cryopreservation of lymphocytes - (Oct/21/2007 )
Dear all,
A couple of months ago we started collection of blood and separation of different populations of lymphocytes. We instantly freeze the fractions of cells according to a protocol, but recently I discovered that I am not able to get viable lymphocytes after thawing. I've looked through and tested many of the suggestions posted here, but without any success and feel frustrated. I post my freeze-thaw protocol in case anyone can see what might be a solution to the problem. According to litterature it should not be that hard to cryopreserve lymphocytes, it is done all the time.
Freezing:
1. Spinn down the population and resuspend in 500ul x-vivo.
2. Add dropvise and slowly 500ul of the freezing solution (80% FCS and 20% DMSO) on ice.
3. Transfere 1ml to each pree-cooled cryotube on ice and have on ice for 10 min.
4. Transfere to -80 for at least 1 hour and then transfere to liquid N2.
Thawing:
1.Thaw the cryotube in a water bath (37 degrees) until almost melted (but have a ice core in the tube).
2. Transfere cooled x-vivo to the tube before transfering everything to a 50ml tube with x-vivo.
3. Spinn down and carefully resuspend the cells in x-vivo.
the lymphocytes are then stimulated with CD3/28 beads and il-2. But without any success! I have tried thawing the cells in warm medium and cooled medium, stimulated the cells immediatly or the next day to see if this double stress could be the reason. I have also tried freezing cells in a more controlled way with isopropanol box (mr frozty) but without any luck. I know that the cells are viable after separations because culturing the cells without cryopreservation is not a problem at all.
I will be really happy for any feedback because this has been driving me crazy for the last couple of weeks!! 
I see that the normal media in a freezing solution is RPMI 1640, could it be something in the x-vivo 15 that kills my cells during the cryopreservation?
Best regards,
Krizzy
In the Freezing:
1. Spinn down the population and resuspend in 500ul x-vivo….instead of x-vivo…it should be FCS.
In step:
3. Transfere 1ml to each pree-cooled cryotube on ice and have on ice for 10 min.
……after adding the Freezing media it should be transferred immediately to –80 C
in a box of isopropanol….or alcohol .not to be incubated in ice.
in Thawing:
2. Transfere cooled x-vivo to the tube before transfering everything to a 50ml tube with x-vivo.
what is the volume of x-vivo used?…I use 10 ml ( to dilute the DMSO to 0.1%).in 15 ml tube .
I stimulate the same day ..of thowing.
the lymphocytes are then stimulated with CD3/28 beads……….what is the count of the lymphocytes….?? Wht is the size of the well???
We had cultured 0.3x 106 in 48 well…but it took long time….but all sudden it increased in number ...
The il2…..i add it the next day or the day after……
I did not understand this…… ( know that the cells are viable after separations because culturing the cells without cryopreservation is not a problem at all.)
At the end could u please let me know how u do u separation of different populations of lymphocytes?????
Haven't heard of x-vivo before but I guess it's your standard culture medium. The following protocols are for B- and T-cell lines but others in the lab use primary T-cells with the same protocol.We've usually used RPMI 1640 plus L-glutamine, pen/strep and 10%FCS to make complete RPMI. Our freezing medium was 10% DMSO, 50%FCS, 40% complete RPMI and we'd resuspend in 1ml of this prior to freezing immediately in a Mr Frosty isopropanol container at -80C at least overnight and then transferred to liquid nitrogen. This should slowly freeze the cells 1 degree C per minute I think.
So I'd agree with Muna on the freezing conditions using an isopropanol container although your freezing medium is pretty much the same in terms of final concentration (500µl xVivo and 500µl 80% FCS/20%DMSO)
To thaw we'd pre-warm our complete RPMI to 37degreesC then quickly thaw our cells again at 37 until there is no ice. Add to a universal and add dropwise 10-20ml warmed RPMI complete quickly to dilute out the DMSO. We then centrifuge the cells, resuspend in fresh RPMI and culture at 37C, 5%CO2. I have no idea what x-vivo is so you probably need t check the recipe. Some groups use 90% FCS 10% DMSO to freeze cells but it's probably more expensive that way.
I was always told to freeze slowly and thaw quickly and not to leave cells in DMSO for any length of time. So of your cells will die so maybe freezing down more cells per vial could help you. Also if you're immediately stimulating you cells directly after thawing it might be an idea to allow them to recover from the thawing process in culture for 24-48 hrs before stimulating them with anything.
Best wishes,
Ceri
muna,
volume of x-vivo used in the thawing is 15ml.
the count of lymphocytes vary between 1-2 million and we normally use a 24 well plate and 2ml media.
Sometimes we culture a fraction of the isolated lymphocytes (CD4, CD8 and CD4CD25) in addition to freezing down the rest, and therefor I know that the separation it self doesn't kill my cells. We use the autoMACS Pro separator from Miltenyi for the isolation. We start with separation of one population and eiter use the negative or the positive fraction for further isolation of the next (depending on whether it is a negative or positive isolation).
