Some researchers suggest equilibrating cells on ice when adding cryosolution with DMSO, while others on the contrary recommend equilibration at room temperature to facilitate transport of DMSO into cells. What are your cryo experiences with eg. clinical doses of MSCs. Importantly, does anyone know a systematic head-to-head comparison reference to be recommended?
As we usualy do, we work rapidly using a vial-box with cold isopropanol. We use a 10% DMSO complete DMEM as cryopreservation medium. Immediately, we store the cell vials at -80°C for three days and then passed to liquid nitrogen.
I usually put the sample into 0 degree C for 10 min and vaporated in LN gas for a few second , then deep frozen in LN
I think you can find the protocol in Caplan's patent. Go to the USPTO site and search under Caplan or Haynesworth as the inventor.
MSCs are generally regarded as rather easy cells to cryopreserve and I would agree based upon my own experience with the earlier answers that adding the DMSO on ice is fine. DMSO permeates cells in seconds so 10 minutes of equilibration should be more than enough. That said I would suggest that you compare protocols from the literature in your own lab to be sure that an unidentified variable is not impacting outcome. Simple things such as the solution into which you put the DMSO as well as cooling rate, whether or not you use nucleation to initiate freezing and storage temperatures can impact cell yield and viability.
We did some experiments two years ago which showed that human MSCs in DMEM + 50% FCS and 10% DMSO had higher viability without equilibration, i.e. our standard 1oC/min temperature decreaase at -80oC and transfer to -150oC freezer . I know other people have a different experience.
Dear Jivko, very interesting observation, did you publish these findings somewhere? Can you maybe upload something???
Hi Guido - it was a MSc student work and we were looking for cryopreservation methods which work for us. Since equilibration didn't bring the expected benefits, we didn't follow. The observation wasn't published unless we count the thesis and this forum now.
Not at the moment as inside are some data we would like to publish. But send me a message with a specific question if you wish, I will be happy to answer.
I resuspend human MSCs in RPMI1640 + 20% FCS first and add 10% DMSO (volume) while shaking the tube in a speed of one drop per second. This is a standard procedure I use in the lab, and produces high viability (>95%) without equilibrating cells. The trick is to reduce the cryo temperature at standard 1 degree C/min in a -80 degree C freezer for 2 hours and transfer immediately to liquid nitrogen. For recovering, the cells are thawed at 37 degree C within 5 minutes after cells are removed from the liquid nitrogen, and replace the storing buffer with fresh culture medium. Place the recovered cells to a 37 degree incubator with 5% CO2 right away.
I note a trend in the answers to using FCS. I always advocate removal of undefined additives such as human or animal sera. In clinical products we worry about transmission of unidentified disease and in research the influence of undefined components on your experimental outcomes. Removal of sera may result in the need for cryoprotectant formulation modification, so try several DMSO concentrations plus hydroxyethyl starch.
Dear Kelvin, good suggestion to remove undefined human or animal sera. Do you think clinical grade human serum albumin would also do as a more defined substitute?
I think clinical grade serum albumin is much better than sera for cell cryopreservation and culture, particularly if your objective is quantification of stem cells by quantitation of colony forming units or clinical use. I still worry about the presence of other contaminating factors that may influence "biology" if the MSCs are subsequently being used to study effects of exogenous reagents.
In heart valve cell and tissue cryopreservation we have found that we could remove FCS. I have attached our most recent manuscript on this topic.
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