Question

Is it necessary for the cell line freezing medium (10% DMSO+ FBS containing medium) to be made fresh when needed?

If yes, why must it be freshly prepared? And how does the commercial product overcome this problem?

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  • Sriram Thoppe Rajendran · K. S. Rangasamy College of Technology
    Usually Freshly prepared is preferred because we can guarantee that its free of contamination, otherwise things will turn bad!
  • Deleted
    Personally, I always prepare 50ml and use it during next several weeks. If you work clean you should not have any problems.
  • Kris Cahyo Mulyatno · Airlangga University
    it's possible to store at -30degree C for next freezing cell!
  • Sven Dittmann · Herz- und Diabeteszentrum Nordrhein-Westfalen
    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.
  • Anis Kurlila · Universitas Islam Negeri Maulana Malik Ibrahim Malang
    Better prepared fresh to avoid contaminants,,
  • Sven Dittmann · Herz- und Diabeteszentrum Nordrhein-Westfalen
    Sorry, in my last posting but should be the word put.
  • Vicente Escamilla-Rivera · Center for Research and Advanced Studies of the National Polytechnic Institute
    I do not think it's necessary, the cryoprotectant activity of this media can last for a few weeks
  • Alasdair Russell · Cancer Research UK
    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
  • Ru-Jeng Teng · Medical College of Wisconsin
    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.
  • Fulvio Celsi · IRCCS Ospedale Infantile Burlo Garofolo
    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...
  • Kenji Okazaki · Kyoto University
    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.
  • Manoharan Parthiban · Tamil Nadu Veterinary and Animal Sciences University
    Freezing medium can be stored and used. So fresh one is not needed
  • Henry Young · Mercer University
    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.
  • Ratneswary Sutharsan · Griffith University
    Cells survive better if you re-suspend them in cold freezing medium.
  • Henry Young · Mercer University
    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.
  • Jose Garcia-Sanz · Spanish National Research Council
    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
  • Daniel Orellana · Fondazione I.R.C.C.S. Istituto Neurologico Carlo Besta
    Really nice advice Jose... thanks.
  • Matthew Lakins · University of Cambridge
    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!
  • Yuriy Fedorov · Longevisys
    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.
  • Chopie Hassan · Leiden University Medical Centre
    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?

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