Freezing and thawing of cells - (Sep/09/2004 )
wht is the best method to freez 300 Million cell from a buffy coat ....like shall i devide it in 3 tubes and freez seperatly ....or in one go?
I would divide them into 3 tubes to reduce the time of thawing. ie. larger volume will require longer time to thaw.
As a complete newbie to the topic, I thought I'd treat you all to a few potentially daft questions. My intended application is the freezing of fragments of umbilical cord tissue (Wharton's Jelly to be precise), followed by thawing and extraction + culturing of mesenchymal stem cells.
My initial plan:
1. Excise the Wharton's Jelly
2. Chop into 2mm cubes/slices under PBS to prevent drying out
3. Add a few lumps to a cryovial with some cryopreservative in it
4. Give it half an hour at 4c to allow the DMSO to penetrate the tissue fragments
5. Stick it in a controlled rate freezer (the standard program on this goes from 4c to -150c over about 40 minutes)
6. Transfer to liquid nitrogen vapour phase.
7. On thawing, use an established protocol (there are plenty about) to get the stem cells out
8. See how many have survived
9. Do some culturing
1. Tissue fragment sizes - is 2mm too big to allow DMSO penetration over 30 mins?
2. How much tissue would you add per 1ml of cryopreservative?
3. My planned cryopreservative will be 10% DMSO, 1% DEXTRAN in a medium which is not yet decided. I've had DPBS suggested to me, as well as DMEM. Does anyone have any thoughts on this? I've seen papers suggesting that the sample should be frozen in the same medium you intend to culture in afterwards. Also, there are copious variants on the basic DMEM recipe (High/low glucose, +/- Glutamine etc etc ) - what's the story here? Whatever medium I use must be serum free and CE marked.
4. Is half an hour at 4c about the right kind of time? I've seen some papers saying room temp, and others suggesting different times
5. I've seen suggestions that you should freeze more slowly than the rate on our controlled rate freezer. This is the rate we use for blood stem cells and it seems fine.
Any help on the above, plus any other hints and tips, would be hugely appreciated.
Hi, I also had a small question regarding freezing, a bit related to Chris' questions I hope. I was freezing my cells today (skin fibroblasts and keratinocytes), which was quite a batch. We have a commonly used protocol here at the lab that works ok. However, because of the amount of cells, some vials were left at RT in their cryomedium for quite some time (~15 min) before freezing. How bad is this for the cells? Would it have been wiser to put the vials in the fridge for the time being?
We use 10 - 20 % serum and 6 % DMSO for freezing.
Thanks for the replies.