The problem of the primary fibroblast (in my experiments) was they resist the cytotoxic of Camptothecin after I stored them in liquid nitro. I'm confused about that and don't know how to solve it. Do you have any tips/advice on this problem or others on primary fiboblast? Can I continue use them as normal cell model in vitro for my experiment (after re-set up the protocol with this primary fibroblast which after freezing store)?
How are you storing camptothecin? The E-ring of CPT will open under basic conditions (pH > 7.2) and that will make it inactive. I store CPT in 100% DMSO at -20oC. Does your CPT still work on unfrozen primary cell lines? How long did you culture the primary cell line with CPT before freezing? If you used low levels of CPT in the cell culture, you could select for CPT resistant cells over a period of weeks/months. But just freezing fresh primary cell lines would not make them resistant to CPT.
I make sure CPT still work well (with the same store condition: in DMSO at -20oC). The problem just because of my primary fibroblast. I just treated fibroblasts with CPT in SRB assay (cytotoxicity assay) and did not store fibroblast after treating with CPT. I mean I afraid of the characteristics of fibroblast maybe change after freezing store. I don't have much experiments on primary fibroblast so I would like to know you have any advices on this. Sorry if I explain not well.
I would also be more concerned with your CPT than with the HFF (human foreskin fibroblast). I work with those cells all the time and have never noticed changes to their drug sensitivity following a freeze/thaw cycle. Have you carefully controlled for the density of your cells. perhaps your cells formed clusters after being thawed and as a result a physical barrier. very unlikely to be changes in your HFFs though.
1) Are your cells passaged after thawing or are they used in your experiments directly after "reviving" them? I would strongly recommend to cultivate the cells for (at least) two passages after thawing.
2) "primary HFF" means which passages after extraction? HFF change during cultivation and it would not suprise me to see differences in HFF passage 3 compared to passage 10. Therefore I would recommend to perform a control experiment comparing cells from the same donor, one set "just" passaged", say testing p3 / p5 / p10 / p20 and another set freeze/thawn treated two passages before use. This might help to discirminate between "passaging" and "freeze/thawn" effects.
I agree with Peter Schroeder. It is not a good idea to use just thawed cells in experiments. With primary cells, you may not have the luxury of passaging two times, but you should at least passage them once before you plate them for experiments.
Peter makes a good point about passaging primaries. If your primary cells are at a high passage number, they may simply have lost their repsonse to CPT. Even with established cell lines, I find that a particular response I am looking for may be lost over many passages.
If HFF change during cultivation (as Peter Schroeder said), I will perform again my experiment follow Peter's suggest. Because I had used HFF to set up SRB on them, then I freezed them. After thawn them, I cultivated them 2-3 passages before using them on SRB assay and I got the CPT resistant from fibroblast cell.
Thank you so much for all your points.
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