Standard Operation Protocols (SOPs)
for the Cryopreservation of Mouse Germplasm
1. Cryopreservation of Mouse Spermatozoa in 1.8ml
cryotubes
2. Cryopreservation of mouse spermatozoa in French
straws
3. Mouse Embryo Cryopreservation in 1,8ml cryotubes
4. Mouse Embryo Cryopreservation in 0.25 ml plastic
straws
5. Superovulation protocol
6. In vitro fertilization
1. Cryopreservation of Mouse Spermatozoa in 1.8ml cryotubes
Equipment
Cryoprotectant agent (CPA)
18% raffinose, (Sigma; R-7630)
3% skim milk, (Difco Betalab 1878-17)
made up in sterile water (Sigma; W-1503).
1.8ml Nunc cryotubes.
Cryotube rack.
Micro centrifuge and 1.5ml microfuge tubes.
35mm culture dishes, (e.g. Falcon 3001).
Deep polystyrene box with lid (suitable for holding liquid nitrogen).
Small dewar for liquid nitrogen.
CO2 incubator (37°C).
Hot block held at 37°C.
Sexually mature male mice at least 8 weeks old (not recently mated).
Method
Place 9ml Sigma H2O (W-1503) in a screw top 15ml Falcon tube (2097) and
equilibrate to 60oC in a water bath. Add 1.8g raffinose and dissolve by gentle
inversion. Add 0.3g skim milk and dissolve by gentle inversion. Make up to 10ml if
necessary. Aliquot into microfuge tubes and centrifuge at 14,000 rpm for 10 min.
Collect supernatant and filter the solution using a 0.45µm syringe end filter. Place
1.1ml aliquots into cryotubes and store at -20oC.
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Cryopreservation method
To prepare the cooling apparatus place a platform (e.g. the insert from a Gilson
yellow tip box) into the polystyrene box. This acts as a support for the cryotube
rack. Carefully pour liquid nitrogen into the polystyrene box to just cover the
platform. Place a cryotube rack on top of the platform so that it is suspended in
liquid nitrogen vapour. Replace the lid on the polystyrene box and allow it to fill with
vapour. Replenish the liquid nitrogen as necessary during the freezing session, but
do not allow the level to rise above the platform.
Thaw one 1.1ml aliquot of cryoprotectant solution for each male mouse and bring to
37°C in the incubator or hot block. Mix by inversion if there is any precipitation.
Pipette 1.0ml CPA into a small culture dish and place on the hot block at 37oC.
Dissect the vasa deferentia and cauda epididymides from the mouse and clean off all
fat and blood. This is easily achieved by placing the organs on a paper tissue and
examining them under a microscope. Transfer the cleaned organs to the dish of CPA
and using watchmaker’s forceps mince the epididymis and squeeze sperm gently out
of the vas deferens. To disperse the sperm, gently shake the culture dish for ~30
seconds. Place the dishes lid on a shelf in the CO2 incubator, then rest the base of
the dish, containing the sperm, at an angle on the lid. Incubate for sperm
preparation for 10 minutes at 37oC.
Keeping the culture dish at an angle, remove the animal tissues from the suspension
by scraping them to one side of the culture dish with a pipette tip. Place 100µl
aliquots into each of 8 cryotubes and tighten the screw cap to seal the cryotube.
Place the cryotubes in a pre-cooled rack, supported in the liquid nitrogen vapour
phase of the freezing apparatus and leave for 10 minutes. Remove the cryotubes
from the freezing apparatus and plunge them into liquid nitrogen. The sperm
samples can now be stored in a liquid nitrogen refrigerator until required.
Thawing mouse spermatozoa frozen in cryotubes
Equipment
Aliquot(s) of frozen sperm.
Water bath at 37°C .
Forceps
Method
Note: Take special care when following this protocol that the cryotubes are not filled
with liquid nitrogen before plunging them into the water bath, such tubes may
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explode. If liquid nitrogen is present in the cryotube, wait for it to evaporate and
escape before plunging the cryotube into the water bath.
