Usually forcing TERT expression is pretty much straightforward.

You can also try single cell cloning. Depending on the maturation level and the passenger mutationa, you can try seeding single cell colonies in RPMI 20%FCS with one cytokine, such as GM-CSF, IL3, IL7, etc. It might take a while until you get a cell line, and you may not get any.

If I were you I would make a PDX instead of a cell line. You loose the clonal complexity in the cell line while the PDX may recapitulate the real Leukaemia

Hasan Issa

Thank you very much for your useful answer

my purpose of cell line generation is development of new line based on rare type of AML and I dont want to do personalized therapy for patients I'd like to know how many passages needed for cells purification under cytokine addition media

Reading old publications where cell lines were established might give some clue. Usually, cells were kept in culture for 6-12 months and observed over the whole time frame. Primary cells can go in sensescence any time, so cell line generation can fail any time, also depends on the cell type, the more cells proliferate the higher chances of success are given. It might make sense to freeze some cells after different time points (like every 2 weeks) to have some backup and also to check if the cell that were frozen can be re-thawn and recultured.

The longer cells will be in culture, the more they will modify their genetic architecture, so it would be wise to take some time point when you are sure that cells grow fine and can be recultured to then immediately make some stock and working bench - so that you can always go back to the initial phenotype, working benches then should only be in culture for approx. 3 months, then a new one should be thawn. And working benches should be prepared out of the stock bench. If cells are growing stable for 2-3 months, it might be a good time point for generation of the stock bench.

Best of luck!