If yes, why must it be freshly prepared? And how does the commercial product overcome this problem?
Hi Hui-Chun
I think this is another one of those things in science that is shrouded in folklore, handed down from postdoc to PhD student without question. Personally I was always taught to make it fresh each time and bin it after a few days (due to stability of the DMSO), however I have known many people (in past and present labs) that make a batch that is kept in the fridge and used over several months. What's more is that I cannot see any obvious detriment to cell viability between the two methods. Later I looked into the stability of DMSO in different solutions and this is clearly not a factor as (in a neutral pH) DMSO is very stable. Further, I very much doubt it's due to 'avoiding contaminants' as there's no reason to believe that this tube of freezing mix will be more susceptible to contamination than your media bottles.
Just to add another twist, I was also taught (by a postdoc while I was a naive and impressionable PhD student) that DMSO is severely toxic to your cells - which I believed up until recently when I met with R. Ian Freshney who literally wrote the book on cell culture (http://eu.wiley.com/WileyCDA/WileyTitle/productCd-0470528125.html). Contrary to what I (and others in the room) believed, Ian informed us that DMSO (at the 6-10% doses we use) is not toxic to most cells at all, and what's more it's historically been used as an additive to cell cultures to induce differentiation (especially in breast cells). So, although not toxic it's still not good for my mammary cells.
Anyway, I hope you get something interesting from this. And before you ask, yes, I still make my freezing mix up fresh each time ;)
Best of luck
Al
I do it like Krzysztof. Another working idea is to but only the 10 % of fresh DMSO to the cells. I mean, you transfer cells with media into kyrotubes and the add only the 10% DMSO, shake 1-2 times and then freeze.
I do not think it's necessary, the cryoprotectant activity of this media can last for a few weeks
Hi Hui-Chun
I think this is another one of those things in science that is shrouded in folklore, handed down from postdoc to PhD student without question. Personally I was always taught to make it fresh each time and bin it after a few days (due to stability of the DMSO), however I have known many people (in past and present labs) that make a batch that is kept in the fridge and used over several months. What's more is that I cannot see any obvious detriment to cell viability between the two methods. Later I looked into the stability of DMSO in different solutions and this is clearly not a factor as (in a neutral pH) DMSO is very stable. Further, I very much doubt it's due to 'avoiding contaminants' as there's no reason to believe that this tube of freezing mix will be more susceptible to contamination than your media bottles.
Just to add another twist, I was also taught (by a postdoc while I was a naive and impressionable PhD student) that DMSO is severely toxic to your cells - which I believed up until recently when I met with R. Ian Freshney who literally wrote the book on cell culture (http://eu.wiley.com/WileyCDA/WileyTitle/productCd-0470528125.html). Contrary to what I (and others in the room) believed, Ian informed us that DMSO (at the 6-10% doses we use) is not toxic to most cells at all, and what's more it's historically been used as an additive to cell cultures to induce differentiation (especially in breast cells). So, although not toxic it's still not good for my mammary cells.
Anyway, I hope you get something interesting from this. And before you ask, yes, I still make my freezing mix up fresh each time ;)
Best of luck
Al
Our lab deal with primary cell culture and we never really use freshly prepared freezing medium. We prepare 50 ml then aliqoute into 10 ml tubes and keep in -20C for weeks. So far it works well.
Just a thought anyway....DMSO is stable in neutral pH, but it must not be exposed for too long to light...so if you prepare a freezing medium stock, then wrap in aluminum fold and freeze at -20...then it will be quite stable...
Frozen aliquotes of cell freezing solution for one-time uses are very nice.
An Important point will be to use "DMSO from a FRESHLY OPEND VIAL"
for the solution preparation, because, in open bottle, DMSO rapidly absorbs H2O included in humid atmospheres, therefore its purity automatically go down from 100% to 95, 90 > > >, while time goes on.
Freezing medium can be stored and used. So fresh one is not needed
Yes, should be made up fresh each time. When you receive your DMSO from company, if not already aliquoted, aliquot into appropriate volumes for one-time use and freeze with dessicant, -20C not bad, but -80C better. DMSO is very hydroscopic, but also exothermic when mixed with an aqueous solution (freezing medium). Also, you need the highest purity of DMSO you can find for best recovery rates. We used 99.99% pure DMSO before dilution for freezing adult stem cells at 7.5% DMSO v/v with 92.5% freezing medium, with a 95-98% viability recovery rate. Remember, DMSO is a universal solvent. So anything less than almost pure, and you are dealing with unknowns that could harm your cells.
Cells survive better if you re-suspend them in cold freezing medium.
Depends on what cells you are trying to freeze and thaw. Clones of our naive uncommitted adult-derived stem cells, derived from single cells by serial dilution clonogenic analysis actually preferred ambient temperature dilution medium (98% viable) rather than 37C (~93% viable) or 4C (~88% viable). Difference was about 5-10% less viability at the other two temperatures. But then again, our cells may be different than yours. Bottom line, you need to "know your model system", i.e., test all your parameters to determine optimal conditions before your experiments start in ernest.
With respect to toxicity, it is more the exothermic properties of DMSO when added to an aqueous solution that will "bake" your cells, if present in the immediate vacinity of the cells, and hence will kill them. This can be circumvented by waiting at least 2-3 minutes after adding the DMSO to the medium before adding your cells prior to freezing.
