I am developing combinations of targeted drugs for cancer therapy.
In order to make sure that the effective combinations of drugs I select in vitro will not turn out to be too toxic in vivo, I want include a few 'normal' cell lines as control. I expect the drugs IC50s to be significantly higher on the normal lines than the cancer lines I use.
So, what would be the best 'normal' control to include in my experiments? Would the usual 3T3 be sufficient ? Or should I tailor the choice of control lines (e.g. of different tissue origin) based on the known in vivo toxicities of the drugs I choose?
Dear Lorenzo, it depends on the specific aim of your experiment. Generally, primary fibroblasts may be sufficient. If possible, it could be interesting to use normal cells as control derived from the same tissue/organ of cancer cell lines you analyze. However, I think that beyond the normal cells you use, in vitro you can obtain only a partial evaluation of toxicity.
I suggest to use human foreskin fibroblast even if the results can be different than a real in vivo situation,as suggested by Massimo Donadelli. Primary cells are usually dificult to keep in culture...I am referring to hepatic cells, due to their low proliferation rate.
Thanks. We choose to add this control because we saw that very effective/synergistic combinations in vitro turned out a bit too toxic in vivo, when given at proven effective concentrations. So we thought that with appropriate controls (maybe normal cell lines originated from the same tissues affected by the drugs in vivo) we might rule out from the beginning combinations that later will turn out to be impossible in vivo. But I'm not sure if how often people do this or the rationale to choose fibroblasts/ epithelial or other cells.
I don't have much confidence in "normal" cell lines. When you look close enough, you usually notice that cells that grow forever are anything but normal even if they originated from a normal piece of tissue. Prime example: The CV-1 cell line. It is often used as a "normal control" because it is easy to grow and originates from the normal kidney of an African green monkey. That's about where the normality of this cell line ends. In most other respects, CV-1 cells are highly abnormal. They are, for example, pseudodiploid, have multiple chromosomal abnormalities, and express very high levels of the multi-drug resistance gene, mdr-1. The overexpression of mdr-1 is particularly troublesome. If your drugs of interest are substrates of the P-glycoprotein, using CV-1 cells as normal controls may give you superb therapeutic indices that have, however, very little bearing on reality.
As a complement to foreskin fibroblasts you may want to consider freshly explanted hematopoietic cells - especially if hematopoietic toxicity is a concern. We have purchased normal human bone marrow cells (mononuclear fraction) from health volunteer donors from a commercial source. Getting the cells from a commercial source is not cheap, but quality has been consistently good, and we always got the correct number of cells delivered at the correct time. We have used these freshly explanted bone marrow cells for in vitro clonal assays of progenitor cells and the flow cytometric analysis of primitive hematopietic cells (e.g. CD34+ cells). One caveat: Hematopoietic progenitor assays may not be practical if your drug combinations require long exposure times.
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