32 replies to this topic

[anilkumarpr]

I dont agree with preethi or anil as  one cannot preserve the cells in glycerol for more than 6-8 months.

Hi all,

When I went through top to bottom of this page, it remind me something I could tell in general

As the wise words (Auguste Rodin) say
"Nothing is a waste of time if you use the experience wisely"

We have -85 Deep Freezer for cell storage in our lab. I got normal revival of cells even after an year, when I preserved them in glycerol. Those were cell lines, not primary cells. The case may be different with primary cells. Yet to get experience with it.

Anil

[nexuson]

The protocol described by soundi is the same as i use and it works well.
Highly suggested...

[HEK-293A]

Hi,

I work with cells not more than half year, but I did met with the similar problem with you. And it's strange that I did all the operation right: thaw quickly, no shaking, and no bubbles when adding medium. But still, I can't get the cell alive, just they don't settle down or very very slow. Plus, I never spin my cell, because I always dilute them into T-75 flask at which concentration DMSO will not be toxic. So the only changes I made I guess is to make sure medium really warm, not just warm them up in 37 degree for 15mins, but 30mins to make sure.

[syty_2000]

Hi, there
I think the cell line is more tough. I do the similar way to freeze and thaw cells as you suggest above. It works fine. But when I come to fresh spleen cells, I have only 10 percent cells viable after freeze and thaw. My question is that if I am not going to culture the cells after I thaw them, instead I want to do FACS staining and cell sorting, should I dilute them in cold media or warm media after I thaw them in the water bath? I always leave cells in 37 degree water bath for just one min, but they are still crystal. I don't know whether I can leave them in the water bath for a little bit longer. Thank you!

[soudi]

Hi, there
    I think the cell line is more tough. I do the similar way to freeze and thaw cells as you suggest above. It works fine. But when I come to fresh spleen cells, I have only 10 percent cells viable after freeze and thaw. My question is that if I am not going to culture the cells after I thaw them, instead I want to do FACS staining and cell sorting, should I dilute them in cold media or warm media after I thaw them in the water bath? I always leave cells in 37 degree water bath for just one min, but they are still crystal. I don't know whether I can leave them in the water bath for a little bit longer. Thank you!

Hi,

Spleen cells could survive freeze and thawing as well. I would freeze the same number of cells in two separate containers and thaw them in warm or room temperature media to see the difference atfer 24 hours in 37C0 incubator. I usually take the tube out of the liquid nitrogen then warm it up in the water bath for may be one minute and while still is half frozen i add 37degree media to dilute the DMSO. If spleen cells have been treated with amonium chloride for lysing red blood cells they will become very sensitive to freez/thawing procedure.

Good luck,
Soudi

[Sg_ACC]

Hi all,

I had read all the suggestions given in the page, thought maybe someone can give me some suggestions to my protocol as well.

I am relatively new in ACC field. Recently i just got 3 vials of Pig bone marrow cells (primary cells). As far as i know, those vials were frozen for about... 5 months in - 80'C. Will that affect the cells viability since they are stored in -80'C for so long and also they were taken in & out of the -80'C occasionally?

When i thawed the cells which i was given and observed them, they were like..... DEAD! omg... i'm not sure whether is the protocol or if is just ME.

i thawed the cells according to the protocol:
take vials out from -80'C
thaw quickly in 37'C water bath
transfer content to 15ml centrifuge tube
add 10ml complete media dropwise to content
spin @1500rpm, 5mins
discard media (with DMSO as cryopreservative)
resuspend pellet with 10ml media
cell count & viability check
seed suspension to T25 and incubate.

When i did a viability check and cell count... i have got something like 45% viability for my cells, but after the incubation overnight... they did not attach to flask and were still in suspension. Its my first time dealing with primary cells and i'm not too sure why they always die after the incubation? Never had such problems with cell lines.

HELP!



Sg_ACC

[humab]

while i think it might be difficult to agree on a procedure that will work best for most, please make sure NOT to

take the viall out still with some liquid nitrogen, walk to the water bath.

