An International Centre for Mouse Genetics An overview of the MRC-Harwell’s cryopreservation...

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


Mary Lyon Centre – high barrier unit


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)


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 service


Host bi-annual cryocourses

Hands on demonstration of: Embryo freezing Sperm freezing In vitro fertilization

Reference point Disseminate skills


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


Transport - Dry shipper

Keep samples at LN2 Temp

Expensive Re-usable Considered safe by IATA Robust


What we cryopreserve

Pre-implantation embryos

Oocytes

Spermatozoa

Ovarian tissue


Cryopreservation of the pre-implantation embryo

Controlled rate freezing

Vitrification


Freezing machines

• Planer Kryo 10

• FTS - BioCool IV


Goblet ClampCBS goblets - nice idea but!


Sperm freezing equipment


Cooling RateMeasuring cooling rates

• Comark thermocouple used to measure freezing rates

• Temperature during transportation not measured a present (Thermoguard or iButtons)


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


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


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

An International Centre for Mouse Genetics An overview of the MRC-Harwell’s cryopreservation...

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