I have sorted cell line but the problem is that it proliferate well upto three or four passages after that their growth slows down making me wait for long time. So i am thinking to make it immortalized. But i don't have any idea how to do it. If anyone have any experience or protocol regarding it, kindly share with me. Thanks
If you are trying to immortalise normal, healthy cells then what you are seeing is normal. An immortalised cell needs to undergo some change that allows it to continually propagate without intervention. For example, in cancer, this would be a mutation in a cell cycle regulation gene. But in normal healthy cells, these changes do not happen quickly. When your cell's growth is slowing down, that is called senescence, and it is normal.
In my old lab, we have had some success in immortalising cells by keeping them in culture, refreshing the media regularly, and "passaging" by trypsinising the cells and putting them back into the flask. You will have to do this for a long time (as in months) before you get a cell that carries the change required for constant growth.
Be aware though, that this cell is no longer completely representative of the original normal cell, as it now contains a mutation or loss in a gene that regulates cell cycle growth.
We considered our cell cultures as cell lines (this is the correct terminology for immortalised cells) once the cells get to passage 10, but this is arbitrary.
Thanks Sophie for your answer and i do agree with you. I have also alternative path so it doesn't matter for me about culturing for months, i will do that if necessary. Actually i have some special cells sorted based on gene expression and i just need them to have expression of my required genes but problem is after very few passages their growth become very slow. So my aim is to make them fast growing with affecting the gene expression of my required genes.
Sarah Yeung
Thanks. Time is ok for me as its my side way work, not my specific project. I also studied about SV40 and hTERT but still not very clear
you can try immortalising your cells (most preferably B-cells) using EBV inoculum but i dont think it will be good choice for already using a cell line. EBV can be used to immortalise B-cells and generate LCL (Lymphoblastoid cell line) these last for pretty long time and it again depends on needs of your study.
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