!!!

most cryovials can - under certain circumstances - let some of the liquid nitrogen leak into them. any cryovial coming from liquid nitrogen storage is therefore to be considered dangerous and explosive for the first few minutes. take it out of the tank using usual safety precautions, place it into a special container, and let it sit for a few minutes. THEN take it to the water bath.

This is not hearsay, it has happened to me more then once!

take care.m

[Sg_ACC]

yup i agree.... it happened to my frd, the vial exploded and injured her face.

[Gerd]

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

For bacteria it's important to freeze quickly.

That's because when freezig slowly, ice cristals may form.

Strange that it's opposite for other cells.

Can someone explain why?

[fred_33]

hi
for general and detailed informations on using cryopreservatives, badcell gave a very good link :
fundamentals of cryobiology

[Radish]

Hi Shilpi

I don't know if this helps at all, usually do it this way:

-freezing medium: DMEM, DMSO,FBS (it must be ice cold when you added to the cells-DMSO is detrimental to cells at room temperature)

after collecting the cells i do snap freeze in dry ice, I then move to -80 where I keep them overnight, and I move them to LN2 the morning after.

-thawing:

warm medium, thaw frozen vial quickly, use a 1 to 10 ratio of cell volume:growth medium to dilute de freezing medium as much as possible, then spin down 5' 1000rpm and plate.

I never had any problem, but it must depend on the cell type you are using.

good luck

[ecgian]

Hello,
I was just reading through this message, and I wanted to add another question to the group. Once cells are frozen down and stored in liquid N2, how long can the frozen stocks be stored there and still be good when thawed?

Thank you.

hi
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?


Thanks
Shipley

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.

Good luck and let us know if it had worked.

[ecgian]

This stream is very helpful. I would like to add a few questions about freezing cells to the group:

1) How important is it for the freezing medium (DMEM complete + 10% DMSO) to be ice-cold v. RT?
2) While I'm aliquotting the cell mixtures into the cryovials, the cells are sitting in the freezing medium at RT (either in the aliquots or in the centrifuge tubes). Is this terrible for the cells? It probably is ~10 minutes??
3) Is it terrible if the cryovials aren't chilled?

Thank you. I hope I am not destroying my cells and the cell stocks.

[squallweathered]

I was just reading through this message, and I wanted to add another question to the group. Once cells are frozen down and stored in liquid N2, how long can the frozen stocks be stored there and still be good when thawed?

The official party line is that frozen correctly and the LN2 is kept stocked, then you can pretty much store them indefinatly. There are some cells that might only last a few years, and if frozen with anything other than DMSO the LN2 life may be reduced as well. But if using standard proceadues then its not unheard of to resusitate cells 15 years after freezing - we did that a few times and the cells were near 100 viable when cultured.

As a general comment, I've found that when I've been freezing and resusitating tough cells like fibroblasts, there is very little that can go wrong. A few extra g on the centrifuge, a little shorter/longer at cold temps, none of these really made a difference. However, when doing the same for B-cells and similar, the forgivness was far less. Nothing but following a proceadure exactly made the cells survive. All the main points have been covered already, but in general, use chilled freezing media (DMSO is less toxic then), use an alcohol based freezing box in the -80deg overnight and then transfer to LN2 (chilling the cells at about 1deg per minute until -80), and when resusitating move fast and efficiently.

Rather than bringing a potentially exploding vial in LN2 to the cell culture room, we usually bring a small tip-box of 37deg water to the cryo room. Take the vial out of the LN2, into the 37deg tip box, move to cell culture and by the time we're there the cells are half thawed and ready to be diluted.

Hope my ramblings help someone!

Happy research!

Edited by squallweathered, 09 April 2010 - 03:14 AM.

[coxki]

Please if anyone can help me with this... I stored by mistake some suspension cells that were sent at -80°C, at -20°C overnight. Does anyone know if they could survive to this treatment? Now I stored them again at -80...
Thanks in advance!
coxki