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Cryopreservation Protocols Contents | ||||||
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Slow freezing procedure for 8-cell mouse embryos
Larry Mobraaten and the Cryopreservation Crew
Contents
I. IntroductionResearch conducted during the past twenty years has led to the development of efficient procedures to cryopreserve mammalian eggs and embryos. Furthermore, this research has also led to rather full understanding of many of the factors that cause damage to embryos when they are cryopreserved. Ova and embryos of fifteen mammalian species have been successfully cryopreserved. Literally, tens of thousands of live young mice, rabbits and cattle have been born from cryopreserved embryos. Live mice have even been born after developing from embryos stored in liquid nitrogen for more than thirteen years. Hundreds, perhaps thousands of children have also now been born after transfer of frozen-thawed embryos. Briefly, fundamental and applied studies of embryo cryopreservation have shown that successful cryopreservation of ova and embryos requires only four steps: (1) Cells are suspended in a concentrated non-physiological solution of a cryoprotectant.
(2) The cells are cooled to very low subzero temperatures in a way that permits them to lose virtually all of their cell water. This water loss may occur just prior to the embryos being cooled, or during the cooling process itself.
(3) For long-term storage in the solid state, the frozen embryos are stored in liquid nitrogen at -196 degrees C. To restore their function, they are warmed to physiological temperatures.
(4) Finally, the cryoprotectant must be removed from the embryos without damaging them either by some inherent toxic effect of the cryoprotectant, or from osmotic shock during its removal.The procedure used for freezing and thawing in the Jackson Laboratory Frozen Embryo Repository is based on that described by Whittingham et al. [Whittingham, D., S. Leibo, et al. (1972). "Survival of mouse embryos frozen to -196?C and -269?C." Science 178: 411-14.]
A. Freezing method 1. Embryos are placed in 0.1 ml of PBS and held on ice until ready for addition of cryoprotectant.
2. An additional 0.1 ml of PBS at 0?C with 2 M dimethyl sulfoxide (DMSO) is gently added to each cryotube to bring the final concentration of DMSO to 1 M.
3. After at least 30 minutes equilibration at 0?C, the cryotubes are transferred to a salt and ice bath made up to maintain a temperature of -6?C.
4. Two minutes after immersion in the ice bath, the contents of the cryotubes are seeded with an ice crystal from the tip of a Pasteur pipette. To prepare the pipets a small amount of PBS is drawn into the tip of a Pasteur pipette by capillary action. The pipette (or pipettes, if more than one seeding is to be done) is then put inside a glass test tube that is partially immersed in a -10?C salt and ice bath. When the embryo culture is ready to be seeded, a pipette with the frozen PBS in the tip is just touched to the surface of the medium containing the embryos. This will bring about the ice nucleation of the medium with minimal damage to the embryos caused by the formation of ice crystals The cryotubes are then capped and transferred to a controlled-rate freezer which has been precooled to -6?C.
5. The temperature in the controlled rate freezer is lowered at the rate of 0.5?C per minute. Temperature changes in the freezing chamber can monitored by the use of a thermocouple inserted in a control cryotube containing 0.2 ml ethanol (95%). The thermocouple is connected to a continuous strip-chart temperature recorder.
6. When the temperature of the freezing chamber has been lowered to -80?C, the cryotubes are removed from the freezing chamber and immediately put into a liquid nitrogen storage container with the location recorded for easy retrieval. This step must be carried out as rapidly as possible so as not to let the cryotubes start warming.
7. The storage location of each tube should be recorded for furture retrieval.A sample of approximately 50 embryos is frozen in each run and afterwards thawed and assayed for viability (as evidenced by cleavage of the embryos) to test the conditions of the freeze run.
B. Thawing method 1. Cryotubes are removed from liquid nitrogen storage and placed on the bench at ambient temperature until thawed, which usually requires from 12 to 15 minutes.
2. When completely thawed, 0.8 ml of PBS is slowly added in a dropwise fashion to dilute the DMSO.
3. The contents of the cryotubes are withdrawn by means of a Selectapette (Clay Adams) and transferred to embryological watchglasses or depression slides where the embryos can be observed.