Using forceps, hold the cryotube in air for 30 seconds and then thaw rapidly by
plunging it into a 37°C water bath. When the sample has thawed, gently agitate the
cryotube to ensure the sperm sample is evenly mixed. Then gently pipette 5μl of
the sperm suspension directly into your in vitro fertilization drop. Sperm motility will
initially appear low but within 10 minutes sperm quality and motility can be
assessed. If necessary add a second aliquot of sperm to the in vitro fertilization
drop. The in vitro fertilization drops are now ready to accept the cumulus-oocyte
masses.
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2. Cryopreservation of mouse spermatozoa in French straws
Equipment
CPA (preparation see below, section MEDIA)
0.9% NaCl
Fine scissors
Fine forceps
Spring scissors
Watchmakers forceps
stereomicroscope
4-well-dishes (Nunc, Wiesbaden, Germany cat. no. 176740)
incubator (37°C, 5% CO2)
0.25 ml French type mini straws (Minitueb, Tiefenbach, Germany, cat. no.
13407/0010)
1 ml syringe
Heat sealing apparatus for plastic films
Flask for liquid nitrogen
Freezing canister: 50 ml-syringe, styrofoam, acrylic bar (50 cm)
Liquid nitrogen tank; cryopreservation cups (Sigma cat. no. C-3928)
Method
To prepare the freezing canister: insert a piece of styrofoam tightly into the bottom
of a 50 ml-syringe, heat seal the outlet of the syringe, and fix the syringe to the
acrylic bar.
Thaw an aliquot of CPA in the incubator. Fill one well of the 4-well dish with 200 µl
0.9% NaCl, another well with 160 µl CPA.
Sacrifice the male mouse by cervical dislocation and dissect the two caudae
epididymides. Place the epididymides into 0.9% NaCl of the 4-well dish (on ice,
alternatively at 37°C). Under a microscope remove all remaining fat and big blood
vessels with a watchmaker forceps and a spring scissor.
Place both caudae epididymides into the CPA in the well of the 4-well dish (on ice).
Cut them several times with a spring scissor and let the spermatozoa swim out for 35 minutes (on ice or at 37°C). Shake the dish carefully until the suspension is
homogenous.
Pipette 10 x 15 µl-aliquots on the lid of a dish. Connect the 1 ml syringe with a 0.25
ml straw, aspirate successively 100 µl HTF medium, an air bubble, one 15 µl-drop,
another air bubble. Heat seal both ends of the straw. Repeat for all drops.
Place samples in the freezing canister and put it in the liquid nitrogen vapour phase
of the flask and leave it there for 10 minutes then plunge the freezing canister
directly in liquid nitrogen (-196°C).
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Place both caudae epididymides into the CPA in the well of the 4-well dish.
Thawing of mouse spermatozoa frozen in French straws
Equipment
Water bath (37°C)
40 mm culture dishes (Nunc Cat. Nr. 150318)
HTF medium (preparation see below, MEDIA) or another IVF medium
Equilibrated mineral oil
Incubator (37°C, 5 % CO2)
Method
1) Place the frozen straw directly into the water bath for 10 minutes.
2) Meanwhile prepare dishes for the IVF, 200µl HTF medium per culture dish
covered with equilibrated mineral oil.
3) Dry the straw with a tissue, keep straw horizontal and cut both ends. Remove
only the sperm suspension and transfer 2-4µl of the suspension to the prepared
medium drops.
4) Place all drops in the incubator for 45 minutes to capacitate the spermatozoa.
5) Evaluate motility of the spermatozoa.
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3. Mouse Embryo Cryopreservation in 1,8ml cryotubes
Equipment
1.8 ml Cryotubes
Embryo flushing concavity dishes
Culture dish
Capillary
Rate controlled freezer
Liquid nitrogen storage container
Media: PBS-BSA
Cryprotectant: DMSO
Mineral oil.
Surgical equipment
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 -6°C and -12°C salt-ice baths.
Set-up the controlled-rate freezer (Temperature in the controlled rate freezer is
lowered at the rate of 0.5°C per min, from -6°C to -80°C).
Transfer cryotubes in the -6°C bath for 2 minutes.
Into the -12°C bath place one 50-ml Falcon tube containing a small amount of PBSBSA 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 -6°C bath.
