a slow freeze/thaw method for cryopreservation of mouse embryos

www.emmanet.org November 2007

A slow freeze/thaw method for cryopreserving

mouse embryos

FESA (Frozen Embryo & Sperm Archive)

Medical Research Council

Harwell, UK

Martin Fray


Mary Lyon Centre


Key aspects of cryopreservation

Cryoprotectant used

Seeding temperature

Freezing rate

Thawing rate

Handling

Data management


Freezing machines

• Freezing machines vary but the

freeze/thaw principles remain the

same

• LN2 as refrigerant

- Planer Kryo 10

• Electrical power to an alcohol bath

- BioCool IV


• Modification of method published by

Renard and Babinet, 1984

• This method is robust and works for

all stages pre-implantation stage

embryos

The method


Embryos are frozen in plastic semen

straws

AB12

Label

Plug

Diluent:

1M Sucrose

Air Air

Embryos in

cryoprotectant:

1.5M ProH

Plug


Preparing the straws

A

A B

75mm

• Push the cotton plug in

until it is 75mm from the end

• Use a metal rod to do this


Labelling the straws

• Label each straw with a unique

identifier

• Labels must be compatible with

LN2 – some adhesive labels peel off!


Marking the straws

Mark straws to indicate the

volumes of sucrose/ProH/air

to be aspirated

Use a ruler and a pre-marked

rack

AB12

1 2 3

20mm 7mm 5mm Label

Cotton/PVA plug


Supporting the straws

• Place straws on a stable

support

• Don’t flick them


Preparatory steps


Preparation precedes performance

Make sure you have all of your supplies before starting

Check media are in date.

Clearly label ProH and wash dishes (1 set/strain)

Dispense ~300 µl of ProH into a dish

Label separate ProH and sucrose dishes for filling straws

Before you start

IVF dish M2 ProH


Switch the machine on in good time

• Check that freezers are working and

programmed correctly

• Check alcohol levels or LN2 levels

where appropriate

• Switch the units on and take them

down to the start Temp

• Chamber start Temp should be -7°C


Pre-filling straws

• Aspirate ProH so that

the sucrose meniscus

reaches the third mark

• Aspirate sucrose to the first mark –

then aspirate air so the sucrose

meniscus reaches the second mark 1

2

3


Sealing the straws

• Aspirate air so that the

sucrose fraction wets the

cotton/PVA plug – this will

seal the straw

• Don’t rush

• Place straws back on a

stable support


8-cell embryo in 1.5M ProH

0 min

Some water out

1 min

ProH in

5 min

Equilibrium reached


Freezing steps


Equilibrating the embryos

Carefully inspect your embryos

before aspirating them

Place embryos selected for

freezing in the drop of ProH

Leave the embryos to equilibrate

in the ProH for 15 minutes


Checking the embryos

• Carefully inspect your embryos

again before loading them into

the straws

• Aim to freeze only first quality

embryos – exceptions always

apply!

• Work in pairs if possible to QC

the process

• Don’t rush


Loading straws

• Trap embryos between small air

bubbles in the pipette – easy to see

• Transfer the embryos along with the

minimum amount of media into the

straws ProH fraction

• Don’t fragment the ProH fragment

by blowing air into the straw

• Load sufficient embryos to recover

the stock


Plugging the straws

• Seal the straws with a capillary

sealant like Critoseal

• Smooth end of sealant with finger

• Place straws on a stable platform

• If you drop them the ProH and

sucrose fractions will mix!


Loading the machine

Check the freezing machine

has reached its holding Temp

of -7ºC

Place the straws in the

freezing machine – ProH

fraction first

Equilibrate the straws for 5

minutes

Get yourself ready to seed the

straws


Seeding the straws

• Pre-cooled a cotton wool bud or pair

of ‘heavy duty’ forceps in LN2

• Draw the seeding tool across the

top of the sucrose fraction

• Ice crystals should form immediately

• Keep re-chilling the seeding tool

• Work efficiently!

• Wait 5 minutes


Checking seeding

• After 5 minutes check that

crystallisation is complete

• Ice crystals should be visible

throughout the sucrose and ProH

fractions

• Keep the ProH fraction cold


• Select ‘ramp 2’ to cool the

embryos -30ºC at 0.5ºC/min.