4. Embryos are washed once in KSOM medium before putting into culture.If a programmed thaw is desired, it can be accomplished by removing the cryotubes from the liquid nitrogen refrigerator and placing them in the freezing chamber of a controlled-rate freezer which has been precooled to -100?C and then warming them at 8?C per minute until they reach +2?C. The controlled warming rate is discontinued and the vials are allowed to warm to about +13?C. The samples are allowed to liquify at ambient temperature for about two minutes before the DMSO is diluted out as described above.
C. Embryo culturing methods
Culture under oil.
Washed oil is used and must be prepared well in advance of embryo culturing. To wash the oil, 500 ml of Aldrich light mineral oil (or equivalent) is placed in a 500 ml Co-Star plastic bottle. 50 ML of MEM plus BSA is added and set up on a magnetic stirrer for vigorous stirring for 48 hours. After that time the oil and medium are allowed to separate and the oil is decanted to a clean 500 ml bottle. A second wash is repeated exactly as above. The final decanted oil is sterilized by filtering through a Nalgene sterilization filter of 0.8?m pore size. The sterilized oil can then be stored in the filter base. Prior to use the oil should be gassed (5% carbon dioxide, 5% oxygen, and 90% nitrogen gas mixture) by using a gas washing apparatus with a disposable pipette and bubbling the gas through the oil for about 10 minutes (the oil may become cloudy in appearance at this point if it was stored cold prior to using). 1. Prior to thawing (can be done the afternoon prior to thawing embryos), 35mm tissue culture dishes (Falcon 3001) are set up with 0.2 to 0.3 ml droplets of media (KSOM) and overlaid with mineral oil. It is important to use fresh media with the pH adjusted. The prepared dishes are placed in a modular incubator chamber and thoroughly gassed with a 5% carbon dioxide, 5% oxygen, and 90% nitrogen gas mixture. The chamber is placed in a 37?C incubator and held until embryos are ready to be introduced into the culture drops.
2. Cryotubes containing the selected embryos are thawed as above. While thawing, the previously prepared culture dishes are placed on a warming tray at 37?C under a plexiglass gassing cover with a continuous gas (same as above) flow.
3. The thawed and washed embryos are introduced into the culture drops. It is important to be sure that the pipette tip is completely inside the culture drop under oil and that injections of bubbles is avoided. Presence of bubbles will make later observation very difficult. When all culture dishes have been loaded they are placed back into the chamber, the chamber is sealed and gassed. The sealing and gassing are critical to maintain proper pH in the KSOM medium.Culture in glass tubes. 1. For an in vitro test of viability, embryos are transferred into 12 x 75 mm tubes containing about 1 ml of KSOM medium that has been gassed for 20 seconds with a gas mixture of 5% carbon dioxide, 5% oxygen, and 90% nitrogen, held at 37?C for about 15 minutes to allow equilibration, and re-gassed just prior to the introduction of embryos. Glass tubes are prepared by rinsing 5 times with distilled water and drying inverted in a 110?F oven for 2 hours. The tubes are rinsed once with 1 ml media before use.
2. After the embryos are introduced, the tubes are gassed one more time and sealed.
3. Embryos are then cultured at 37?C in the test tubes for 48 hours and their viability, as judged by continued development to the blastocyst and expanded blastocysts stages, is ascertained following the 48 hour period.Top
- We no longer use washed oil, but instead buy "embryo tested" oil from Sigma.
- Note that the medium we call "PBS" is actually PBS with BSA added.
- There are many other methods to cryopreserve embryos; this is simply the one we use. Others use different cryoprotectants, different containers (straws, for example), different freezing and thawing protocols, and different equipment. The protocol here works well as described. It should work if straws are substituted for vials, or if a different controlled-rate freezer is used, but if this is done, there very likely will need to be adjustments in timing and or freezing and thawing rates, because of changes in the heat transfer rate.
