Cryopreserving earthworms with antifreeze agents at -20°C

[Gaijin]

im doing a science project on cryonics and preserving live tissues and simple live organisms. i am experimenting with finding ways to keep Earthworms alive for extended amounts of time(months even) in sub zero environments(-20°C or lower). it is a common fact that Earthworms can survive for a long time in cool environments for a month or two longer by slowing their metabolism, but I am am trying to see if i can induce a much more deep(colder and longer) hybernation. I am trying various biological antifreeze agents such as Glycol and some organic alcohols. Anyone have any suggestions, or links to relevant sites.

Also information on how live tissues are frozen, would be pertinent too, for that is another goal of the project. Post here with any suggestions or ideas. Thanks

 

[quetzalcoatl9]

DMSO is frequently used to freeze cells for later culturing. Unfortunately, it can be toxic to cells in high enough concentration.

A less toxic method you may want to try is to use glucose, which will lower the freezing point of the cytosol in the cells.

Animals that can survive freezing have novel methods of anti-nucleation and usually physiological means of compartmentalizing the frozen water (such as in a fat layer).

 

[DocToxyn]

As quetzalcoatl9 has suggested, and you may have already done, there are numerous animals species that have adapted to survive extreme cold and even freezing. Some of the coldwater fish species, winter flounder or the notothenoid sp. are quite well studied in this respect. Also our local wood frog, Rana sylvatica, has been studied for its ability to tolerate body freezing.

 

[Gaijin]

k, thanks

what exactly is DMSO

and does anyone know what is most effective as a cyroprotectant, glycol or glyceren, or any other non toxic chemicals. like various sugars, glucose fructose, or alcohols..

has anyone done any studies or experimens here useing these or any other methods of live freezing or know of any links to sites where i could find them.

 

[DocToxyn]

k, thanks

what exactly is DMSO

and does anyone know what is most effective as a cyroprotectant, glycol or glyceren, or any other non toxic chemicals. like various sugars, glucose fructose, or alcohols..

has anyone done any studies or experimens here useing these or any other methods of live freezing or know of any links to sites where i could find them.

DMSO= dimethyl sulfoxide, a common solvent used in biochemistry that is rather well tolerated by living tissues/cells. You can't use it straight-up, significant dilution (nothing more than 10% DMSO and that would be high) into some physiological medium is required.

Check out pubmed and use a search term like "cryopreservation". I did this and got almost 250 hits, not all look like they will relate to your questions, but you'll find things like this - Cryopreservation of a whole ovary as a strategy for restoring ovarian function. That should get you started, if you come across anything confusing, someone here can probably help.

 

[Moonbear]

The method you use is going to depend on what you are trying to freeze/preserve.
When I store cells frozen, I just mix them with 50% sterile glycerol, but to prevent brain tissue from being damaged, we have a more complex cryoprotectant that uses sucrose and ethylene glycol in buffer, but that's not live tissue, just tissue where we don't want freezing artifacts. If you look up literature on sperm cryopreservation (for artificial insemination), you'll see that different formulations are used for every species, and the differences are important for keeping the sperm alive. So, there's no easy answer on this.