It is quite possible that the cells respond to a treatment differently depending from the cell confluence. Whether the cells have free space to grow or are in contact with each other is having a tremendous effect on their cell signaling cascades and gene expression patterns. Typical example is the well-known contact inhibition phenomenon:

http://en.wikipedia.org/wiki/Contact_inhibition

Consequently the sensitivity to pharmacological treatments could be also dramatically different depending on cell confluency. Well known example in this respect is some cancer chemotherapy agents that selectively kill actively dividing cells (sub-confluent cell culture conditions) while sparing cells that are not dividing (confluent cell culture conditions). Since the gene expression patterns of growing and cell cycle arrested cells are different, this can also result into selective expression of membrane transporters, metabolizing enzymes, or binding proteins that could affect your pharmacological treatment. On the other side: you can also have drugs that are having very similar effect in confluent as well as in sub-confluent conditions. One just needs to make experiments to find out if particular chemical treatment is affected from cell number conditions or not. Anyway, to assure a good reproducibility of the experimental data I can highly recommend always using the same cell confluency conditions.

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It depends. Generally you can use the same concentration of the drug. 

The only way to answer your question is to test it. Since drugs and small chemical inhibitors all work differently in different cell types, your best bet is to try and use the same concentration in a larger density of cells. I don't imagine that the cell density should impact the effect of the drug. I usually place larger density of cells into more media, therefore the amount of chemical inhibitor will increase in order to maintain the same concentration. I would recommend plating both cell densities and testing the same concentration, and then measure your out-put (p53 expression). Good luck

It depends of the type of the drug. If it is a competitor for a membrane transporter or for an intracellular enzyme, the concentration could vary in function of the cell number depending on the expression of transporter or the enzyme.  There are some example of drug for which is known that the activity id cell number -dependent. So try different concentration on the same number of the cells and then try the best concentration for your assay with different number of the cells. Be sure to have a good assay as readout of your drug effect

bye

It is quite possible that the cells respond to a treatment differently depending from the cell confluence. Whether the cells have free space to grow or are in contact with each other is having a tremendous effect on their cell signaling cascades and gene expression patterns. Typical example is the well-known contact inhibition phenomenon:

http://en.wikipedia.org/wiki/Contact_inhibition

Consequently the sensitivity to pharmacological treatments could be also dramatically different depending on cell confluency. Well known example in this respect is some cancer chemotherapy agents that selectively kill actively dividing cells (sub-confluent cell culture conditions) while sparing cells that are not dividing (confluent cell culture conditions). Since the gene expression patterns of growing and cell cycle arrested cells are different, this can also result into selective expression of membrane transporters, metabolizing enzymes, or binding proteins that could affect your pharmacological treatment. On the other side: you can also have drugs that are having very similar effect in confluent as well as in sub-confluent conditions. One just needs to make experiments to find out if particular chemical treatment is affected from cell number conditions or not. Anyway, to assure a good reproducibility of the experimental data I can highly recommend always using the same cell confluency conditions.

--

http://about.me/Atanas_At

"Inoculum effect" leading to decrease of potency of a drug when cell density increases do exist particularly with antibacterials (beta-lactams for instance). MICs are always measured using a standardized amount of bacteria (5.10^5 cells / mL) to be able to compare results from different cpds / laboratory. The "inoculum effect" also happen with antiviral cpds. Therefore, since this phenomenon is drug class dependent, test  your cpd at different cell density: 5.10^5, 1^106, 5^106, 1^107 to have an idea.

Before 1998, as part of my "Habilitation" on mechanisms of P-Gp related MDR at University of Hamburg, I had a systematic experimental look into the problem. this was not published in English, since it was preparatory work. The main findings, as i can recall from memory:

Methods: Transport substrates doxorubicin, daunorubicin, ciclosporin, and dexniguldipine (a then hopeful mdr-modulator). Cells used: F4-6 mouse erythroleukemia, gastric carcinoma (Dietel) and maybe others in their usual incubation medium (5*10^5 - 1*1^07) at multiple dfug concentrations. Details of methods are published. Let me know if you want to repeat any experiments. [email protected]

Findings:

1. With substrates that accumulate to a high degree inside cells (all of the above), the actual intracellular content is a direct, nonlinear function of the inverse of cell density. The effect is seen over a wide range of substrate concentrations. Some compounds bind extensively to Eppendorf vials, so precautions need to be taken.

2. Accordingly, the actual concentration gradient between medium and cells increases according to a similar function. Lower cell density = higher concentration gradient.

3. Basically the same relationship is found in mdr-positive cells, albeit on a lesser slope. Again, the effect of the mdr exporter (in lowering the ic concn expressed in % of non-resistant) gets more impressive at lower cell density.

Some original "concentration-response curves" can be found as hardcopy at "Staatsbibliothek Hamburg". I hope this is helpful for concept building.

This depends also on the type of culture. We have seen that density of peripheral blood lymphocytes is of great importance for the response to the same concentrations of immunomodulatory molecules. We think that T- cells respond to the molecules first but then depending on their activity there is (high cell density) or there is not (low cell density) further response by B-cells.

It depends on the cell type, normal or transformed. Neoplastic (cancer) transformed cells or normal fibroblasts produce different growth factors into the medium very often. In conditions of high cell density the concentration of this type of molecules will be more, then in low cell density, so that the result could be different. 

