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Subculture issue of primary cell


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#1 stephanie_ym

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Posted 16 January 2013 - 11:19 AM

Hello everyone,

I am culturing primary cell isolated from human prostate cancer tissue. Now it is on passage 14. The first 7 passages grew as monolayer very fast, about 2 days per passage, and the cell looked very bright and 3D. Starting from the 9th passage, the cell condition went down. Vacuoles and granules occurred within the cytoplasm and it became more and more severe as I passaged, and the growing speed went down too. I have to passage at 1:2 instead of 1:5 before, about 4-5 days per passage. The cells are now bigger and flat. Could someone please tell me the reason caused this problem and how to treat it? Is it natural cell aging or lack of nutrients in the growth medium or contamination?

Culture medium used: LONZA PrEBM( prostate epithelial basal medium with no FBS) supplemented with BPE, hydrocortisone, hEGF, epinephrine, insulin, triiodothronine, transferrin, gentamicin/amphotericin-B and retinoic acid.

Thank you very much.

#2 bob1

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Posted 16 January 2013 - 12:18 PM

Google for Hayflick Limit.

#3 stephanie_ym

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Posted 16 January 2013 - 07:17 PM

But do cancer cells obey Hayflick Limit?

#4 leelee

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Posted 16 January 2013 - 10:41 PM

But do cancer cells obey Hayflick Limit?


I don't know the answer to this- but I do suspect that not all cancer cells would be able to divide indefinitely. I'm sure someone else has a better answer.

I was wondering- do you know that your original cells were only cancer cells, that it was a pure culture to begin with? Perhaps in the beginning, a number of different primary cells formed part of your sample and what you are seeing now is those dying out as they reach their Hayflick limit, leaving only the cancer cells behind?

Just a thought... (I have no experience with culturing cancer cells)

#5 bob1

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Posted 17 January 2013 - 12:03 AM

Cancer cells may or may not be immortal - the transformation process is quite rare, so rare that before HeLa cells were developed, many many tumours had already been tried for this process.

#6 stephanie_ym

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Posted 17 January 2013 - 12:25 AM

Cancer cells may or may not be immortal - the transformation process is quite rare, so rare that before HeLa cells were developed, many many tumours had already been tried for this process.


By "transformation" you mean the transformation process from being mortal to immortal? At this time there is nothing I can do, just let the cells go?

Edited by stephanie_ym, 17 January 2013 - 12:46 AM.


#7 bob1

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Posted 17 January 2013 - 11:59 AM

Yes, going from mortal to immortal.

You can try to keep passaging them, but the spontaneous transformation rate is very low. Transfecting (stable transfection possibly) with viral genes such as Ad5 E1A or HPV e6/e7 can increase the transformation rate, but it is still quite low.




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