I usually find that till the centrifugation step I can see the pellet, but after that even when I'm being really careful, I lose major amount of cells. Is there any way I can save them from getting discarded? (I have freezed cells in 5% DMSO and 95% FBS) , also for how long can I store them before reviving them (mine were freezed a year back).
Hello Prashsti Chadha
There are two schools of thought when it comes to removing the cryoprotectant from the cells during the process of thawing.
1. Generally, the cell suspension is centrifuged and the supernatant containing the cryoprotectant (DMSO) is discarded, and the cell pellet is resuspended in fresh cell growth medium prior to placing the cells in the culture vessel.
2. However, in case of fragile cells, you need not centrifuge after thawing because the centrifugation process may be detrimental to the cells thereby increasing cell death. Therefore, in such a case, it is recommended that the cells be plated without centrifugation and that the growth medium be changed after 24 hours or even earlier once the cells have attached to the substratum to get rid of the cryoprotectant.
I would suggest that you thaw SH-SY5Y cells just before you are done for the day, leave the cells in the incubator for the night and change the medium with fresh medium first thing the next morning.
The freezing medium for SH-SY5Y cells is complete growth medium containing 10% FBS and supplemented with 5% DMSO. You may also use freezing medium containing 95% FBS and 5% DMSO.
Whenever you freeze mammalian cells, you should follow the gradual freezing technique (at the rate of -1°C /min) by using Mr. Frosty freezing container which is a system designed to achieve a rate of cooling very close to -1°C/min, the optimal rate for cell preservation. During the freezing process, after transferring 1 ml resuspend cells in each vial, keep vials at 4°C for 10 minutes, then at -20°C for further 10 minutes. Then move the cells to -80°C overnight and then transfer the vials for final storage in liquid nitrogen the next day.
Also, you should harvest the cells during their maximum growth phase (or log phase) and should have greater than 80% confluency before freezing. Additionally, it is important to consider the optimal concentration for freezing cells which may vary depending on cell type. Typically, the concentration of cells in the cryogenic vial should be within a general range of 1x10^5 - 1x10^6 cells/ml. If these above factors are taken into consideration, you may be able to store the cells in liquid nitrogen for several years.
Best.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line