Freezing and thawing of cells - (Sep/09/2004 )

Reduce your centrifugation speed to 600 rpm for 5 minutes. Also, when DMSO contacts fresh medium, it will heat up. This vcan kill your cells. so take 5 ml medium in a tube first. Then add your thawed cells to the medium drop by drop as slowly as u can. Then spin as above. It is also good if u change the medium once again the next day.

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Hi Shipley,

Here is what I do for all the cells I am working with (cell lines, primary cells, human, rodent...)

When cells are confluent I trypsinize them. After inhibiting trypsin with complete medium (what you use to grow cell in) I centrifuge slowly (what you did is fine, 1500rpm for 5 mins) to pellet them. I then resuspend the cells in freezing medium (compete medium, with 10% DMSO and 40% FBS). I distribute the cells in freezing vials (around 2 000 000 cells per vial) then freeze them. The way I freeze them , I either wrap the vials in kimwipes and put them in a polystyren box overnight at -80 degrees, this will allow the cells to freeze slowly or I use a freezing Box containing isopropanol (same thing O/N at -80). The day after, I put them back in nitrogen.

To thaw the cells: Do it quickly at 37 C get your medium warmed up before starting. If you have cells lines you culture them without pelletting them. If they are primary cells , you follow your protocol you add your cells to fresh warm medium, centrifuge and get rid of the DMSO, you can give them a second wash with warm PBS (optional). Resuspend cells in complete medium and they should be fine.

Soudi protocol is also a very good one.

Good luck.

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In my experience, centrifugation at 1500rpm X 5min is too high (it further depends on the "g" you are getting in your centrifuge, it might dehydrate the cells), infact you don't need to centrifuge just plate them as such after thawing and if you want you can change the medium after 2-3 hours if you are concerned about DMSO though the cencentration would be much less.

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My suggestion is try to thaw more cell stocks and pool them together into a relatively smaller plate, let cells stick to plates overnight and then change fresh warm medium.

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Hi ...about cell thowing...what type of cells are you using?and does the freesing media contain DMSO?

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can anyone send me information explaining the effect of pen/strep in growth media..
thank you so much
antoine

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I think you should add the medium drop by drop to the thaw cell and dilute to 20x with the medium within 2 min, instead of directly add the medium to the thaw cell. Then incubate at room temp for 5 min, followed by centrifuge the cell and resuspend it in culture medium to remove any DMSO added during freezing the cell.

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I think pen/strp in the growth medium is to prevent the growth of bacteria / contamination in our cell culture.

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hi
r u freezing the cells correctly ?? if not follow the protocol of labrat has given u ..and try with this procedure dont add the cells to medium (take cells into a nunc tube after thawing from 37 add drop wise media ml/min to the cells ~ 5ml mix gently while adding the media, after 5min spin at 1000rpm for 5 min

All the best

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There are a few things you can do to improve the recovery of your cells. Remember each cell type and each cell line will have its tricks and moods but with one or two adaptations a general protocol should work fine:

1) Freezing. Work quickly. Use 10% DMSO with no less than 20% serum. Media (e.g. DMEM) is cheaper than serum so you can use that to make up the rest of the freezing media. Don't leave your cells in freezing media more than 20 minutes when aliquoting. Place your vials on ice until you take them to the freezers. Use ice cold freezing media. Resuspend to an apropriate dilution (too many cells will not freeze properly, too few will not recover to decent numbers - what you freeze is always more than what you can recover). Lower temperature slowly (about 1 degree per minute). If you have a freezing box with isopropanol, precool to 4C then place your vials inside and the box at -80 overnight. That will garantee the right rate of freezing. If not, use the cheap method: place at -20C for about 2 hours, then at -80 overnight, then transfer to liquid N2 for long term storage. Your cells will take a hit at -80 and after 6 months you might have lost the batch. You can also place the vials inside a styrofoam box with the lid almost closed at -20 then -80. This will garantee a more homogeneous temperature drop. Just make sure you cool the box before and don't close the lid completely.

2) Thawing: there are different ways of thawing, and it's all up to debate, but you need to go with whatever works best for you or whatever you are most comfortable with. The important part is to bring the cells back to 37C as quickly as possible. Freeze slow, thaw quick - rule of thumb! You can either place your vials in a 37C water bath briefly until you see it melt, use warm media that you pipet into the vial, or warm it up in your hands. Whatever you do, make sure you don't let the cells stay in liquid freezing media below 37C for too long. You also want to remove DMSO - someone said they didn't understand the need to spin down. Here's why: DMSO has an effect on cells, than can go from promoting replication, to promoting diferentiation, to affecting cell survival, for better and worse. You freeze at 10%. Let's say you thaw adding an extra 9mL. That's still 1% DMSO in the media. Well above innoccuous levels. I know many people leave it until the next day after they thaw because they don't see an effect on their cells, but there can be an effect and some cells are more sensitive than others. To give you an example, if i don't remove it from my stem cell lines they will die. And if i leave it at more than 1/500 i will affect the cell fate significantly.

3) Centrifugation - and this goes for everyone: stop thinking RPMs and start thinking G force. It doesn't matter how fast or slow you spin the rotor, it's all about the force applied to the cells which has to do with the radius and incline of the rotor. So if your cells are really fragile, try 200 G (I use two centrifuges, one will spin at 1000rpms the other at 2000rpms for the same 200 Gs, see the problem?). If they are tougher, try 500 G, even 800 G. If you don't know the conversion for your equipment, go find out, call the rep, find it online. Again, as long as you get a nice pellet at the bottom after about 5 minutes, not so compact that you rip every membrane and not so loose than you will suck them off the bottom, use the least G force you can. That will also keep debris and dead cells afloat better and you will get rid of those as well.

4) Ressuspension: be gentle! Don't worry about resuspending too well, 5-6 times until you don't see any chunks, let your cells recover first, you just want to get them in suspension and plate them out. They will spread out just enough overnight. Again, the way you freeze will affect the recovery, if you're freezing dead cells or compromised cells or killing your cells during the freeze process, you can't expect too many to live the next day. Also, don't trash your plate the next day just because you see a lot of floating dead cells, change media and give it another day or two, you might be surprised at how many cultures will eventually come out nicelly after a couple days. Another example, with human stem cells you will get easily 90% dead cells with the best protocols. So sometimes you can be working with the toughest cell line and there is only so much you can do.

Practice makes perfect. Try a couple things out, see what works for your cells and for you. Then go with that, it's like a superstition, if it works, stick to it. Admiting you need to improve your technique, coming here and asking for help is a great start so you're clearly on the right track! Good job!

Good luck to everyone on their work and keep up the good science!!

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