Hi Kamila Mustafina

You can take back your existing cell line from a long passage number into 0 or 1 by making single-cell cloning. Each clone means here the number of cells produced from single-origin and carrying consistent characteristics. With increasing passage numbers, cells acquire versatile characteristics during long periods of passaging. I attached an image here descring the procedure of single-cell cloning.

Hello,

To make a cell line you start with a primary cell line; an organ (kidney for example) or an embryo (chicken) is minced, treated with proteases to individualize the cells and put in culture... after a variable time (days) some cells (like fibroblasts) make clones while the others die. When the dish is full the cells are trypsinized/splited 1in five , diluted and put in a new dish; this is passage 1!!! if you keep all the cells (possibly after a few passages so you increase the number of cells by 5 at each passages (amplification better than PCR!!!)) you can have many tubes that you keep in liquid nitrogen; this is your seed stock passage n. After several passages (n+x variable depending on the cells and the lab!!!) your cells lose their characteristics or die... so you defrost a tube of your seed stock and start again at passage n. When you see that your stock at passage n starts to be low, you make a stock at passage n+1; that will be your new seed stock. Keep some of the stock n if ever you have to start again one day from exactly the same cells. You can also redo primary cells (as people working with chicken fibroblasts do each 2 weeks, for example, because the number of possible passages is reduced, whereas with monkey kidney cells (type CV1)you are almost not limited by the number of possible passages( 30-40).

Sometimes spontaneously or if you add mutagens or after transfection with an oncogene (as antigen T of SV40; Cos cells) or infection with some viruses (voluntary or not (Hela cells) you will obtain an immortal line which can be passage an infinite number of times... the passage number is frequently forgotten or it only indicates the passage number in the lab.

This is the exact question I had two years ago. The passage numbers are arbitrarily/wrongly attributed to cell lines. Cells can not be P0 or P1 for stocks being sold from the cell banks as they would have to be multiplied. Another explanation I received is that when you defrost an aliquot of cells that are considered P0.

I use the passage number as an indicator of cell condition. I don't use cells less than P4 or higher than P20 for experiments. P4-20, from my experience, show consistent results between the experiments. Also from the experience, cells frozen at higher passages do not perform like one you multiplied and froze after you got a new batch of cells. I also noticed inconsistencies within the same cell line bought from the same distributor within 2 months period. They performed differently from each other even they were declared the same and bought with that in mind.

Mateo Glumac

Thank you for your response! It's good to know that even cell banks have batch-to-batch variation. Also, that confirms that they have a renewable source of P0s and P1s. I also saw people assigning P0 to freshly thawed cells, but it doesn't make sense to me at all - if cells accumulated mutations over multiple passages, they are not going to magically recover after freezing.

Salequl's answer above explains how P0s can be made.

Salequl Islam Didier Poncet Thank you very much for your answers!

Didier, I see what you mean, but I was wondering how can anyone get any P0 or P1 cells. Just out of curiosity - like how can cell banks have so many of P1 or P0s, they should be renewable in that case. But when I read descriptions of cell lines, they usually come from some person or a mouse in the 70s and I don't think they could freeze so many of those to last for over 50 years

for me P0 is a primary cell line; if you start with enough monkey kidney or chicken embryo you can make a big seed stock... where did you bought passage 0 cells?

Ah I see. Yes, actually in my lab in Japan we had P2s or P3s of HEK293 cell stocks. I haven't seen a tube of P0 myself, but assumed it was possible to order. Now I am not sure. So in your experience, the lowest passage number we can get commercially is P1?

yes but you can by primary cells (P0) . about your HEK P2 or P3; these cells were originally isolated and grown by Dutch biologist Alex Van der Eb in the early 1970s... so I doubt that 50 years later your lab still have P2 or P3... the ATCC does not indicate any passage number for the HEK it sells.

Didier Poncet oh I see. So that was exactly my question: it means that there is no P0s or P1s for old cells lines? And if, as you say, ATCC doesn't assign the passage number to some of their cell lines, how come most protocols recommend using "low passage" cells? How can we be sure it's low passage cells if we don't even know the passage number when we get it from ATCC? Or does it just refer to "low passage" relative to the point when we receive it from the cell bank?

My guess that in my lab we called P1 whatever we got from ATCC (or other cell bank) and just counted from there.