EMMA SOPs - Mouse Embryo Freezing

 

Mouse Embryo Cryopreservation in 1.8ml cryotubes

 

Equipment

Method

Transfer embryos recovered from each female in

a) PBS-BSA (0.1 ml) in a 2-ml cryotube, precooled on ice
b) M2 medium, RT

Add gently 0.1ml of cryoprotectant solution (2M DMSO in PBS-BSA: final concentration of DMSO will be 1M, precooled on ice). Let cryotubes to stand at least 30 minutes on ice (equilibration phase).

Prepare two different -6oC and -12oC salt-ice baths.

Set-up the controlled-rate freezer (Temperature in the controlled rate freezer is lowered at the rate of 0.5oC per min, from -6oC to -80oC).

Transfer cryotubes in the -6oC bath for 2 minutes.

Into the -12oC bath place one 50-ml Falcon tube containing a small amount of PBS-BSA and one pasteur glass pipette (prepare one pipette for each cryotube) and wait for PBS-BSA freezing (a small amount of PBS is drawn into the tip of pipette by capillary action).
Seed the top of the embryo suspension in each cryotube with a frozen PBS-BSA crystal at the tip of a pasteur pipette by carefully touching the surface of the medium containing embryos

Cap the seeded cryotubes in -6oC bath.

Transfer cryotubes to the controlled-rate freezer which has been precooled to -6oC

When the temperature of the freezing chamber has been lowered to -80oC, remove the cryotubes and immediately put them into a liquid nitrogen storage container (this step must be carried as rapidly as possible to prevent the cryotubes warming up).

The storage location of each tube is recorded for future retrieval.

Control samples: freeze one control vial (about 50 embryos) for each freezing process. Thaw this vial to assay for viability (test embryos in culture and by embryo transfer).

Freeze 1-2 control vials (about 30-50 embryos/vial) for each strain; to be thawed and assayed for viability and fertility of F1 crossing.

Thawing embryos frozen in 1.8 ml cryotubes

Equipment

Method

Remove cryotubes from liquid nitrogen and place them on the bench at room temperature until thawed (usually it requires about 10-15 min).
When completely thawed, slowly add 0.8 ml of PBS in a drop wise fashion to dilute the DMSO.
Collect the content of each cryotube with a pipette and transfer to embryology watch glasses to observe the embryos.
Wash embryos once in KSOM or M16 medium before any subsequent manipulation.

 

Mouse Embryo Cryopreservation in 0.25ml plastic straws

Equipment

Method

Collect and transfer the embryos into a 35mm culture dish containing 2ml M2 and screen embryos carefully for abnormalities. When all the embryos have been collected, wash them thoroughly through three dishes of M2.

To prepare the straws, take a 133mm plastic straw and using a metal rod push the plug from position A to B.

how to prepare the straws 1   how to prepare the straws 2

Label the straw as appropriate With a marker pen make 3 marks on each straw.

how to prepare the straws 3

1st mark 20mm from the plug.
2nd mark 7mm from mark 1.
3rd mark 5mm from mark 2.

To fill the straws fit a 1.0 ml disposable syringe to the labelled end of the straw and aspirate sucrose to mark 3. Aspirate air so that the sucrose meniscus reaches mark 2. Aspirate 1.5M propylene glycol so that the sucrose meniscus reaches mark 1. Aspirate air until the column of sucrose reaches half way up to the plug and forms a seal with the polyvinyl alcohol plug.

Before loading the embryos into the straws allow them to equilibrate for 15 minutes in a drop of 1.5M propylene glycol, at room temperature. Seal the straws using "Cristaseal"or a heat sealer.

Freezing Method

Load the straws into the programmable freezer and cool to -7oC (cooling rate not critical). Wait 5 minutes to equilibrate and then seed the sucrose fraction by touching it near the plug with the tips of forceps cooled in liquid nitrogen. Wait 5 minutes, then check that ice crystals have migrated to the embryo fraction. Cool at 0.3oC/min to -30oC and then plunge the straws directly into liquid nitrogen.

Freezing Machine

Freezing Machine

Thawing embryos frozen in 0.25 ml plastic straws

Using forceps, hold the straw in air for 30 seconds and then place in a water bath, at room temperature until the contents of the straw have thawed. Dry the straw with a tissue and cut off the seal and also cut through the PVA plug leaving about half the plug in place to act as a plunger. Using a metal rod, expel the entire liquid contents of the straw into a 35mm culture dish. Wait 5 minutes, during this time the embryos will shrink considerably. Transfer the embryos to a drop of M2. They will rapidly take up water and assume a normal appearance. After 5 minutes place the embryos in a second drop of M2 and assess their quality.