A Slow Freeze/Thaw Method for Cryopreservation of Mouse Embryos

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

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A Slow Freeze/Thaw Method for Cryopreservation of Mouse Embryos

  1. 1. A slow freeze/thaw method for cryopreserving mouse embryos FESA (Frozen Embryo & Sperm Archive) Medical Research Council Harwell, UK Martin Fraywww.emmanet.org November 2007
  2. 2. Mary Lyon Centrewww.emmanet.org November 2007
  3. 3. Key aspects of cryopreservation  Cryoprotectant used  Seeding temperature  Freezing rate  Thawing rate  Handling  Data managementwww.emmanet.org November 2007
  4. 4. 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 IVwww.emmanet.org November 2007
  5. 5. The method • Modification of method published by Renard and Babinet, 1984 • This method is robust and works for all stages pre-implantation stage embryoswww.emmanet.org November 2007
  6. 6. Embryos are frozen in plastic semen straws AB12 Air Air Plug Plug Label Embryos in Diluent: cryoprotectant: 1M Sucrose 1.5M ProHwww.emmanet.org November 2007
  7. 7. 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 75mmwww.emmanet.org November 2007
  8. 8. Labelling the straws • Label each straw with a unique identifier • Labels must be compatible with LN2 – some adhesive labels peel off!www.emmanet.org November 2007
  9. 9. Marking the straws  Mark straws to indicate the Cotton/PVA plug 1 2 3AB12 volumes of sucrose/ProH/airLabel 20mm 7mm 5mm to be aspirated  Use a ruler and a pre-marked rackwww.emmanet.org November 2007
  10. 10. Supporting the straws • Place straws on a stable support • Don’t flick themwww.emmanet.org November 2007
  11. 11. Preparatory stepswww.emmanet.org November 2007
  12. 12. 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 strawswww.emmanet.org November 2007
  13. 13. 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°Cwww.emmanet.org November 2007
  14. 14. 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 markwww.emmanet.org November 2007
  15. 15. 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 supportwww.emmanet.org November 2007
  16. 16. 8-cell embryo in 1.5M ProH Some water out Equilibrium reached ProH in 0 min 1 min 5 minwww.emmanet.org November 2007
  17. 17. Freezing stepswww.emmanet.org November 2007
  18. 18. 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 minuteswww.emmanet.org November 2007
  19. 19. 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 rushwww.emmanet.org November 2007
  20. 20. 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 stockwww.emmanet.org November 2007
  21. 21. 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!www.emmanet.org November 2007
  22. 22. 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 strawswww.emmanet.org November 2007
  23. 23. 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 minuteswww.emmanet.org November 2007
  24. 24. 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 • Work efficiently! • Select ‘ramp 2’ to cool the embryos -30ºC at 0.5ºC/min.www.emmanet.org November 2007
  25. 25. 8-cell embryos cooled to -300C at 0.50C/min.Ice Extra-cellular Most water out solutes very concentratedwww.emmanet.org November 2007
  26. 26. 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 • Work efficiently!www.emmanet.org November 2007
  27. 27. 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 www.emmanet.org November 2007
  28. 28. Storagewww.emmanet.org November 2007
  29. 29. 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 radiationwww.emmanet.org November 2007
  30. 30. 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 testswww.emmanet.org November 2007
  31. 31. 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 forcepswww.emmanet.org November 2007
  32. 32. Store stocks in duplicatewww.emmanet.org November 2007
  33. 33. 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.www.emmanet.org November 2007
  34. 34. Storage in cassettes and boxes • Storage in cassettes and boxes is an alternative • Ideal for use with large bulk storage tanks • Method used by TJLwww.emmanet.org November 2007
  35. 35. Shipment – use dry shippers• Keep samples at LN2 Temp• Re-usable• Considered safe by IATA• Robustwww.emmanet.org November 2007
  36. 36. Thawing strawswww.emmanet.org November 2007
  37. 37. 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)www.emmanet.org November 2007
  38. 38. Getting ready• Prepare work area in advance• Label one empty dish with thestraws identity• Place two drops (~200µl) ofM2 in a second dish• Don’t rush - thaw one straw ata time• Wear safety glasseswww.emmanet.org November 2007
  39. 39. Thawing straws Hold straw in air for 40 sec Plunge straw in water bath at room Temp (20 -25ºC) Wipe straw carefullywww.emmanet.org November 2007
  40. 40. Unsealing the straws  Cut off capillary sealant - don’t flick the straw  Bisect the cotton/PVA plug - don’t flick the strawwww.emmanet.org November 2007
  41. 41. 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 minuteswww.emmanet.org November 2007
  42. 42. 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.www.emmanet.org November 2007
  43. 43. 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 culturewww.emmanet.org November 2007
  44. 44. Embryo freezing dynamics 120 100 % of initial cell volume 80 60 40 CPA Freeze Thaw Diluant Media 20 0 Timewww.emmanet.org November 2007
  45. 45. www.emmanet.org November 2007

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