Hello everyone,
I am working on a project where I receive daily samples to freeze at -80 °C. Specifically they are peripheral blood mononuclear cell (PBMC), until now I was using CryoStor® CS10, but it is very expensive, especially considering the amount of samples.
I have been looking for an alternative medium that I can prepare myself and I have seen this:
- 70 % Dulbecco's Modified Eagle Medium (DMEM)
- 20 % Fetal Bovine Serum (FBS)
- 10 % Dimethyl sulfoxide (DMSO)
The problem is that, from what I have been told, it must be prepared fresh, but I would be very keen to be able to prepare aliquots (even partial ones) in order to simplify the work of the technicians.
So my questions are:
- What is the problem with mixing it all together and freezing it? I guess here that the problem is that each component is normally kept at different temperatures (room temperature for DMSO, 4 degrees for DMEM and minus 20 for FBS). But I really don't understand the chemical reason.
- Is there any way? Maybe by preparing partial aliquots or using another mixture...
Thank you in advance and best regards,
Oscar
Dear Dr. Oscar Crespo-Salvador
1. What is the problem with mixing it all together and freezing it?
Preparing freezing media fresh could be because of two reasons.
1. Preparing aliquots of freezing media for use in future could increase the risk of contamination. When freezing media is prepared fresh one may be confident that it is contamination-free. However, if you are to prepare aliquots of freezing media for future use, ensure that they are kept sterile and for one time use only because such aliquots are more susceptible to contamination.
2. What is the effectiveness of DMSO in performing its function (reducing the water content inside the cell thereby preventing intracellular ice formation), if it is present in the mixture and frozen, compared to when it is prepared fresh? I do not know! Because I always prepare fresh freezing media. You may give it a thought.
2. Is there any way? Maybe by preparing partial aliquots or using another mixture...
For freezing PBMCs, I would prefer to use 90%FBS + 10% DMSO.
Regards,
Malcolm Nobre
In my experience, I've found that using a freezing solution can effectively preserve samples kept at 4 degrees Celsius for up to 7-8 days. This method has consistently yielded positive results, maintaining the integrity of the samples during storage.
I would like to draw your attention to this thread:
https://www.researchgate.net/post/Is_it_necessary_for_the_cell_line_freezing_medium_10_DMSO_FBS_containing_medium_to_be_made_fresh_when_needed
Thank you very much to all three of you for your answers!
Dear Dr. Malcolm Nobre I'm a bit intrigued why you prefer 90% FBS + 10% DMSO, have you experienced better results than with the one I said? Do you freeze it or do you also keep it for a few days at 4 °C as Dr. Dhananjay Kumar says?
The thing is that I would actually prefer to use that formula as it is easier to prepare. And also, because for the PBMC extraction process I already have to use FBS, of which I make 1 mL aliquots that I freeze (when we get the FBS bottle, usually 500 mL, we have to make aliquots anyway, so I take the chance)... so doing it for this would not be much different contamination wise.
Dear Dr. Dhananjay Kumar , then I could prepare the solution (with or without DMEM) once a week, it seems much more practical than every day, thank you. But you have never tried to freeze it? it seems strange to me that it is not normally done, considering how much easier it would make the process.
I have read more about DMSO and I see that it actually freezes at only 19 °C, so if the mixture is kept at 4 °C it is already below its freezing point... I guess at 10% it might hold liquid.
Interesting thread Dr. Sabine Strehl , very related to my topic and quite educational. I see that the mixture could be kept at 4 °C for quite some time then, which is strange to me because as Dr. Malcolm Nobre says, one of the things I would be concerned about is contamination (of the FBS).
I'm also relieved to know about toxicity, because I'm usually in a big hurry to get the cells into the freezer once I resuspend them in the medium.
It even seems to me that Dr Matthew Lakins does precisely what I am proposing, if I understand correctly:
"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)."
Although it is not clear to me whether he means BEFORE using it with the cells.
Kind regards,
Oscar
Repeated freezing and thawing of freezing solutions can damage components like proteins, affecting their functionality. It introduces variability and potential inconsistency in the solution's composition, impacting experimental reliability. Thus, it's generally not recommended to freeze and thaw solutions repeatedly.
My idea here would be to thaw the FBS to make aliquots by mixing it with the DMSO (and maybe PMEM) and then again to use it to freeze the cells - it should be fine, right?
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