Transfer cryotubes to the controlled-rate freezer which has been precooled to -6°C
When the temperature of the freezing chamber has been lowered to -80°C, 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).
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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
Material
Embryo flushing concavity dishes
Culture dish
Capillary
Media: PBS-BSA, M16, KSOM
Cryoprotectant: DMSO
Mineral oil.
Surgical 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.
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4. Mouse Embryo Cryopreservation in 0.25 ml plastic straws
Equipment
Programmable freezing machine
35mm culture dishes
Cristaseal or heat sealer to seal straws
0.25 ml plastic semen straws (IMV; Catalogue Number FZA201)
1.5M propylene glycol solution (Sigma; Catalogue Number. P-1009) made up in M2.
1.0M sucrose solution (BDH; Catalogue Number 102745C) made up in M2.
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.
Label the straw as appropriate
With a marker pen make 3 marks on each straw.
1st mark 20mm from the plug.
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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.
Load the straws into the programmable freezer and cool to -7°C (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
0.3°C/min to -30°C and then plunge the straws directly into liquid nitrogen.
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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.
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5. Superovulation protocol
Inject 20-25 days-old females with 2.5-10 IU of PMSG between 3.00-6.00 pm
(hormone dose depends on the age of the animal and on the mouse strain). 46-48
hours after PMSG administration, inject 2.5-5.0 IU of hCG (hormone dose depends
on the age of the animal and on the mouse strain). The females should then be
mated to selected males and plug checked the following morning (20 –24 hours
post-hCG injection i.e. 0.5 day post coitum, dpc).
Collect embryos from
superovulated females 1.5 to 3.5 dpc.
6. In vitro fertilization
Equipment
Culture dish
Media: HTF, MEM, M16, KSOM, PBS-BSA
Mineral oil
Capillary
CO2 incubator
Method
72 hours before collecting oocytes:
Inject 20-25 days-old females with 2.5-10 IU PMSG (carry out treatment at 3.304.00 pm); actual hormone dose and animal age depending on mouse strain; e.g.:
5.0 IU PMSG for C57BL/6J, 21 days-old.
48 hours post-PMSG injection:
Inject 20-25 days-old females with 2.5-10 IU HCG (carry out treatment at 6.00-6.30
pm) actual hormone dose and animal age depending on mouse strain; e.g.: 5.0 IU
HCG for C57BL/6J, 21 days-old. During the same day, also prepare an appropriate
number 35x10 mm of petri-dishes for the IVF and embryo culture.
Three types of dishes needed:
a) Sperm dish with a 900 ml (single drop) of MEM or HTF, under oil.
b) IVF dish with a 500 ml (single drop) of MEM or HTF, under oil
c) Wash dish with four drops (100 ml each) of MEM or HTF, under oil.
All dishes must be stored in the 37°C in a CO2 incubator overnight.
The IVF procedure must be carried out within 12-14 hours post-HCG injection.
a) Sacrifice 1-3 sexually mature males, which have not been mated for at least 5
days. Extract the sperm (see sperm freezing procedure), using the sperm dish (wash
the cauda epididymides and vasa deferentia before transferring them to the PBS
solution).
b) Alternatively, thaw frozen sperms (see sperm thawing procedure)
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c) Sacrifice the superovulated females and take out the oviducts.
Wash oviducts in PBS solution and transfer to an IVF dish.
Release the
oocyte/cumulus cell masses from the oviducts (take care to avoid blood
contamination of IVF culture media).
d) Transfer oocyte/cumulus cell masses to the IVF dishes.
e) Add fresh or thawed sperms. 10-20l of fresh sperm or 40-50l of thawed sperm
suspension should be sufficient. Place IVF dish in 37°C CO2 incubator for 4-6 hours
to allow fertilization to occur. Then transfer the fertilized eggs from the IVF dishes
into one of the four drops of the wash dish.
Repeat this operation three at least times, then put wash dish in 37°C CO 2 incubator
overnight.
Prepare a culture dish with four drops (100l each) of M16 or KSOM medium and
put it in the 37°C incubator. The next day transfer the embryos to the culture dish
and wash them in two or three drops of M16 or KSOM medium and culture until
ready to use.
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