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
European Mouse Mutant Archive - EMMA
CNR/IBC
Istituto di Biologia Cellulare, Monterotondo, Italy
CNRS/CDTA
Centre de Distribution, de Typage et d'Archivage animal, Orléans, France
MRC/MGU
Mammalian Genetics Unit, Harwell, UK
Karolinska Institutet
Karolinska Institutet, Stockholm, Sweden
FCG/IGC
Instituto Gulbenkian de Ciência , Oeiras, Portugal
HMGU/IEG
Institute of Experimental Genetics, Munich, Germany
EMBL/EBI
European Bioinformatics Institute, Hinxton, UK
GIE-CERBM/ICS
Institut Clinique de la Souris, Illkirch/Strasbourg, France
Sanger Institute
Wellcome Trust Sanger Institute, Hinxton, UK
CNB/CSIC
Centro Nacional de Biotecnología, Madrid, Spain
Fleming
Biomedical Sciences Research Centre Al. Fleming, Athens, Greece
OULU
University of Oulu, Oulu, Finland
BIAT
Vetmeduni Vienna, Biomodels Austria, Vienna, Austria
IMG
Institute of Molecular Genetics, Prague, Czech Republic
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
50.0
0.0
1
2
3
4
5
6
7
8
9
-50.0
1
0
1
1
-100.0
Dry Shipper
-150.0
-200.0
50
0
1
2
3
-50
-100
Dry-Shipper
-150
-200
An International Centre for Mouse Genetics
4
5
6
What we cryopreserve
 Pre-implantation embryos
 Oocytes
 Spermatozoa
 Ovarian tissue
An International Centre for Mouse Genetics
Cryopreservation of the preimplantation 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
Clamp
CBS goblets
- nice
idea but!
An International Centre for Mouse Genetics
Sperm freezing equipment
An International Centre for Mouse Genetics
Cooling Rate
Measuring 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
Temperature (°C)
Rate box
Cooling rates – Cooling
Thermosafe
50.00
Time (min)
0.00
2
4
6
8
-50.00
10
3Layer
2Layer
-100.00
1Layer
-150.00
Sperm straw in container
Temperature (°C/min)
-200.00
50.00
Styrofoam
Time (min)
Liquid nitrogen
0.00
2
4
-50.00
6
8
10
3Layer
2Layer
1Layer
-100.00
-150.00
Mean±SD (n=3)
An International Centre for Mouse Genetics
2.5 cm
6-9 cm
Cooling
Rate
Cooling rates
– dewar
Time (min)
Temperature (°C)
50
0
0
2
4
6
8
10
35CM
-50
30CM
25CM
20CM
-100
15CM
10CM
-150
Cooling Rate (°C/min)
-200
Time (min)
50
0
0
2
4
6
8
10
35CM
-50
30CM
h
25CM
20CM
-100
15CM
10CM
-150
-200
Mean±SD (n=5)
An International Centre for Mouse Genetics
LN2
Cooling rates –Cooling
effectsRon fertility
Groups
BOX
35CM
30CM
25CM
Cooling rate (°C/min)
-37.40
-70
-115.88
-145.88
Motile
27.25
23.25
46.75
55.25
Progressive
10.50
8.25
12.25
14.75
Rapid
14.25
13.50
19.75
23.00
50.00
BOX
35CM
30CM
25CM
40.00
30.00
20.00
10.00
0.00
Motile
Progressive
An International Centre for Mouse Genetics
Rapid
Fertilization Rate (%)
Sperm Viability (%)
60.00
40.00
30.00
20.00
10.00
0.00
BOX
35CM
30CM
25CM