A Slow Freeze/Thaw Method for Cryopreservation of Mouse Embryos

A slow freeze/thaw method for cryopreserving
mouse embryos

FESA (Frozen Embryo & Sperm Archive)
Medical Research Council
Harwell, UK

Martin Fray

www.emmanet.org November 2007

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


The method

• Modification of method published by
Renard and Babinet, 1984

• This method is robust and works for
all stages pre-implantation stage
embryos


Embryos are frozen in plastic semen
straws

AB12

Air Air
Plug
Plug
Label
Embryos in
Diluent:
cryoprotectant:
1M Sucrose
1.5M ProH


Preparing the straws

• Push the cotton plug in
until it is 75mm from the end

• Use a metal rod to do this

A

A B

75mm


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
Cotton/PVA plug 1 2 3
AB12
volumes of sucrose/ProH/air
Label 20mm 7mm 5mm to be aspirated

 Use a ruler and a pre-marked
rack


Supporting the straws

• Place straws on a stable
support

• Don’t flick them


Preparatory steps


Before you start

 Preparation precedes performance

 Make sure you have all of your
supplies before starting
IVF dish M2 ProH
 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


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 sucrose to the first mark –
then aspirate air so the sucrose
2 meniscus reaches the second mark
1

• Aspirate ProH so that
3
the sucrose meniscus
reaches the third mark


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

Some water out Equilibrium reached

ProH in

0 min 1 min 5 min


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.

Ice

Extra-cellular
Most water out 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)

100
90 Cooled at 1.7 C/min
80
Survival rate (%)

70
60
50
40
30
20
10 Cooled at 0.18 C/min
0
0.1 1 10 100 1000

Warming rate (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

1.0M Sucrose
No Sucrose (non-permeating solute)
ProH 1 min.

Rapidly swollen embryo
containing ProH and water
(damaged)
Isotonic solution.
5 min.


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

120

100
% of initial cell volume

80

60

40

CPA Freeze Thaw Diluant Media
20

0
Time


A Slow Freeze/Thaw Method for Cryopreservation of Mouse Embryos

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A Slow Freeze/Thaw Method for Cryopreservation of Mouse Embryos