I established primary normal cell cultures from human foreskin. Then identifying these cells by ICC method with anti-Vimentin and anti-Cytokeratin19 antibodies. The cell showed positive signal to Vimentin and negative signal to Cytokeratin19 (they was expecting results, because only cancer cells show Cytokeratin19). However, I don't know if the ICC's results are enough to demonstrate normal cells? Are these results enough proof to provide to research journals when I would like to submit a paper?
By "normal" do you mean non-neoplastic? You could attempt to demonstrate this functionally, by determining if the cells grow in anchorage-free conditions, such as soft-agar, ultra-low attachment surface plates, etc. These assays have been historically used in fibroblasts and epithelial cells to demonstrate some measure of neoplastic transformation (vs. "immortalization", if applicable). Failure to grow without solid surface contact is some, although not definitive, evidence of "normalcy". It would likely strengthen whatever conclusions you make from marker expression analysis.
"Normal" cells should express the same cariotype from the original tissue.
Also, you can follow the pattern of markers from the original tissue .
To discart cell trasformation you can tranplant the cells into nude mice and see if they grow as tumoral cells
Dear Colleague,
First, at which passage are you on your "primary" culture cell. Because, if superior to 40 ie, they have no more the characteristics of normal cells originated from your tissue, because they have acquired one of the characteristic of tumoral cells: immortality.
To my mind, it's always difficult to affirm that cells in culture have the same characteristics than in vivo; and as said by Annegret, often, when you establish primary cell culture, the most "resistant" one are fibroblasts, which survive more than epithelail cells, and can also contaminate other cultures. Moreover, fibroblasts are no more well recognized in vitro because changing their apparence. In the past, when we haven't not the actual mean of cell identification in vitro, a lot of researchers thought to have established new primary cell culture, but it was fibroblast. I don't say that is the case for you, but to be well define. And also remember that some actual research conduct to obtain stem cells from fibroblast (de-differentaition) , to diffrentiate them after in other cells usefull for an eventual cell therapy.
Best regards
Didier
I think your question is well answered. Ninety nine percent chance is that you have got fibroblast.During cell isolation from foreskin, if you don't get good separation of dermal and epidermal layer by dispase treatment, fibroblast will out grow and elminate epithelial cells. Look at the morphology and density. Fibroblast tends to grow in multilayers whereas epithelial cells will restrict to monolayer. You should get CK19+, if you have isolated keratinocytes.
Thank you so much for your answers!
"Normal"cells mean non-cancer cells. Because I would like to use them in order to compare some bio-effects to cancer cells.
You may check the proper compartmentisation of various cytoskeleton compartments.
- Badges
- Science topic
- Similar topics
- Methodology
- Research Methodology
- Research Papers