Usually in our laboratory we obtain permanent cell lines from primary cell cultures by continuous cell cultivation. We do not use special immortalization agents because for our purposes we need less manipulated cells.

If you continue culturing the cells and if you are lucky, after 80-100 passages you will have a permanent cell line. It is long way and you will meet a lot of challenges: possible microbial contamination (if you work with  colorectal or gastric cancer for example), presence of too much fibroblasts (the serum in culture medium can also stimulate their growth), the cells can stop their proliferation, etc. The cell culture origin is very important – it can determine some specific challenges that you can meet.

At the beginning you can try to find in the literature experience of other scientists  in establishing the cell line  of the same origin like yours. It will be very helpful for you but do not forget that each cell line is completely unique.

If you have more cells, you can try to “check” different culture condition in order to fix the  most appropriate for your cells.

In order to reduce the risk for contamination or some other falls, it is better to have at least two repetitions of your cell culture – passage them completely separately, using independent sets of reagents (culture media, enzymes…).  

Freeze and store cells at different passages. If for some reasons the cultures stop growing you can use the cells from this reserve for a “second chance”. Something more, if you  obtain a  permanent cell line, the frozen cells at different passages  will allow you to follow the immortalization steps of your cells – from primary culture to the permanent cell line.

And finally, do not forget to record everything in your working diary, even the most “insignificant”  data. Later, it can be very helpful for you.

The establishment of a new permanent cell line is not an easy task. But it is very exciting! If the first try is not successful, try again! Good luck!

Use an immortalization kit

There are two ways, either your cells automatically transform into a cell line, that is the spontaneous transformation and believe me it is the best. Or you can use the immortalization kit. 

Usually in our laboratory we obtain permanent cell lines from primary cell cultures by continuous cell cultivation. We do not use special immortalization agents because for our purposes we need less manipulated cells.

If you continue culturing the cells and if you are lucky, after 80-100 passages you will have a permanent cell line. It is long way and you will meet a lot of challenges: possible microbial contamination (if you work with  colorectal or gastric cancer for example), presence of too much fibroblasts (the serum in culture medium can also stimulate their growth), the cells can stop their proliferation, etc. The cell culture origin is very important – it can determine some specific challenges that you can meet.

At the beginning you can try to find in the literature experience of other scientists  in establishing the cell line  of the same origin like yours. It will be very helpful for you but do not forget that each cell line is completely unique.

If you have more cells, you can try to “check” different culture condition in order to fix the  most appropriate for your cells.

In order to reduce the risk for contamination or some other falls, it is better to have at least two repetitions of your cell culture – passage them completely separately, using independent sets of reagents (culture media, enzymes…).  

Freeze and store cells at different passages. If for some reasons the cultures stop growing you can use the cells from this reserve for a “second chance”. Something more, if you  obtain a  permanent cell line, the frozen cells at different passages  will allow you to follow the immortalization steps of your cells – from primary culture to the permanent cell line.

And finally, do not forget to record everything in your working diary, even the most “insignificant”  data. Later, it can be very helpful for you.

The establishment of a new permanent cell line is not an easy task. But it is very exciting! If the first try is not successful, try again! Good luck!

Dear all

Thanks a lot for your beneficial answers.