I froze the NT2 cells last week, but when I try to defrost them, they don't attach to the flask surface. This problem has happened several times in the past, even trying it in different labs I still have the same problem. I would be really thankful if any one has any helpful suggestion?
Check this out:
http://www.med.uio.no/imb/english/research/groups/chromatin-regulation-stem-cells/documents/isolation-somatic-cell-nuclei.pdf
Dear Ewalds-Kvist
Unfortunately, this protocol didnt work for me, anyway thanks!
Perhaps there are some steps that were hurried during the cryopreservation.
Can you provide more details of the protocol you used so we can see where to make amends if necessary.
Hi, as pointed out it is better to know your protocol to freeze and to thaw your cells. anyway in general, after provide the correct freezing media for your cells (with the correct amount of DMSO), two simple rules are freeze slowly, thaw quickly.
In general I collect my adherence cells, I centrifuge low speed and remove the media, resuspend in freezing medium (and the correct concentration of cells), and put in a freezing cells container. If you don't have one of this I also used to envelop the cryovials in a lot of paper towel and put in a box at -80°C. Wneh you thaw I put the cryovials in a bath at 37°C, if you want to eliminate immediately the freezing medium, you have to add some ml of NT2 cells media (usually use for proliferation), centrifuge at low speed and seed in your flask.
This is my protocol in brief, it is important that
- you have correct freezing medium composition for your cells
- you have correct number of cells being frozen
- you freeze cells that grow wells, without any contamination
- you freeze very slowly (1°C/minute)
- you thaw quickly your cells
- you remove DMSO with centrifugation (in my experience I only diluite the freezing media when I seeded my cells and change the medium the day after)
Do you have this problem only with NT2 cells, or are other cell lines also growing poorly after a freeze thaw cycle?
I believe there may be problem with freezing media and freezing steps. Probably cells are not alive due to this. You should try with fresh cell line now
.
Thanks Everyone for the comments,
The following protocol in my lab is performed for cell lines such as HEK-293T, PC12, MCF7, MDA231,.. and It works well for them, but it doesn't work for NT2 cells
For Freezing
The cells were split by trypsinization when they reached to 85%–90% confluence,
centrifuged @1000rpm for 5min
Mix (450 micro of cells + 450 micro of FBS) and transfer them into cryovial and finally adding 100 micro DMSO
(OR, sometimes we solve the cells in 900 micro of FBS and then adding 100 micro of DMSO)
Wrapping the cryovial with cotton and put it in -80 freezer for sevral days
Put the cryovial in the nitrogen tank
For Defreezing
the freezed cells are melted by rubbing the cryovial in the hand,for a few minutes and then 1 ml DMEM is added to the freezed cells to dilute the DMSO
Then, We either centrifuge the cells or directly transfer them into a new T25 FLASK and changing the medium after 24hrs.
Hi, I used the same protocol with the only difference that I used 5% of DMSO instead of 10%, and I use the 37°C bath (or the incubator) to defrost the cells.
The problems were about some passage between the trypsinization (maybe too long?), and the freezing, if you are sure that the cells were in good proliferative condition, I thought about the DMSO (old or new?), you can control if you do not have problems with the freezer temperature (were they wrapped well in cotton?), or with nitrogen tank (do you control the nitrogen level, problems with other cells in the same box?). You talked about same problem in different labs, can you remember if you use the same freezer, same medium or nitrogen tank? If you find the "common denominator" maybe you can understand the problem.
In the meantime, to perform your experiment, it is better to ask your lab for another cell aliquote, if the problems were something in the freezing process you could have death cells in all the other aliquotes.
Dear Valeria
You mentioned helpful points. However, I don't see any problem with the reagents & chemicals, because we use same reagents for freezing other cell lines. Moreover, the protocol is performed carefully, even in another lab with different reagents,
Actually,your suggestion regarding checking the nitrogen level sounds important, in addition, the proliferative condition of NT2 cells is another good point that demands more consideration. ( By the way, is there any way or approaches to check their proliferative state of those cells?)
I will double-check your points and take them into my consideration to see whether there is any changes or not.
Two comments:
1) Thawing the frozen cells by rubbing the vial in your hand might be too slow. We always agitate the vial in a 37 degree water bath. As soon as the cell suspension has thawed, we dilute it without delay into a fairly large volume (at least 10 ml) of HEPES-buffered tissue culture medium that is supplemented with about 10% of fetal bovine serum. We want to substantially lower the DMSO concentration as quickly as possible and before we expose cells to centrifugation.
2) Freezing: We have tried 3 methods. a) The one you describe. Results were inconsistent and often poor. (Cells would not grow after thawing). b) Programmable cell freezer. Results were consistently good, but the initial investment was high. c) A controlled rate cell freezer (simple mechanical device constructed of aluminum and plastic) that is plugged into the neck of the liquid nitrogen tank (gas phase). Results are surprisingly good and consistent. The device was was moderately priced (I don't recall the exact price) and purchased from the manufacturer of the liquid nitrogen tank.
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research
- Cancer Biology
- Cancer Cells
- Cancer Stem Cells