Also, we wanted to keep our naive uncommitted stem cells uncommitted and naive. Hence we needed to remove the DMSO ASAP before any induction by the DMSO could occur in our stem cell populations. So, as soon as the freezing medium changed color from yellow (frozen) to salmon (thawed) we added the cell + DMSO + freezing medium suspension to our ambient temperature dilution medium. Inverted a few times to mix and immediately centrifuged the mixture at ambient temperature to separate the stem cells (in the pellet) from the DMSO (in the supernatant). The supernatant was removed and the stem cell pellet resuspended in plating medium for cell counting, dilution, and plating.
One of the first cytotoxic cell lines obtained was frozen in 14%DMSO in PBS.... no sera, no media, it was the best for being able to recover them viable (if yo try to do this with fibroblasts the viability will be 0%, but with lympocytes works very well). I think it is important to make initial aliquots of the DMSO, and then the second important thing is to freeze the cells in small volumes (we usually make the cell freezings in 200 µl instead of the usual 1ml). The main reason is that the bottleneck in the thawing is the time where the cells spend from -40ºC to +4ºC (when ice cristals can form). Decreasing the volume you can thaw the cells much faster and the cells spend less time at the critical temperatures, thus increasing viability
Just to reiterate the most important point here, which Henry Young goes in to detail above, is the quality of the DMSO.
It is imperative that the DMSO is fresh and of high quality, however, once mixed in to the freeze media, it can be frozen at -20 in aliquots (I use 5ml aliquots).
The best DMSO that I have found works really well, is D2650-5X5ML from Sigma, and comes in 5 glass vials of 5ml aliquots. The glass vials are sealed (you have to snap them to open them) which means the DMSO never takes up water no matter how long they stay on the shelf!
If you are going to freeze cells once a month or even less frequently - prepare fresh each time. Keep DMSO frozen -20 or -80 in between for 3 months or so. After that you may use it for compounds but not for freezing.
If you freeze your cells once a week you can make a batch of freezing media for a month , divide into 1 week portions it and keep it at -80. Works fine with primary human fibroblasts and alike.
I have a question about freezing long fibroblast cells, has any one experience in freezing these cells in 20% DMSO and 30% FCS+ FGM2 medium? Are the cells viable after thawing them?
The idea that DMSO will be less effective at driving water out of the cells during freezing, therefore DMSO should be fresh is reasonable, but I must say, I have been using the same bottle of DMSO since 1997 with no problem whatsoever with my cell lines. We always freeze in a final concentration of 10% DMSO/40% FBS/50%media containing cells. We always prepare the DMSO/FBS fresh on ice and freeze either with one of those alcohol freezing containers or with styrofoam containers with equal success.
I have always aliquotted cells in 1 ml aliquots, and when thawing, I don't bother to spin them down to remove the DMSO, but rather simply change the media the next morning. Works quite well for HIT-T15, INS-1, MIN6, beta-HC alpha-TC1-6 and 1-9, Beta-TC6.
Is it important to keep cells on -20c before -80C for long time storage??
@Chopie Hassan - I routinely freeze lung fibroblasts in 10% DMSO, 40% FCS, 50% Full DMEM media (which itself contains 10%FCS). And the cells are perfectly viable once thawed. (not sure about your specific makeup of freeze media, but this works)
@Fahran Haider - No. Cells, once in the freeze media, should be transferred as quickly as possible to the -80C in a suitable freezing container that cools at around -1C/min. Then, once frozen, should be transferred to liquid nitrogen for long term storage. (Something like this Mr Frosty in the link provided..)
http://www.thermoscientific.com/content/tfs/en/product/mr-frosty-freezing-container.html
@ Matthew Lakins
Thank you so much for kind feedback. Can we store cell in freezing media for long time on -20 C only?
@Farhan I must confess I have never even tried to store at -20C so can't answer that one. I was always advised not to!
@Farhan based on my experience, you can store cells in -20 maximum overnight, then viability drops down...Some protocols suggest that you cool cell @+4 for 1h, then trasfer to -20 for 3-4 hours (max) and then -80...In any case, I agree with Mattew..better not to do..
@ye jj, DMSO prevents formation of ice crystals which otherwise lyses the cells during thawing. 7.5-10% DMSO is usually used to feezing cells. You used low concentration of DMSO means cell may not recover well while Thawing, because of ice crystal formation..Rather adding DMSO in cryo tubes, start a new cell line storage.
I am looking for a protocol to freeze PBMC and find this interesting discussion. I have another question. If the 6-10% DMSO is not toxic to most of the cells, and if there is no stability issue, why all the protocol suggest to "add dropwise 20% DMSO/FBS to the equal volume of cell suspension, to make final concentration of 10% DMSO solution"? why can't we resuspend cells in 10% DMSO/FBS directly?
Thank you very much
Cheng-Ming, if you have very sensitive cells, they might get osmotic trouble if you drop them into a medium of different composition. So it can make sense to change the medium slowly. On the other hand, this always happens, when you wash your cells with PBS or another buffer, so it depends on trial and error and comparative experiments (e.g checking for survival rates unter different conditions). I would not be overly concerned unless there is scientific evidence. Life scientists tend to do stuff out of believe and/or unproven assumptions every now and then.
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