How to freeze a cryo stock from your cell lines

The longer you culture your cells, the more they change and collect mutations. To track the quality of your cells it is important to write down their passage number, on every cell culture flask and in your lab book!

Cells have the best quality if they are freshly bought and thawn. You can define these cells as passage 1 (p1). After each splitting the passage number increases by 1.

after the 1. splitting:    p2

after the 2. splitting:    p3

after the 3. splitting:    p4

after the 4. splitting:    p5

after the 5. splitting:    p6 ...


Of course, this is not absolutely correct if you bought a cell line as they were in culture before. But usually you dont get to know their true passage number. Therefore, your numbering system just refers to your own handling of these cells but it is still a pretty good estimation.


If you isolate your own primary cells the passage numbering is completely correct. The isolated cells are p0, after each splitting they become p1, p2, p3 ...

Prior to starting with any single experiment, you HAVE to freeze a sufficiently big stock of each cell line you work with. It is very important that you freeze cells with a low passage number before they acquire any weird changes during culturing or even get contaminated. This system is absolutely necessary for a successful cell culture!



After thawing new cells for the first time, you multiply them as much as possible. And then you freeze all of them just leaving one flask in culture for your experiments. This will generate a low passage stock of your cells that will last well into your PostDoc time as you will establish a snow ball system.



How you freeze a proper cryo stock and establish the snow ball system, I will show you in the PDF that you find under this article.

Count in 2 weeks to freeze your cryo stock before you are allowed to start with any experiments!


  • It guarantees a long-term high quality of your cells.
  • You have a backup of fresh cells for your entire graduate studies.
  • You dont need to be scared of contaminations any more, as you can thaw fresh cells any time.

Dont get pushed by your supervisor to start experiments too early!

These 2 weeks to freeze your cryo stock are your investment into a successful cell culture for the next 5 years of your Ph.D.!


How many scientists do this? Surprisingly ... not that many.

Most people thaw cells, do their experiments and then freeze the remaining cells once they dont need them in culture any more. Maybe freezing 3 vials at the beginning as a contamination backup.

Just test it. The next time you get cells from a collaborating lab, just ask them for the passage number of the cells. Most often your collegues will not know it as they got the cells from another lab or because the responsible Post Doc left the lab a while ago.

Cancer cell lines are often cultured over many years. Passage numbers above 50 are common.

If you want to establish reliable cell culture experiments, you should consider carefully if you really want to get cells from the neighboring lab with an undefined passage number. You dont know how the cells were treated, how many starvations and dilutions they went through and in which condition they are right now.



I highly suggest you to buy those cell lines that you need for important experiments. Their quality is good and they are mycoplasm-free!! If you take good care of them and you establish a low-passage cryo stock you will enjoy a long-lasting good cell quality. And you can at least trust this part of your experiment.

I also made these mistakes over years, saving money at the wrong end. But this doesnt pay off. In the worst case it takes you endless hours of work to do mycoplam tests or figure out cell identities.

How do you convince your boss to buy the cell lines? Well, lets talk about this during coaching.

Cells should not be kept in culture for more than 4 months in a row as they change more and more. This means that you should throw your cells 3x a year and thaw new ones. Yes, its only 3x a year, thats not that often! But you should really do this. Again ... to keep up the quality of your cells.



By the way:

I am a big fan of culturing cells without antibiotics. But in critical situations like thawing new cells I do recommend to use antibiotics just for 3 passages till you froze your cryo stock. Its not fun to get a contamination in new cells without backup. It just gets expensive as you have to buy new cells.



Bonus: How to build up a cryo-stock from your cell line