I have used 10%DMSO+90%FBS mixture for freezing cancerous human cells for years with no problem. However, since I began to establish my own laboratory, I am having an isssue that seems to be unsolvable.
I use the protocol below. Can you spot a mistake? Thanks.
I aspirate the media off.
Wash the cells with PBS. Aspire it.
Add adequate amount of trypsin (1mL for T25, 2-3 mL for T75 flasks).
Incubate the flasks at 37 for 5 minutes.
Add at least double amount of medium (supplemented with FBS) to stop trypsinization.
Transfer the mixture to an autoclaved glass tube.
Centrifuge it for 5 min at 1000 rpm.
Aspirate the supernatant.
Dissolve the cell pellet in 1mL of freezing medium.
Take them into a cryovial tube on ice and do not let them stay there for more than 5 minutes.
Immdeiately take the cells to -80 (when our -80 was broken I actually moved them to -20, too).
Transfer them to liquid nitrogen next day (sometimes I move the tubes to liquid nitrogen after 4-5 hours).
I filter my freezing media through 0.2 um filter in case of contamination during the process. I pre-chill the cryovial tubes and sometimes freezing medium, as well.
Apparently you're doing everything right. What kind of problem are you having?
I don't see any problem with your protocol. however, I normally add 80% of full media+10% FBS+10% DMSO for freezing media.
Hi, I am intersted in your frozen medium amount.
As you mentioned flask T25,when it's cell at confluency, you may could get about 3 x 10 6 cells (T75 could get about 4 times larger cells numbers than T25) , and 1 ml of freezing medium usually frozen cells between 1 x 106 cells to 1 x 107 cells. so I think if you could make cells feel not so "crowed", they may "happy" during frozen.
Every thing looks okey except -80 and -20 steps. First of all I recomend you to use cryobox like mr frosty or similar one in -80 for 24 hrs then move cryovial to liquit nitrogen. You should place the cryovial in mr frosty then put them in -80. DON'T USE -20
Although, the protocol is almost same as any generalised cell freezing protocol, I have few suggestions:
1. After adding Trypsin, we do not need 5 minutes incubation for all the cell lines. For some cell types, even 1-2 minutes incubation is more than sufficient. It also depends on the Trypsin stock concentrations (we use 0.025% trypsin)
2. After Trypsin incubation, we centrifuge cells for 5 min at 1000 rpm, always at 4 degree celsius.
I think, if you elaborate about the specific issue that you are facing while cell freezing, it would be of great help to all of us.
Thanks!
Everything seem ok but some steps u should taken care
1 steps u r doing for making cell suspension are ok but the freezing media u r using may have some problems. Use 60% serum; 30% media without serum; 10% DMSO. Never filter your media again. Specially when serum is added.
2. place the cryovial In a cryobox and first place it in -20 for around 2-3 hours. Then place it in -80 . Next day transfer it to the liquid nitrogen. Keep the cell number as 1 million cells per vial in 1 ml media.
3. Thawing is also very important when u are checking your freezdown. Follow the steps for thawing.
after removing the vial from liquid nitrogen , quickly thaw the vial in 1-2 minutes After removal. thaw until it becomes liquid . pour the cells in tube containing 10 ml medium . Centrifuge and resuspend the pellet in 5 ml media and pour it into a T 25. Replace the media on second day if suffici numbe of cells have attached to the surface.
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