Hi,
I have some problem with the viability of my primary epithelial cells after thawing. I have used two protocols (centrifuging or only diluting 10 times more and changing the media the day after). Regardless I only have 30% of my cells alive. I was wondering if the procedure of diluting DMSO might be my problem. My question is that: When I want to thaw the cells, should I dilute the DMSO slowly in pre-warmed media or cold media. The same question when I want to re suspend them and put them in the incubator. I put too much time to isolate these epithelial cells from the gingival tissue and any help in rescuing them after freezing is much more appreciated.
My freezing media is 10%DMSO+80%FBS+10%DMEM
Thanks
Go back to freezing process. When you mix your cells with medium containing 10%DMSO, do not wait at room temperature, within 5 minutes get cell-cryotubes to cooling process. You do not want cells staying in DMSO at RT for long time.
Thawing: Take frozen tubes out, place at 37C waterbath, keep on shaking tube so that you watch melting. The moment the contents are fully melt or almost melt, Wipe the exterior surface of tube with ethanol to avoid contamination from water bath. Throw all contents of tube in large cell culture dish with medium. And leave it for culture. DMSO in large culture medium will not hurt the cells. Spinning thawed cells can hurt cells more. This procedure has worked for me for all cells I used.
If you have any question, let me know.
I agree with Subhash in the freezing and thawing procedures, and I believe your problem was probably at the cryopreservation not at the thawing procedure. The only things I would add are two things. After mixing your cryopreservation solution it will heat due to the dmso dilution. Prepare it a few minutes before so it reaches at least room temperature or 4-8º C is even better. And the other thing is the cooling rate of your samples. If you use a controlled freezer or a Mr frosty you should´t have any problems. If you are doing a manual freezing please comment how you do it, the problem could be there.
Hi, the freezing of the cell should be slow, while the thawing fast. I agree with the fact that for some cells the centrifugation step di worse than the dilution of DMSO and changing media the day after.
For the freezing procedure you have to pay attention to the number of your cells (at least 1 million, less it is worse), you can also try to reduce to 5% the DMSO for some primary cells is better than 10%. I use always the freezing media at RT, put in freezing container (it is better have one with primay cells and rigorously at RT when you put the cells in!!!!) and at -80°C. You can wait a day (or less) and then immediately in liquid nitrogen.
Thanks everyone for the comments. For the thawing procedure, as you also suggested, I tried both protocol but non gave me a good yield. But for the freezing procedure I might messed up with pre-warming my freezing media. Since I have aliquot of the freezing media in the freezer, I warmed it up before re suspending my cells. Do you think I have to make fresh one each time? Next time I will re suspend my cells in a cold freezing media to see if I get better results and also will thaw them in a cold media too.
I believe it is not a problem to store freezing medium for short times (at 4 to 8 ºC), probably not for months but weeks is ok (I always prepare it just before freezing but I know of other people that prepare freezing medium and maintain it at cold). You don´t need to prepare every time if you freeze often. If you prewarm your freezing medium that might be the problem, short times at room temperature are not toxic, but at higher temperatures it is toxic to your cells.
Hi, the principle of freezing, when you using the freezing container, is to freeze the cell -1°C /1min, so I start with freezing media and container at RT temperature.
I don't recommend you to freeze and, in particular, thaw with cold media.
I find some discussion on researchgate and on web about people that have problem with the yield of different type of epithelial cells, and epithelial cells from different tissue had different response. Maybe these cells are particular sensitive
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