I heard that we have to passage until 50 passages in order to claim that the primary breast cancer cells become an immortalized cell line. Is this correct? Where can I find this information?
I have successfully cultured primary breast cancer cells from very small biopsy tissue (less than 0.5 cm) from human patient. However, I need to prove that this is cancer epithelial cells but it is still confusing what markers to use. One paper mentioned CD44+, CD24+ and CD227+ (MUC1). One supervisor said it is same as Breast Cancer Stem cells markers such as CD44+, CD24- and CD45-. In another paper it mentioned CD44+, CD24+ and CD227+ plus to check Cytokeratin 14, 18, 19, as well as telomerase activity (TRAP assay).
If you guys have done this, appreciate sharing of your knowledge on this.
You can look up the protocol NIH used to make the immortal 3T3 fibroblast line. I believe it was 50 passages, but you can follow their protocol to make a line immortal.
I am not an expert on breast cancer, but perhaps this paper on HPV-induced immortalization will be of your interest:
http://www.academia.edu/download/42838099/Steenbergen_RDM_Walboomers_JMM_Meijer_CJ20160219-31463-18wyfd1.pdf
Take care.
You should first show that they are epithelial in nature, for that u can use markers like E-cadherin and cytokeretin5/8. In order to demonstrate the cancerous property, you can go with the telomerase activity and immortalization in culture. very importantly you should also show the negative fibroblast contamination in your culture because you have derived it from primary cultures.for fibroblast contamination you can go for Vimentin, and fibroblast surface marker antibody.
Thank you guys. Ankit Mathur, I have question about detecting about Vimentin. Are you telling that if there are vimentins, the epithelial cells are contaminated with fibroblasts?
For cytokeratin 5/8, are there any reasons of not detecting 14/18/ and 19 instead? I noted that E-cadherin expression level is not much different from breast tumor and normal breast cells. E-adherin level reduce when breast cancer become invasive, or last stage of the breast cancer too. So, do you think it is still worth to test E-cadherin? I am confused.
Why can't we just go straight to test CD44/CD24 and CD227 too also? Are there any others cells that have such similar markers?
Roland van leeuwen, the link is not intact and cannot reach to the page. Pls send it again. thanks
You are looking for the cells to live beyond the "Hayflick Limit' (established in the mid 1960s), It can vary a little by cell type but the limit usually lies somewhere between 40-60 passages. Its now known to be related to the shortening of telomeres (the 2009 Nobel Prize (Medicine or Physiology) was given for the identification of telomeres, the seminal work reflected the ideas of Hayflick)...Bob
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