Method

16.2.2.1. Freezing the Embryo

1. Prepare two 35-mm tissue-culture dishes containing freezing solution 1, one dish containing freezing solution 2, and one containing freezing solution 3. Incubate at room temperature.

2. Transfer the mouse embryos (eight cell to blastocyst stage) from M16 to freezing solution 1. Rinse briefly.

3. Transfer the embryos to the second dish of freezing solution 1 and rinse.

4. Place the embryos in a dish containing freezing solution 2, and incubate at room temperature for 10 min.

5. Transfer the embryos to freezing solution 3, and incubate at room temperature for 10 min. During this period, label the straws, load them with embryos, and seal the straws.

6. Begin cooling within 10-30 min from when the embryos are first exposed to freezing solution 3. Cool the straws in the programmable freezing machine from room temperature to -6°C at -2°C/min and hold at -6°C for 5 min.

7. Remove the straws from the freezing machine. Hold the straws in a vertical position, and vertically seed them by grasping the straws at a point above the embryos with a pair of forceps dipped in liquid nitrogen. Hold for a few seconds until the liquid at the point of contact freezes.

8. Return the straws to the freezing machine and maintain at -6°C for 10 min.

10. Finally plunge the straws into liquid nitrogen and store.

16.2.2.2. Thawing the Frozen Embryos

1. Prepare one 35-mm tissue-culture dish with freezing solution 4, two other dishes containing freezing solution 1, and two containing M16 medium. Incubate at room temperature.

2. Remove the straws quickly from the liquid nitrogen, and hold at room temperature for 30 s before placing in a 37°C water bath until the ice melts.

3. Expel the embryos into the dish of freezing solution 4, and incubate at room temperature for exactly 10 min.

4. Rinse the embryos twice in two dishes of freezing solution 1.

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

6. Culture the embryos, or transfer them into recipients.

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