an international centre for mouse genetics an overview of the mrc-harwell’s cryopreservation...
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An International Centre for Mouse Genetics
An overview of the MRC-Harwell’s cryopreservation service
Martin Fray
Frozen Embryo & Sperm Archive – (FESA)Medical Research Council
Harwell, UK
An International Centre for Mouse Genetics
Mary Lyon Centre – high barrier unit
An International Centre for Mouse Genetics
The MRC frozen embryo archive
Worldwide Genetic Resource of mouse models of human disease
• ~1450 stocks, >470,000 embryos– Includes transgenic, mutants, chromosome anomalies
& inbred strains
Sole UK archiving centre http://www.har.mrc.ac.uk EMMA (European Mouse Mutant Archive)
An International Centre for Mouse Genetics
CNR/IBCIstituto di Biologia Cellulare, Monterotondo, ItalyCNRS/CDTACentre de Distribution, de Typage et d'Archivage animal, Orléans, FranceMRC/MGUMammalian Genetics Unit, Harwell, UKKarolinska InstitutetKarolinska Institutet, Stockholm, SwedenFCG/IGCInstituto Gulbenkian de Ciência , Oeiras, PortugalHMGU/IEG Institute of Experimental Genetics, Munich, GermanyEMBL/EBIEuropean Bioinformatics Institute, Hinxton, UK GIE-CERBM/ICSInstitut Clinique de la Souris, Illkirch/Strasbourg, France Sanger InstituteWellcome Trust Sanger Institute, Hinxton, UK CNB/CSICCentro Nacional de Biotecnología, Madrid, SpainFlemingBiomedical Sciences Research Centre Al. Fleming, Athens, GreeceOULUUniversity of Oulu, Oulu, FinlandBIATVetmeduni Vienna, Biomodels Austria, Vienna, AustriaIMGInstitute of Molecular Genetics, Prague, Czech Republic
European Mouse Mutant Archive - EMMA
An International Centre for Mouse Genetics
An international service
An International Centre for Mouse Genetics
Host bi-annual cryocourses
Hands on demonstration of: Embryo freezing Sperm freezing In vitro fertilization
Reference point Disseminate skills
An International Centre for Mouse Genetics
Liquid nitrogen storage
Samples retained in mobile dewars (n=35)
Bulk (Chart) LN2 tanks (n=10)
3 x LN2 rooms all with an independent LN2 supply
BOC and Cryo-service deliver to site
An International Centre for Mouse Genetics
Transport - Dry shipper
Keep samples at LN2 Temp
Expensive Re-usable Considered safe by IATA Robust
An International Centre for Mouse Genetics
What we cryopreserve
Pre-implantation embryos
Oocytes
Spermatozoa
Ovarian tissue
An International Centre for Mouse Genetics
Cryopreservation of the pre-implantation embryo
Controlled rate freezing
Vitrification
An International Centre for Mouse Genetics
Freezing machines
• Planer Kryo 10
• FTS - BioCool IV
An International Centre for Mouse Genetics
Goblet ClampCBS goblets - nice idea but!
An International Centre for Mouse Genetics
Sperm freezing equipment
An International Centre for Mouse Genetics
Cooling RateMeasuring cooling rates
• Comark thermocouple used to measure freezing rates
• Temperature during transportation not measured a present (Thermoguard or iButtons)
An International Centre for Mouse Genetics
Mean±SD (n=3)
Cooling Rate
-200.00
-150.00
-100.00
-50.00
0.00
50.00
2 4 6 8 10
Tem
pera
ture
(°C) Time (min)
3Layer
2Layer
1Layer
-150.00
-100.00
-50.00
0.00
50.00
2 4 6 8 10
Te
mp
era
ture
(°C/m
in)
Time (min)
3Layer
2Layer
1Layer
2.5 cm
6-9 cmLiquid nitrogen
Styrofoam
Sperm straw in container
Cooling rates – Thermosafe box
An International Centre for Mouse Genetics
Mean±SD (n=5)
Cooling Rate
-200
-150
-100
-50
0
50
0 2 4 6 8 10
Tem
pera
ture
(°C)
Time (min)
35CM
30CM
25CM
20CM
15CM
10CM
-200
-150
-100
-50
0
50
0 2 4 6 8 10
Co
oli
ng
Rate
(°C
/min
) Time (min)
35CM
30CM
25CM
20CM
15CM
10CM LN2
h
Cooling rates – dewar
An International Centre for Mouse Genetics
Groups Cooling rate (°C/min) Motile Progressive RapidBOX -37.40 27.25 10.50 14.2535CM -70 23.25 8.25 13.5030CM -115.88 46.75 12.25 19.7525CM -145.88 55.25 14.75 23.00
Cooling R
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Motile Progressive Rapid
Sperm Viability (%
)
BOX 35CM30CM 25CM
0.00
10.00
20.00
30.00
40.00
BOX 35CM 30CM 25CM
Fertilizati
on Rate (%
)
Cooling rates – effects on fertility
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