Methods

16.3.2.1. Freezing Embryos

1. Prepare two 35-mm tissue-culture dish with freezing solution 1 and one with freezing solution 5. Incubate at room temperature.

2. Transfer the mouse embryos (eight cells to the morula stage or 2V2 d post-hCG) from M16 culture medium to one of the dishes containing freezing solution 1.

3. Rinse the embryos twice in the second dish of freezing solution 1.

4. Place the embryos in the dish of freezing solution 5, and incubate at room temperature for 20 min.

5. Load the embryos into prelabeled straws, and seal.

6. Keep the embryos at room temperature, so that the total exposure to freezing solution 5 is 20 min.

7. Place the straws horizontally onto the bottom of the -60 to -80°C freezer.

8. Cool the straws for 5-15 min.

9. Plunge the straws into liquid nitrogen using cooled forceps as before.

16.3.2.2. Thawing Embryos

1. Prepare two 35-mm tissue-culture dish containing M16 medium, and warm in a 37°C incubator pregassed at 5% CO2. Also, prepare two dishes containing freezing solution 1 and one containing freezing solution 6. Incubate at room temperature.

2. Remove the straws quickly from the liquid nitrogen, and place in a 25°C water bath until the ice melts.

3. Expel the embryos into the dish of freezing solution 6, and incubate at room temperature for exactly 12 min.

4. Transfer the embryos into a dish of freezing solution 1, and rinse twice in the second dish of freezing solution 1.

5. Transfer the embryos into the prewarmed M16 medium, and rinse twice.

6. Culture the embryos or transfer them into recipients.

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