I would like some advice regarding THP-1 cells after freezing stock vials. I was growing these cells perfectly; they were behaving wonderfully to the letter. I was using the ATCC recommended medium without antibiotics and would seed at 200,000 cells/ml grow them for two to three days and pass them at 800,000 cells/ml. I did go beyond this number a few times but when I reseeded them at 200,000 cells they would still grow as they did before. So all was well and good at this point.
I expanded the cells until I had enough for roughly 20 vials of cells for freezing at 8.5 - 9.5 million cells per vial (as ATCC suggests). I measured the cell number by haemocytometer so I am not certain that the number is correct. But the cells were frozen down slowly in complete medium containing 5% DMSO.
After I froze the cells I read that DMSO can differentiate the cells to Macrophage like cells and that it should be avoided. I also read that just freeze thawing these cells can sometimes kickstart differentiation. Anyway I have thawed some two vials now and I am noticing that they are no longer behaving as they did before.
1. They are no longer growing as fast as they did.
2. A small number of the cells are clumping together.
3 A small number of the cells have differentiated and are adhered to the flask surface.
I think that I may just be noticing these behaviours because I am over anxious and looking for them; they were possibly there before but I did not see them.
So if anyone has had an issue with THP-1 cells behaving differently after freeze thawing then please, any advice would be greatly appreciated?
Hi Robin,
From the sound of it, the THP-1 cells are acting like THP-1 cells. You did not mention the initial cell concentration after thawing or the current cell number/ viability. I'm not sure how you thawed them, but this is how my lab thaws THP-1 and other cell types that tend to differentiate with DMSO.
For one vial of cells we rapidly thaw them in a water bath and add 1ml of cells to 25-30 ml of the medium in an up right T-25 flask. The cells are placed in the incubator in the upright position (cap up) for 3-4 hrs. This will allow the cells to settle to the bottom of the flask. After several hours, the flask is carefully removed from the incubator and ~20 ml of the "old" medium is removed, and replaced with 5-10 ml fresh medium. The flask is then returned to the incubator in the upright position for several more days.
First, this method allows the reduction and removal of the DMSO in a cell friendly manner. Second, by growing the cells for several days in the upright flasks allow the cells to remain in close contact and improves autocrine and paracrine signaling, which improves cll viability and growth. After several days, they should be ready to go and can be handled in the normal fashion.
I hope this info. helps.
Good luck and CULTURE ON!
Hi Robin,
From the sound of it, the THP-1 cells are acting like THP-1 cells. You did not mention the initial cell concentration after thawing or the current cell number/ viability. I'm not sure how you thawed them, but this is how my lab thaws THP-1 and other cell types that tend to differentiate with DMSO.
For one vial of cells we rapidly thaw them in a water bath and add 1ml of cells to 25-30 ml of the medium in an up right T-25 flask. The cells are placed in the incubator in the upright position (cap up) for 3-4 hrs. This will allow the cells to settle to the bottom of the flask. After several hours, the flask is carefully removed from the incubator and ~20 ml of the "old" medium is removed, and replaced with 5-10 ml fresh medium. The flask is then returned to the incubator in the upright position for several more days.
First, this method allows the reduction and removal of the DMSO in a cell friendly manner. Second, by growing the cells for several days in the upright flasks allow the cells to remain in close contact and improves autocrine and paracrine signaling, which improves cll viability and growth. After several days, they should be ready to go and can be handled in the normal fashion.
I hope this info. helps.
Good luck and CULTURE ON!
Thank you both Daniel and John. There are some very interesting points you both raise here. I never would have considered placing the flask in an upright position but now that you have explained it does make sense.
When I revive the cells I thaw at 37 degrees until I have a small amount of frozen solution floating in the vial. I then place the cell solution in a 50 ml falcon tube and resuspend the 1 ml (very slowly) in 9 ml medium and centrifuge at 1000x ( maybe a little fast compared to you John). I then I then pour of the super and resuspend in 10ml (1ml and mix then 9ml) medium. I assume at this point I have removed the DMSO successfully, but maybe not? i then leave the cells to grow over night until I count the next day.
The counts I performed the day after thaw were at the top end of log ~ 800,000 cells/ml and viability was ~87% (not great). I then passed them, seeding at 200,000 cells/ml and now they have slowed right down I.e. they don't need passing after three days, more like every 4-5 days. The growth rate is much slower than pre-frozen cells.
I think maybe I have been a bit hasty with how I have handelled the cells. Judging by both your answers they should be allowed to settle for several day. I will try this with my next thaw.
Sorry, just a couple more questions.
Would you do a count with the initial thawed vial before leaving them to aclimitise?
By several days do you mean 4-5 day?
Thanks guys for passing on your experience. These cells seem to be quite fussy and particular. Your wisdom in these matters is greatly appreciated. :)
Robin,
I would do the cell count either just before or just after you change the media (day of thaw). I have found that the cell viability will decline over the first several hours. i attribute this decline to delayed thaw induced damage. Yes, by several days I mean 4-5. After 2 days, if the media begins to look yellow and the cells look happy under the microscope you can transfer the to a larger T-75 flask (upright) and add 5-10 ml of fresh media. I suggest you start with 5ml. You don't want to over-dilute them. If they look add another 5 ml. This way you keep the cells happy and slowly increase their number.
I just want to second what Daniel and John have said.
Though I will say I never left the THP-1 cells alone for several days post-thaw (maybe it would have helped). What I always did was the thaw as you described, pelleted the cells at ~500xg to remove DMSO, and then resuspended the cells in 5mL fresh medium. I would put them in a T25, laying down, and leave them for 2 days, and then add another 5 mL, provided their viable counts weren't terrible and the cells looked happy. After another 2 days I'd add 10mL of fresh medium and transfer to a T75 (again, laying it down). I never seemed to have an issue with the cells early on - I always had issues when they got around passage 30.
Hi Kenneth,
I see you graduated from URI. I graduated from URI in 1990, Ph.D. Small world. By the way, you culture your cells for 30 passages! Are you CRAZY!!!. Some friendly advice, you should only use your cells for 5-7 passages. Contrary to popular belief, cell lines are Heterogeneous and they due change over time. I have several cell lines that after 7-8 passages show a 5-8x increase in the IC50 value of drugs. This, despite them being treated with TLC.
Hi Daniel,
I saw that as well. A particular Disney song got stuck in my head after seeing that.
As for the cells, we stopped using them a while back. Too much trouble and not enough established data for what we were using them with. Moved onward (and hopefully upward) to HL60s with an easily track-able marker set to keep an eye on how much they change. Thanks for the advice, though! I'll definitely keep it in mind.
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