We are evaluating a new anti-cancer drug that, among other things, blocks cells in G2. We need to test its non specific effects on normal cells but in regular cultures these are also replicating and so are also affected. The cells can be grown to near confluence to halt replication but then if grown for a further 48-72 hrs for the drug exposure experiment, the control cultures also loose significant viabilility. Can anyone suggest a culture protocol that will maintain the cells in good viability for this period even though they're not dividing? That is, without using chemicals to inhibit cell cycle.
Dear Michael,
monocytes isolated from PBMC by plastic adherence to culture plates and culture in presence of GM-CSF is possible. This is also the basis of monocyte-derived dendritic cells, 7-9 days of culture, only differentiation with good viability and without proliferation
Dear Michael,
you can use lymphocytes, you take them from a buffy coat or from mouse lymph nodes and add the drug. The cells do not divide unless you activate them. You can check for viability, or you can determine the frequency of cells able to get activated (by limiting dilution) in the presence or absence of the drug
Dear Michael.
if you are interested in adherent cells, such as epithelial cells, and do not want to add mitotic blockers, you might try low serum conditions, This will slow the mitotic index considerably, but will not stop it.
Thank you all for your answers.
I should have mentioned that we are already tested several types of hematopoeitic cells and that came out OK. We need to test several types of adherent cells such s epithelial, prostate, lung etc.
Lauren, we had thought of growing them to near confluence, wasing and growing again in SFM or such DMEM (although this would remove any effect of serum proteins on the drugs). Do you have experience with this method?
Michael
Dear Michael,
you can do it with NIH 3T3 although you should pay attention to the clone of NIH3T3 that you use, first make sure that they are mycoplasma free and second check that the cells stop proliferation after serum deprival (washing the media out a couple of times to make sure that there is no serum left). If the clone you use works well, you should be able to maintain the cells for 72-96 hours deprived of serum and treating them with your drug. Afterwards you can go back to fresh media with serum and without your drug to see how many remain alive.
But as I mentioned, it will work with some clones and not with others, you may have to test several
Dear Michael.
We have experience with different types of human epithelial cells and fibroblasts. In general, the tumor-derived cells are grown in serum-supplemented medium, whereas different types of mortal cells will have SFM. The trouble with the tumor cells is that you have to carefully screen those that are still relatively well defined such that they are sensitive to confluence. Many of the tumor cells have lost this sensitivity to varying degree. Alternatively, you could do some growth curves determining the change in mitotic rates in cultures with 10% serum that has been reduced to 1%, and then back to 10%. There exists a strong body of literature describing different cell lines in depleted serum conditions. Once you find the best fit, you can do as Jose mentioned above, which is comparing controls against your anti-cancer agent treatments.
Dear Michael,
if your drugs acts on proliferating cells and cause G2 arrest, could be useful to block cell proliferation not in G1 because this drugs might cause, as you have already observed. apoptosis. An alternative could be an arrest at G1/S transition. For this aim you can use the double thymidine block that arrests several types of adherent cells at G1/S.
Dear Michael, Your problem may be your culture medium. If you use serum in your medium, most sera contain inductive factors for cellular proliferation. We use heat inactivated serum (Atlas Biologicals, Fort Collins, CO) in our media (Opti-MEM + Glutamax (GIBCO) + HI, pH 7.4). Our cultures maintain a quiescent state with no proliferation. However, you will need to change the medium to keep your cultures viable. Our medium is normally salmon in color. A color change to orange means the cultures need to be fed. If the color turns yellow, there is too much lactic acid in the medium and the medium needs to be changed ASAP before the cultures die. If you want to have your cultures proliferate, add PDGF-BB in nanomolar quantities (titrate for desired proliferation rate). The PDGF-BB has only a single inductive activity, and that is to stimulate cellular proliferation. You want to maintain a proliferation rate of greater than once every 12-14 hours. Less time than that and the DNA-checking enzymes do not have enough time to ensure that there are no mutations in the cell's genome that will lead to permanent mutations, i.e., turn off the p53 gatekeeper gene, that would allowed uncontrolled cellular proliferation.
Dear Henry,
A tip: X-VIVO from Lonza, Belgium. This is a GMP-medium useful for many different cell types including DC, Mph, T cells, tumor cell lines and PBMC. This medium can be used without any serum components or plasma or glutamine. We use this medium since many years for culture of different cells including differentiation of very sensitive naïve cord blood-derived T cells and CD34+ stem cells and monocyte-derived DC cultures for clinical use.
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