help with cryopreservation - (Feb/12/2002 )
I would just try the tried-and-tested method (maybe vary your DMSO / Serum mix) and do some trypan blue on resuscitating the cells?
if i cultivate my primary cultured cells under serum free growth media, is it still the similar cryopreservation protocols will be used? or the 10% DMSO and 90% serum can not be used for the purpose if i intend goin to cryopreserve my cells ?
at this point you can store you cells in Serum free media + 10%DMSO
I general freezing cells using styrfoam boxes work fine however, results gets better if you do not keep your cells at 4C rather start from room temperature, then cover the styrofoam with another and place a rubber band, place it now in -20 C for 1-2 hour and then over night in -80c and then too liquid nitrogen. Higher serum content in freezing medium helps. You can also add old medium in which cells were growing that way when you will thaw they will have the natural growth factors available.
Interesting suggestion, Sia. I've never heard of using old, used medium to improve after-thawing culture. Has anybody else tried it?
I find very interesting all the contributions. About this "old" medium, I bet it would help. I have used it for helping cells grow in very low density conditions, after transfection, and at least it is sure that it wont cause any harm if you give it a try. It is just media used during 24-48 hours (depends on cell density) by actively growing cells, it should not be yellowish and you can use it during maximum of 2 weeks, fresh is of course better.
I hope this does not offend anyone, it might be too obvious, but maybe the problem is in the way you thaw your cells, instead of how you freeze em. A fast thawing with fast removement of DMSO is very important, as I have experienced.
Yes. We use old medium for freezing not for thawing. This is the composition for preparing the "conditioned freezing medium":
50% fresh medium + 42.5% conditioned medium i.e. old medium from cultured cells + 7.5% DMSO.
I think this is also used for preparing the PCB / MCB
I am confused about the freezing of my cells. I'm currently freezing human lymphocytes and also cell lines (MCF-7, MDA, PC3). I place the cells in Mr. Frosty ( -80 ) overnight and then place them in liquid nitrogen.
For the lymphocytes, I have been instructed to use freezing media (90% FCS + 10% DMSO ) whereas for the cell lines I'm using 10% FCS growth media + 7.5% DMSO.
But unfortunately, my human lymphocytes are dying even after 1 week.
I would be very happy if someone could please help me out.
I don't know about lymphocytes but I freeze my MCF-7 and MD-MBA-231 cells in 100 microliters of unsupplimented medium, 1.8ml FCS and 100 microliters of DMSO. We have pretty good viability for the breast cancer cells, but other people in the lab have been complaining of problems with LNCuP cells which show very poor viability when they are frozen down.
I use 5% DMSO and 20% serum to freeze down. Wrap the cryovials in some blue roll and freeze in -80 overnight. Transfer to liquid nitrogen and they come out a treat when you recover them (works well with macrophages)