I am facing problem of cell revival. When I freeze my clone in liquid nitrogen it looks very good. After 10 days when I check in for revival it is not looking good but revival occured. I used media containing 10% DMSO and 90% FBS. Can anyone tell me how to overcome this problem?
There are sever points to be considered before you do freezing of cells
1. Dont overgrow the cells on flask before freezing around 70-80% confluency is preferable.
2. Freezing medium differs cell to cell so refer the data sheet from where you brought cell line. I generally use 70% FBS + 20%DMEM+ 10%DMSO(CELL CULTURE GRADE) for HEK293T cell line.
3. DONT freeze cells directly to liquid nitrogen. Slowly freeze them giving a overnight incubation in freeze tank with isopropanol(available commercially ) at -80C before in liquid nitrogen. Isopropanol will help to rise slowly the freezing temperature at 1oC/min.
4. While thawing back. DO FAST THAWING. Prepare every stuff before removing the vial and then keep a water bath at 37C ready. Now once removed from liquid nitrogen without wasting time thaw them in water bath once ice is gone now dilute them in complete medium (DMEM + FBS) so that your DMSO gets diluted to 0.1% and then centrifuge and pellet the cells and drain the medium and resuspend the cells in fresh complete medium and do less and soft pipetting and plate them onto flask.
There are sever points to be considered before you do freezing of cells
1. Dont overgrow the cells on flask before freezing around 70-80% confluency is preferable.
2. Freezing medium differs cell to cell so refer the data sheet from where you brought cell line. I generally use 70% FBS + 20%DMEM+ 10%DMSO(CELL CULTURE GRADE) for HEK293T cell line.
3. DONT freeze cells directly to liquid nitrogen. Slowly freeze them giving a overnight incubation in freeze tank with isopropanol(available commercially ) at -80C before in liquid nitrogen. Isopropanol will help to rise slowly the freezing temperature at 1oC/min.
4. While thawing back. DO FAST THAWING. Prepare every stuff before removing the vial and then keep a water bath at 37C ready. Now once removed from liquid nitrogen without wasting time thaw them in water bath once ice is gone now dilute them in complete medium (DMEM + FBS) so that your DMSO gets diluted to 0.1% and then centrifuge and pellet the cells and drain the medium and resuspend the cells in fresh complete medium and do less and soft pipetting and plate them onto flask.
Lijo has explained it well. I would like to add that the freezing medium (10% DMSO) should also work. We use similar freezing medium for many cell types. Its more about the procedure and cell type which u use as Lijo mentioned
Thanks for suggestion Lijo and Manoj. My problem is not about medium composition and I am following same procedure as lijo said. I had previously also freezed and revived cell using same composition and protocol before some day I had doubt about contamination and did fumigation and treatment with mycoplamsma spray, major contaminant in mammalian cell culture. row i am able to freeze cell but they were not revived.
Okay if you had problem of contamination previously, Then it would be better if you check for mycoplasm contamination by dapi or pcr method and confirm first your cell are good and then go for freezing. Try if you have some old stock freezed in liquid nitrogen before this contamination problem and try them. Generally when I thaw the cells I do the myco test in passage 1 and freeze them in that passage only rather than waiting for later passage for freezing the cells.
I think that your problems could be your cells for diffrent reasons: the serum is diffrent, the passage is too high ecc...try to thaw another batch of your cells (the earliest passage you have, if it is possible), assure their viability and quality control tests (mycoplasma; time of dupilcation) and then freeze them again slowly (4h to an overnight incubation) at -80C before to put them in liquid nitrogen.
DONT freeze cells directly to liquid nitrogen. First freeze slowly with isopropanol at -80C (it may stay several days) and then freeze i liquid nitrogen.
Freezing and thawing should be fast. Your freezing medium (%90 FBS+%10 DMSO) is fairly ptotective. I agree with friends, Don't freeze cells directly to liquid nitrogen. You can freeze in liquid nitrogen, After several days or weeks with isopropanol at -80C. In addition, Your cells can be sensitive to mechanich effect (santrifuge). After melting of vial in water bath (37C) , Resuspend the cells in fresh medium with %10 FBS and do less and soft pipetting and plate them directly onto flask.After attachment of cells (about 3 or 4 hours) change medium again.
I am doing same procedure for freezing as maria ruiz, sukran and maria.i am incubating my cell as -80 degree for overnight and then transfer it to liquid nitrogen.
I'm using the same procedure: 10% DMSO, incubation at -70 degree even for a week, and then liquid nitrogen.
I agree that fast thawing is very important
Some loss of viability during cryopreservation is unavoidable. But I think the main reason for the observed poor viability at thaw is due to the thaw procedure itself. The presence of serum (which is protective) in thaw medium should be helpful. But even without serum, cells can be thawed with minimum loss. The key is to minimize the exposure to DMSO post thaw. In general I found verious cell lines can tolerate up to 0.5% DMSO, which means you need to quickly dilute the cell suspension by 20 fold (10% DMSO down to 0.5%), then rapidly spin down (at reasonable g force to minimize shear) to get rid of DMSO. Alternatively, if the centrifugation shear to fragile cells proves to be damaging, you can thaw vials directly by adding 19 volumes of thawing medium (1:20 dilution) so that there is a maximum of 0.5% DMSO, then cultivate the cells under the normal conditions. Frequently, I saw that cells grew well even in the first passage. Good luck.
You seem to be doing everything right. Just one question - when you put your cells in the -80 freezer, do you use a freezing container? It is important to freeze the cells gradually. The easiest way is to use a product called "COOLCELL -1C/MIN FREEZER" - very easy to use and very efficient. Otherwise, this may just be an instance when something did not go well during your freezing. Mycoplasma will not affect the cells so suddenly.
i put my cell in -1C container and first i will put it in -20C for 1 hour then overnight in -80C. next day in liquid nitrogen. although my problem was solved when i did fumigation again in last week.
Hello frnds...even i was facing the same problem of cells not reviving properly,but now when i am cryopreserving the cells,i first put my cells in 4 degree ,for half an hour,then shift to -20 degree for overnight,then finally to liquid nitrogen.
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