cryoprotectant solutions. Since these are normally stored at 2ē to 8ēC, leave for 10 minutes to allow to reach ambient temperature.

Label the appropriate number of straws and plugs (patient’s name/unit number/date of birth/ date of freezing etc). Straws can contain one or more embryos (the number in each must be clearly documented).

Switch on the Biotronics freezer and fill the chamber with liquid nitrogen as per instruction manual. Select the required program and press start. This will set and hold the temperature at 22ēC (program paused - the pause button will be flashing). The alarm will sound and can be silenced by pressing the silent button.

This is carried out at ambient temperature.

Before starting the freezing, place the dish containing the embryos to be frozen on a surface at room temperature for 1 minute. At the same time warm the dish containing the first cryoprotectant solution for 1 minute at 37ēC. This will result in smaller increments of temperature change for embryos transferred from culture medium to cryoprotectant.

Transfer the embryos to the relevant cryoprotectant solutions for the required amount of time as per your laboratory protocols. During the incubation step of embryos in the final cryoprotectant solution, load the embryos into the straws as follows:

Draw up approx 1cm cryoprotectant solution followed by approx 0.5cm air. Draw up the embryo(s) in a long bubble of cryoprotectant (4 - 5cm) followed by another 0.5cm air bubble. Finally draw up more medium until the cotton plug has been wetted. Insert the plug. Check the position of embryos under the microscope – they should be near the centre of the long freezing medium bubble. Embryos must NOT be near the plug end of the bubble where seeding will be initiated (placement can be adjusted by standing the straw vertically). Once the embryos are positioned correctly place the straws in the Biotronics freezer.

Running the freezing program

When the embryos have been in the final cryoprotectant solution for the required amount of time start the program running by cancelling pause (press the pause button). Check that the program is running (temperature falling on display).

When the alarm sounds (can be silenced – press the silent button) seed each straw by touching the meniscus of the medium containing the embryos at the plug end with a cotton bud soaked in liquid nitrogen. Visually check that ice formation has been initiated. The program pauses at this point, so after seeding has been completed cancel pause by pressing the pause button. Check that the program has resumed (hold time falling on display).

At the end of the program plunge the straws into liquid nitrogen immediately and store as per your laboratory protocols. Press the finish button.

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