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Tips on freezing and thawing of cells




I am facing problems in thawing of cells. The procedure which we follow is- we take stock from liq. N2 then though it in water bath at 37 degree. Then we take medium in falcon (5ml) then add thawed cells dropwise into it. We then centrifuge it at 1500rpm for 5 mins.
but the problem is that after thawing i am unable to get viable cells.
Almost all cells are dead. So what all modification shall i make ? Its really hard to revive cell line from frozen stock?


The general rules for freezing and thawing cells is: freeze cells slowly but thaw cells quickly. It seems to me that you have not frozen your cells in a gradual manner. There are many ways for achieving gradual decrease in temperature. Check the section for cell preservation on this site and you will find protocols of different flavors for this purpose.
http://www.protocol-...tion/index.html

One thing you could possibly try is not to spin your cells straight away, just add the warm medium, and let them sit over a few hours to overnight, then wash out DMSOcontaining medium, and feed with normal medium plus serum (increase serum conc to 10 or even 20% for a little while). Sometimes brittle cells get sheared while centrifuging, so that's why the modified protocol. DMSO is toxic though, so change medium as soon as you see under the microscope that your cells have settled.
If that's the problem you could instead decrease your spin time- even 1min at 1500 should pellet enough cells.
Also, I add medium to my cells, no vice versa, I'm not sure if that would make any difference at all.
As for freezing cells- I do it your way, it seems fine, but your cells could be very sensitive, so could be the problem.

Though my experience of handling the cells is less than 2years. I would like to suggest you some things. Even though if you leave DMSO in the media after thawing the cells the dmso in low cocentration is not toxic 2 cells more over once you dilute the small volume of cell suspension in 5-10 ml of media, it further dilutes the dmso. It looks to me like the protocol you are following may be causing the cells to die, I think you recognise the fact that centrifuging at high speed can lead to cell necrosis. One method is to lower the speed of centrifuge maybe 900-1000 rpm for 4-5 min should be adiquate.
Alternatively you should also consider the method you are using for preserving the cells. If you freeze the cells directly in liquid nitrogen this can also lead to cold shock induced cell lysis or damage the membrane, one way of preventing this is gradually lowering the temperature such as storing 4 degrees for few hours then transfering to -20 degree for overnight and then storing in -70 degrees for an couple of days then transfering to liquid nitrogen tanks, by following this method u r not only making the cells to adjust to the lower temp you are also ensuring that they donot undergo cold induced necrosis leading to cell membrane damage and lysis.


This is how to freeze and thaw cells:

Always check the cell viability before freezing. They should be highly viable: about 95%. keep your freezing media ( 10% DMSO in FBS) cold. Centrifuge cells for 1000 RMP for 5 minutes. Prepare labeled cryogenic vials. Cell concentration should be about for e.g. collected from a 50 ml flask, about 50 million cells. You can have 10 vials and about 0.5 milion cells per vial. After you centrifuge, get rid of media completely, gently tap the pellet to make it loose. Add 5 mls of cold freezing media resuspend with a pipet and transfer 0.5ml to each vial. Close the cap tight and place cryogenic tubes in special cryogenic container that has alcohol at the bottom and cool down gradually, they usually hold up to 20 vials. Close the top and transfer the container to -70 freezer. Wait 24 hours and no longer than 15 days before transferring them out of the container into the liquid nitrogen boxes.

To thaw the cells, be very quick, take the viall out still with some liquid nitrogen, walk to the water bath. Take the vial, make sure the cap is very tight, sometimes it becomes loose. thaw the vial holding the opening upward so the water from the water bath does not contaminate cells or if there is some detegent in the water it doesn't become in contact with the inside of the vial . When frozen cell media is almost half thawed (about few minutes), take it under the hood and add 0.5 warm media to the cells and immediately transfer to a flask with about 10 to 15ml warm media in it. DMSO will be diluted and you can check the viability.

1 Comments

Dear,
I faced very serious problem here with colorimetric caspase-3 kit. I followed the pamphlet recommendations and made some modifications as following:
1. Weighed 10-50mg liver tissue (not cell culture tissue).
2. Added 250μL cell lysis buffer for each sample.
3. Homogenization manually by glass Dounce homogenizer. (Do not make ice incubation for 10 minutes, I am not sure if it necessary by my modification).
4. Centrifugation.
5. Measured protein by Biuret Assay. The protein results were ranged from 2g/dL to 12g/dL.
6. Diluted protein by cell lysis buffer to range 50-200μg protein/50μL cell lysis buffer in wells plate.
7. Added 20μL DTT to 2mL 2x reaction buffer vial before use.
8. Added 5μL 4mM pNA-DEVD substrate.
9. Waited to 2 hours for color development in dark.

My problem is the kit not working, no color at all. I made 4 trials to ensure that. I followed the GENERAL TROUBLESHOOTING GUIDE FOR CASPASE COLORIMETRIC KITS from kit pamphlet. I checked my cellular extract under microscope as troubleshooting guide said. I found precipitated content had slightly intact cellular content, but the supernatant was slightly clear and no intact cells.

Please send me as soon as possible; your illustrations are very essential and crucial for my research work. I still am waiting to solve this, whatever it takes. If you had answer for my request send me immediately. Notice I received my kit since 1 month, I do not think in expiry.

Sincerely;
Amr G. Elsayed
Applied Medical Chemistry Department
Medical Research Institute
Alexandria University
Egypt
E-mail: amrbiochemistry@gmail.com
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