I need to freeze isolated islets from human pancreas. The islets grown in CMRL 1066 medium supplemented with added vitamin E, nicotinamide, heparin, albumin, and ciprofloxacin. Some authors use RPMI 1640 with FCS and 10% DMSO, others use Medium 199 with added FCS and 10% DMSO. With my islets growing in CMRL 1066, I would like to know if I can use the same medium for freezing just by adding FCS and DMSO.
I have not worked with islet cells but I have frozen down primary human cells in the past and one thing that affects primary cell viability is the order the solutions are added rather than the media, and the rate of the change in temperature during freezing. I used M199 a lot with primary cells, but I would use the same media that they were grown in rather than switching.
I generally add the freezing media in two steps with solution A that contains Media plus FBS. Solution B contains Media, FBS, and DMSO. Solution A + Solution B should yield 10% DMSO, 20% serum, and 70% Media.
After pelleting the cells at 1000 rpm after trypsinization, I resuspend the cells in 1/2 volume of the final freezing media with just media + 20% serum (Solution A). After the cells are fully resuspended, I add the rest of the freezing media components (DMSO diluted in media + serum) Solution B. I add the DMSO containing media to the cells very slowly as it seems to preserve cell viability to do it that way. Adding the DMSO component of the freezing media slowly allows the DMSO concentration to increase slowly to limit rate of DMSO's effects on the cell membrane. DMSO does have some effect on cell membrane fluidity and integrity so after adding the DMSO solution, cells should also be pipetted slowly into the cryovials to prevent cell lysing, especially if you are using a micropipettor with a fine tip. The final concentrations of serum and DMSO should be 20% and 10% respectively.
The actual freezing part should be done slowly in a controlled rate of about 1C/min. For thawing, it should be done as fast as possible so putting them in a 37C sterile water bath works well. Primary cell resuspension and growth should be done so you add the media slowly to the cells to limit the change in the rate in DMSO concentration. Adding regular media into cells containing 10% DMSO too quickly can kill the cells. Then spin the cells and wash a few times with media to remove the DMSO, and place them in growth media in a flask. Complete removal of DMSO is important for many primary or stem cell lines as it can alter their gene expression or induce differentiation.
After we receive human islets we culture them O/N in Miami Media. Next day we handpick all healthy islets and use them in different experimental protocols. If we need them for expression analysis, we handpick islets with its media (on ice), wash in PBS (x2) and quickly snap-freeze in liquid nitrogen.
Freezing is easy but the main question is what is the purpose of freezing; do you want to recover viable cells after thawing or are the islets frozen for convenience of delayed analysis (such as total contents of proteins or hormones or their mRNA)?
If the latter, snap freezing in liquid nitrogen followed by storage in LN2 or -80C will do nicely. However, since you mentioned something about DMSO, my guess is that you are interested in recovering viable islets. This is going to be quite tricky and variable because islets are clusters of cells that range in size so simple rules of diffusion do not apply. If islet function preservation is the goal, results are quite poor (in our hands as well as those of others) and variable. Hence, the need of freshly prepared islets...
If you develop a consistent and reliable method, I am sure that many labs will be very interested in learning your method. By the way, what works for rodents, doesn't work for human islets so there is also that!
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