cryoprotectans

[huig]

Hi Amy,

I have been thinking about cryopresevatoin of trochophores as well, I thought I was alone. Part of my work is cryopreservation of mamalian cells for longterm storage and reviving them as needed. Did you use a cryoprotectans (DMSO or glycerol)? and at what rate did the cooling take place? It could be that cells ruptured due to the formation of large kristals which occur when cooling occurs to fast.

greetings

[oceanarus]

Originally posted by huig
It could be that cells ruptured due to the formation of large kristals which occur when cooling occurs to fast.

Sounds like you are more of an expert in this topic than I, but I would suspect that your quote above sums up the problem I experienced. The only process I used was to simply dunk the trochophores into liquid nitrogen. It wasn't untill after my attempt at cryopreserving that I looked into the process and found out that it was beyond the capabilities of the equipment available to me. I kept the discussion of cryopreserving trocophores in the article short so I wouldn't end up boring the vast majority of the people that will be reading it.

Good luck with your cryopreserving efforts, and be sure to keep us here on Reef Central informed, there are a few of us that certainly be interested in hearing it.

Amy

[huig]

hi

Well that won't be too soon as I am renovating my house and I've very little time.
My idea was collecting some oysters during breeding season and force them to spawn and freezing the larvae. This eliminates the need for a holding facility and the large amount of algae needed.

My protocol would have been:
1. concentrating the trochophores (centrifuging, seeving or ?)
2. adding glycerol 8% concentration
3. aliqouts of 2-3 ml in vials placed in isopropyl alcohol (this slows down the rate of freezing) overnight in a regular freezer
4. store in a -70°C at least 12 h
5. submerging in liquid nitrogen

for reviving:
1. adding seawater (15°C) to the vials and transferring to 50ml flask.
2. seeving and rinsing with seawater (to remove glycerol)

maybe next summer I'll try and put my hands on it.

greetings

[EdKruzel]

Hello Amy,
I seldom visit this area of RC as I have my hands full with a few forums and the mayhem that occurs.

I just read the article on rearing YWG's and would like to applaud you and your husband on achieving such a note worthy goal.

I am an advocate and fully support tank bred and raised livestock. I truly believe it is our future for this hobby.

Congratulations on your success,

Ed

[oceanarus]

Huig - Sounds like a good protocol. I may give it a try one of these days. Will definately keep it in mind incase I try to raise a fish that is just too small for the smallest rotifers. One question, what would you use to maintain the temperature at -70C?

Ed - Thanks for the kind words.

[huig]

Hi Amy

I'd use a - 70°C freezer luckily there is one at work. If you don't have one just skip this step, it could work. I' ve got a cell culture of a mouse celline going so if I find some spare time (next week) I'll see if it works. I know that moluscan larvae are completly different but the principles of freezing and reviving are the same.

btw. you've done a very nice job raising those gobies!!!!