I don't understand why the lymphocytes die after cryopreservation, it really drives me crazy. I have to get viable cells for different projects in the future. I am currently testing out the freezing solution but it sounds strange that something in the x-vivo kills the cells in the freezing process.
Krizzy
You can also try to freeze the cells in 10% DMSO 90% FBS. Then there is no questionable effect of the medium. We do this routinely in our lab (not with lymphocytes though)
Mr Frosty rulez 
Quick thawing is also important. I usually thaw the cells in a 37 C waterbath, then wash the cryotube with ethanol, and finally (when there is still a little ice left) pour them in plenty of medium. Some people say it is good to dilute it slowly, but i never had a problem with this either (>90% viability, not lymphocytes). Then I plate them out, and change medium the next day or after they attached.
[quote name='Ceri' date='Oct 22 2007, 02:29 PM' post='114827']
Haven't heard of x-vivo before but I guess it's your standard culture medium. The following protocols are for B- and T-cell lines but others in the lab use primary T-cells with the same protocol.We've usually used RPMI 1640 plus L-glutamine, pen/strep and 10%FCS to make complete RPMI. Our freezing medium was 10% DMSO, 50%FCS, 40% complete RPMI and we'd resuspend in 1ml of this prior to freezing immediately in a Mr Frosty isopropanol container at -80C at least overnight and then transferred to liquid nitrogen. This should slowly freeze the cells 1 degree C per minute I think.
So I'd agree with Muna on the freezing conditions using an isopropanol container although your freezing medium is pretty much the same in terms of final concentration (500µl xVivo and 500µl 80% FCS/20%DMSO)
To thaw we'd pre-warm our complete RPMI to 37degreesC then quickly thaw our cells again at 37 until there is no ice. Add to a universal and add dropwise 10-20ml warmed RPMI complete quickly to dilute out the DMSO. We then centrifuge the cells, resuspend in fresh RPMI and culture at 37C, 5%CO2. I have no idea what x-vivo is so you probably need t check the recipe. Some groups use 90% FCS 10% DMSO to freeze cells but it's probably more expensive that way.
I was always told to freeze slowly and thaw quickly and not to leave cells in DMSO for any length of time. So of your cells will die so maybe freezing down more cells per vial could help you. Also if you're immediately stimulating you cells directly after thawing it might be an idea to allow them to recover from the thawing process in culture for 24-48 hrs before stimulating them with anything.
Best wishes,
Ceri
[Do you normally heat inactivate FBS before freezing. In if so, what is the purpose of doing so?
We will try to follow your instructions today and simply put the cryovials directly in -80 degrees for 24 hours and the to liquid N2.
Do you think it is necessary to add the freezing solution in a slow manner, or do you simply add it to the cells in one go? It is hard to predict what can be the reason for the poor cell viability, but sometimes the sum of all small things can help.
What is really strange is that I am able to get some fractions from some patients to proliferate, but that is usually not the case. And by doing the same thing with all the lymphocytes from all the samples, using all the same reagents, this doesn't make any sense.
Thanks for your help, I really appreciate it! 
Krizzy/quote]
the following is my protocol for freezing keritinocytes (rho zero's mainly - v fragile cells)
there is no media in the freezing mixture and when i replate i thaw fast (in my hand - waterbaths are dirty) use about 20ml media (instead of 12ml) to cancel out the dmso and change the media the next day.
btw this whole carefull dripping thing - maybe its specific to your type of cells but i'd never bother - plonk the freezing soln onto the pellet of cells and give it a shake (or suck up and down the pipette - nothing with too small a hole of course)
untill their first split they may be a bit off - small flasks can help slow growers (proximity of cells)
-Freezing cells (one flask = one ampoule)
1 aspirate old media from cell culture flask
2 wash with PBS (aspirate)
3 add 2ml trypsin, leave in incubator for 5-10 min
4 knocking of flask may aid removal of cells
5 neutralise trypsin with 5ml DMEM (+FCS,P/S)
6 transfer to universal
7 use another 3ml DMEM (+FCS,P/S) to wash out remaining cells
8 centrifuge for five min at 1000 rpm
9 aspirate, add 1ml FCS containing 10% DMSO
10 transfer to sterile cryoampoule
11 freeze (upright) in -80 overnight
12 transfer to liquid nitrogen
Dear Ceri
you are right …..with freezing down more cells per vial could help .
krizzy
I use 10% DMSO 90% FBS.
I think the freezing process…like everything should be cold to freezing media the vial
But the cells should not be kept in ice for long tome ….
Another thing…when did you freez it ..i mean after how many days from last stimulation..??
Dear Muna, Dominic, Kupac and Ceri!
Finally we have managed to get viable lymphocytes after freezing in liq N2, just by following your instructions on the slow freezing in isopropanol box and using your tips on freezing solution.
Thanks a lot for feedback, I really appreciate it!!
Krizzy
oh... ..good it worked