8-cell embryos cooled to -300C at

0.50C/min.

Most water out

Ice

Extra-cellular

solutes very

concentrated


Ending the freeze session

• Set a timer once the freeze

machine is in ‘ramp 2’

• ~46 minutes to reach -30ºC

• Plunge the straws in LN2 once

the freezer has reached -30ºC



Data management

Accurate records for data capture/retrieval

Record

• Stock details

• Sample id

• Contents of each cryovial/straw

• Sample location

• Freeze/thaw protocol

• Parental genotype


Storage


Stability of the mouse genome

Embryos stored under low-dose irradiation to

simulate long-term storage

• No effect of irradiation found on: • Morphological appearance after thawing

• Survival to blastocyst after overnight culture

• Survival of foetuses and live-born after transfer

• Offspring bred normally and showed no evidence of genetic

defects

• Simulated storage of up to 2000 yr. under normal

levels of background radiation


Recovery of genetic variants

Various mouse stocks recovered after embryo

cryopreservation: • Inbred strain (CBA/CaH)

• Inbred strain + translocation (CBA/H-T6)

• Dominant sex-linked gene (Modp)

• Multiple recessive stocks:

• PT (aa bb cchcch dd pp ss sese)

• HT (aa bpbp fzfz lnln papa pepe)

• XO (tagged with Ta & Moblo)

• Some strains/mutations freeze poorly

• Set up viability tests


Protecting your samples

Yours samples are only safe if they are handled properly

The straws have a ‘small’ thermal mass and will warm up

very quickly

Keep straws submerged in LN2

Handle straws by the end furthest from the ProH fraction

Handle the straws with pre-cooled forceps


Store stocks in duplicate


Storage in canes and goblets

• Only one cane/stock in each freezer

compartment

Only one code/canister

• The straws for each stock are kept

accessible until the stock is fully

archived and a viability test has been

performed.

• Straws are fully submersed in LN2.

• Storage in vapors is NOT advised.


Storage in cassettes and boxes

• Storage in cassettes and

boxes is an alternative

• Ideal for use with large bulk

storage tanks

• Method used by TJL


Shipment – use dry shippers

• Keep samples at LN2 Temp

• Re-usable

• Considered safe by IATA

• Robust


Thawing straws


Effect of warming rate - Whittingham et al

(1972)

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

Warming rate (C/min)

Su

rviv

al ra

te (

%)

Cooled at 0.18 C/min

Cooled at 1.7 C/min


Getting ready

• Prepare work area in advance

• Label one empty dish with the

straws identity

• Place two drops (~200µl) of

M2 in a second dish

• Don’t rush - thaw one straw at

a time

• Wear safety glasses


Thawing straws

Hold straw in air for 40 sec

Plunge straw in water bath at room Temp (20 -25ºC)

Wipe straw carefully


Unsealing the straws

Cut off capillary sealant

- don’t flick the straw

Bisect the cotton/PVA plug

- don’t flick the straw


Emptying the straws

• Expel contents into dish by

pushing remnant of the plug with

a metal rod

• Don’t touch the expelled

contents with the straw

• Don’t push plug into the dish

• Allow ProH and sucrose

solutions to mix for 5 minutes


Embryo shortly after rapid warming

from -1960C

No Sucrose

Rapidly swollen embryo containing ProH and water

(damaged)

1.0M Sucrose (non-permeating solute)

5 min.

Isotonic solution.

1 min. ProH


Washing the embryos

Two x 5 minutes washes in M2

Inspect embryo quality

Low health status stocks may require up to 10 x washes (1:100 dilution/wash)

The embryos are now ready for transfer or culture


Embryo freezing dynamics

0

20

40

60

80

100

120

Time

% o

f in

itia

l ce

ll vo

lum

e

CPA Freeze Thaw Diluant Media

a slow freeze/thaw method for cryopreservation of mouse embryos

Business

straws place straws

straws mark straws

straws label

straws trap embryos

intothe straws

aspirate proh

straws aspirate air

finger place straws