From our hands-on experience using adherent tumor cell lines or primary tumour cells treated with anti-tumoral drugs, the effect depended on the cell density. Moreover the effect dependent whether the cells were seed and adhered at a certain density or were let to grow reaching to the same density. The microenvironment in the mentioned cases were  different, hence a different effect of the same concentration drug.

Hi,

Cell density almost always has an effect on drug outcome. There are many reasons for that. Cell cycle (time and phase), drug uptake ability, media metabolism, intracellular signaling such as contact inhibition and etc., 

There is no way to go about it except making sure that the start point of experiments is consistent.

Cheers

P

Some agents, e.g., methyl mercury, have effects that depend on both drug and cell concentration. This is often because the drug concentration, unless in gross excess, is reduced by the cells and more cells can metabolize more drug, reducing the concentration other see. Hence, it is critical to keep cell concentration constant as drug concentration is varied, and drug concentration as cell concentration is varied. This lets one plot a 3D plot of drug vs cell concentration vs (z-axis) biological endpoint.

Another problem is the level of endogenous drug/agent, e.g., erythropoietin, when assaying the effect of the same agent given exogenously and the effect follows a sigmoid response curve. This is because if the endogenous level is high, the maximum effect occurs at a lower exogenous dose than when the endogenous level is low. This can lead to a false conclusion that the agent has little or no effect, when in fact the effect was saturated by the endogenous activity. JW Byron demonstrated this in the 1960s, showing that most of the early erythropoietin studies were invalid due to this effect.

In your example there are two main aspects to consider: the dose of the drug administered and the behavior of the cells in sub-confluent or confluent cultures. Talking about the dose, 10 uM of drug administered to  5 x 10^5 cells means the same number of drug molecules for 2,5 x more cells than 2 x 10^5. You have to check several concentrations to find the optimal dose for the desired effect. 

It is not good scientific practice to simultaneously investigate the effects of two variables (drug dose and cell density) on a pharmacological effect. In laboratory in vitro studies on pharmacological effects of drugs, as opposed to pharmacotherapeutic efficacy studies in natural settings, it is possible and desirable to control for the potential effect of different variables on a specific response. You should keep one of the two variables constant while investigating effect of the other. Eg determine the dose-dependent effect of the drug on a response (here p53 expression for example) on cultures with different cell densities in separate experiments or if you wish to work with one specific dose of drug, then investigate its effect on p53 expression in cell cultures of different densities.

Sharmila, I believe that we are saying the same thing. One should keep drug concentration constant while measuring the effect of cell density and one should keep cell density constant while measuring drug dose effects. However, at some point one may wish to have a map of the interactions that become visible on the 3D plot of all of these data,

Christofer, you are right.

I also had the same experience while I was using taxol and platinum compounds. It seems the drug e effectiveness tends to be lower  at more confluent  condition. 

No question that cell density is a key variable in cell culture experiments.  Can anyone predict if your drug's effect will be influenced by cell density?  Absolutely not!!! You just need to do the experiment.  If you have seen this effect and are just asking if it makes sense:  Absolutely. I think Peter gave a good list of some of the variables that are influenced by cell density. 

One thing you need to remember is that cell culture is a tool that should be tailored to the questions you are trying to answer. For example, you should use a cell concentration that provides a state of proliferation and differentiation that is relevant to the in vivo situation if you are trying to develop a pharmacological agent for medical applications.  If you are pursuing a more mechanistic goal, then you might want to titter cell density to see the maximal response.  If you are trying to compare it to an other compound, then the impact of cell densities on this other compound's effects should also be tested to insure that you are not creating a favorable bias for your compound.

Absolutely correct! We have to try several concentrations of cells and drugs in our culture experiments every time, when we set up new group of experiments. These two preliminary series of titrations are useful to make a choice.  

It is always better to maintain the cell density same for your study. It is often visible that in lower cell density the anticancer substance show more effect in quick time and tends to follow the apoptotic pathway. In that case it may confuse you.

Yes, in low cell density the anticancer drugs are working stronger, but this is a good way preliminary to see the effect and compare different drugs. Sure, you have to check the effects in conditions of medium confluency and keep the same conditions, when you repeat your experiments.

Cell density can definitely affect cell response. Changes in cell density can change cell decision-making processes. We have studied the response of cells to ionizing radiation. In many cell lines (MCF10A, HEK293, NIH3T3, U2OS, Hep3B, and others) sparsely plated cells undergo apoptosis in response to IR, whereas in densely plated cells apoptosis is inhibited. We found that this is at least partly due to the Hippo pathway, which is activated at high cell density, and can inhibit apoptosis at high cell density. See http://www.ncbi.nlm.nih.gov/pubmed/23852372

Nina, thanks for your good ref.

With ionizing radiation, cell density is important if it leads to cell:cell contact (gap junctions) and thus the "Bystander Effect" in which cells that neighbor those that have been irradiated (i.e., have had a photon or particle deposit energy in them) can also lose the ability to form clones. This is even more evident in 3D cultures than in 2D cultures (more contacts). Drugs that block gap junctions (lindane, octanol) remove this effect. A similar "Bystander Effect" is also seen in some irradiated cell lines but it works by the release of compounds (cytokines?) into the medium and then that medium can be added to unirradiated cultures, shutting down their ability to form clones. Cell density is especially important for the latter mechanism. These mechanisms are separate from those Nina Reuven describes above.