Dear Researchers,
We bought a new human cell line from ATCC and we stock them up in cryotube vials. The cryotube vials were preserved in liquid nitrogen; however they took inside liquid nitrogen. Therefore, the cells attach on cell culture flasks but not spread, grow up or proliferate. Do you have any advice to save the cells??
Sincerely
Dear Mehmet,
Actually this is one of the common things, that I also observed before. After you gather the cells from ATCC they some how need more time then you expect to become and act normal.
I suggest you to wait and check them carefully (change the culture media, be sure that you are using the media that ATCC suggest for these cells and if you add some additives to your media check them as well whether they are in well condition or not etc.). They will recover if they do not have any other problem like contamination or else.
For the Liquid nitrogen issue, this is normal you can see some liquid nitrogen inside the vials. According to general though, this might be a problem if you have a mycoplasma contaminated cells in your liquid nitrogen container, then these might have also infect your new cells. After contamination this would be another layer of concern about your cells health.
I hope you will solve this soon.
Best,
Yunus
Hi Mehmet,
It can depend on the cell line. Some cell lines are OK when the vials are stored immersed in liquid nitrogen, whereas others are not, and really do require storage in the vapour phase. There may be information on the datasheet as to how critical vapour phase storage is for your particular cell line, or you could ask ATCC directly.
At any rate, Yunus is correct, when you first receive a cell line it can be very slow to recover and proliferate. Change the media once the cells have attached if there are a lot of dead ones in suspension, to minimise the amount of dead cells in the culture. Use the recommended media because this can make a difference. Finally, be patient! It can sometimes take several days for the cells to "get going". If the attached cells are very sparse because not many survived, consider re-seeding them into a smaller flask.
Probably the worst experience of this type I had was with a particular mouse cell line, there were so few cells which attached after thawing that I re-seeded them from a T-75 into a single well of a plate (a 6-well plate I think) and had to be patient, waiting for them to go from there up to enough to seed a T-25, then a T-75. That's perhaps an extreme example but be patient and give them a chance. Hope they recover successfully, but it might take a week or two. And also, Yunus made a good point about mycoplasma, get them checked once you have them growing well.
All the best,
Mark
Dear Mehmet,
I never experienced before with the cells from ATCC, but once my cell also acts the same.
Try to observed the morphology of the cells, if they are good (have big dendrites) it means they are okay. Try to use the recommended medium (contain FBS) and change every time the color get changed without washing with PBS.
If they are rounded, it means something happen with the environment. Try to check the humidity and CO2 levels. Don't open the incubator frequently.
I hope the cells can survive. Wish you all the best.
Sandra.
Dear researchers;
First of all, I would like to thank all of you for your valuable answers. However, I would like to be more clear about my problem. After we bought cell line from ATCC, the cells growth and proliferated to passage 15 and we stocked them up (approximately 40 cryotube vials), the cryotubes took inside liguid nitrogen approximately 500 microliters due to the vials have defects on their caps. Thus, liguid nitrogen quickly evaporate when we took out vials.
Hi Mehmet, OK, I see what you mean. The cells may recover if you give them the correct media and be patient. If they are very sparse, consider re-seeding them into a smaller growth area which will help them "pick up" more quickly. If they don't recover well you will just have to ditch your stocks and buy another vial from ATCC...
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