Freezing and thawing of cells - (Sep/09/2004 )
really- your opinions? then why do they read like they were COPIED off the following link from Corning?
Is it just a coincidence that you keep offering references but not actually doing that.
shame shame shame
The following comments are primarily my opinions, but if one wanted citations, I could find where I had read these things.
Freezing mixtures: To be honest, it shouldn't really matter what you are freezing the cells in as long as it is a good pH and balanced osmotically. I wouldn't just use water or something with DMSO in it, but between FBS and media plus FBS I can see no difference. I prefer media plus FBS as I am sure the cells are comfortable with the salt levels contained therein. The cells are in the freezing media for so short a time, I can't see that there would be any sort of advantage to have the extra factors available in the serum. I use media with 10% serum, then add 7.5%DMSO to that. Works every time.
Glycerol: Glycerol is used instead of DMSO when you are interested in less toxicity. For instance, cells that are to be used therapeutically (cord blood, etc.) can't be frozen in DMSO as you can just go around giving that to people. In cultured cells there is also less toxicity, but it doen't go into the cells as well as DMSO. So, as long as you don't leave your cells sitting around in the DMSO on the way out of the freezer, I think DMSO is the better option.
If anyone really wants to read something other than my opinions, I am sure that given some time I can come up with references for this stuff.
Welcome to the forum. I am glad that you are using your time here well and that your first post was relevant to the topic at hand.
For the most part I post in the forum while I am doing something that does not require all my concentration, i.e. waiting on hold, centrifuging samples, or the like. I do not have time to spend with lengthy literature searches so I can add references to everything I post. So, instead of saying "This is absolutely fact", I said it was my opinion, so no one would take the things I posted as hard fact, since I didn't have time to find where I had read these things.
But since it appears to be an issue:
Corning does supply a large amount of information about cell culture and I recommend their web seminars to everyone, even if you are experienced with cell culture. This information can also be found from a variety of other sources.
The infomation I provided may have sounded like it was copied from Corning because both are correct. These other sources also say the same thing. They are probably not copied from Corning either.
Next time I will be sure to take the time to say, "These statements are things I have read in a number of different places and found to be true in practice as well."
If you have more to say about my posts, please pm me instead of taking up space that could be used for useful discussion. I would be more than happy to provide you more "citations".
Hi, of course the thawing procedures may vary depending on cells type, but here what we effectively have been using for years in our Lab:
from liquid nitrogen to a 37 degree water bath
take the cells out of the vial and mix with 5 mL of a preheated 10% FBS cell culture medium (the less pipeting up and down the better, no vortexing!!!no shaking!!!, even no tickling!!!, )
spin down at 500 rpm for 5 minutes
asperate the supernatant and resuspend in the medium for culturing
I am starting to work on cell cultures- hybridomas. Recently I tried to thaw a vial. My method: Thaw the vial placed in liquid N2 at 37 degrees centigrade in about 2 minutes. In sterile conditions, add contents of the vial(1ml) to 23 ml of medium+serum(in Tflask) drop by drop. The serum and medium are already at RT. Place the T-flask in incubator, also at 37 degrees.
When I added cells to Tflask, the viability was aroun 30% but when I checked after 2 days , all the cells were dead. The protocol provided by the company said it is not necessary to remove DMSO when transferring cells to medium.
What could have been the reson for the cells dying?
Here are a couple points to consider (the short version of the pm)
1. cells must be thawed quickly. I always take my cells out of the water bath when there is still a small chip of ice in the tube.
2. No matter what the protocol says I always wash the freezing medium off the cells.
3. Cell viability after freezing can be affected by time out of the freezer (on dry ice, etc.). For example if the cells were mailed to you or something. If the cells were mailed less than 30 days prior to thawing, I would call the company and ask for a new vial.
Let me know if I can be of more help,
Let me see if i could be of any help. First of all, i dont see any logic in wasting time adding the frozenmix drop wise. The moment cryovial becomes liq, drop them into 5ml media and IMMEDIATELY try spinning at 1200rpm, 3 minutes. Remove sup and give them love (an extra ml ) of media.
Hope u get a good thaw. I get 80-90% recovery.
WE are also maintaining cell in our lab now for the last 1 year.
We never spin the cell before plating and it work fine for us.
One think is sure media change after one day is help to increase cell no.
Now the thawing issue seems to have been resolved.
My main concern is that if I want to centrifuge the thawed cells after keeping overnight, what is the rpm I should do it at? I am handling Hybridomas(animal cells, that grow in suspension).
The instruction sheet that accompanied the first cells says that approximately 125xg (=500 rpm) should be tried.
My previous co-worker used to cetrifuge at 1100 rpm despite the instructions.
And the lab assistant feels that it should be around 1000 xg (=3000rpm).
I am confused about which one to follow. Personally, I would go with the instruction sheet, but would that cause the cells to not get separated properly?
What's your opinion?
As far as centrifugation of cells is concerned, I always feel that less is more, so to speak. I pellet my tissue culture cells at 200xg (1000rpm in our table top). You can always tell if the centrifuge is not set high enough because the cells won't pellet. =) (Or you just spin longer at that low speed.) If the cells pellet, it must be enough g. I feel there is no reason to pellet cells at super high speed. Even bacteria. I just pellet those at a lower speed for longer. Then I don't have to spend all that time trying to resuspend them from that tight hard little lump. And eukaryotic cells... there is no point to subject them to extra stress they don't need.
I agree with Beverly here. In our lab we use 110Xg for 5 minutes. works great