Overview
Measurement of cell viability is an early determination in most in vitro screens of a compound's toxicity, either as a stand-alone assay or more likely as part of a high-content screen. Understanding how specific cell viability assays work, and being able to integrate the data from these measurements with other determinations of toxicity in the same concentration range on the same cells, adds considerable power to what can be learned about a drug's effects.

Assay Details
Cell viability as monitored by CV1 is a combined measurement of the number of cells present and their molecular competence. When determined as an IC₅₀ for any compound the assay provides the dose range at which toxicity, whether mitochondrial or through other reactions, overwhelms the cell. In many instances this is an additive effect of several sites of action. The assay itself involves spectrophotometric determination of the reduction of a dye at 490nm in a 96-well format. The colorimetric change from yellow to purple is due to reduction of the dye by cellular dehydrogenases found mostly inside mitochondria. In principal a compound can alter the levels, as well as the activity of dehydrogenases, and/or progressively reduce cell number by killing the cells.

For the most part cell death is the predominant event reported, but as shown in the figure below, the response of a cell to a compound can be complicated. The data shown are an IC₅₀ determination of the effect of chloramphenicol, an antibiotic known to inhibit mitochondrial biogenesis by blocking mitochondrial protein synthesis. The data were collected with HepG2 cells after growing them for 7 days in the continual presence of the drug. As shown, this drug increases “viability” at low concentrations (nanomolar range) but then inhibits it in the low micromolar range, which is the same range at which it blocks mitochondrial protein synthesis.

Figure 1. Effect of chloramphenicol on HepG2 cell growth.  EC₅₀= 0.5nM;  IC₅₀= 1.5µM.

Frequently Asked Questions

Q1. Can CV1 be performed using many different cell types?
A1. Yes, but there are some important considerations when planning experiments because different cells behave differently and overall features of the viability data depend on substrate and other growing conditions. The important take-home message is that data obtained in different cell lines are not directly comparable AND when cells are stressed by a drug they can adapt by changing metabolism, particularly energy metabolism, and this is different between cell types. Therefore multiple events can go on at once, some compensatory as shown in the example of chloramphenicol above. Nevertheless important information is obtained form this rapid and simple assay, particularly when done in the context of other assays on the same cell line.

Q2. How can cells have greater viability as measured with the screen used here than a control line grown in DMSO?
A1. Cells often adapt to drugs by increasing mitochondrial biogenesis. They may also alter other processes such as increasing free radical scavengers. An increase in mitochondrial biogenesis is evident in the data shown in Figure 2, which reports the levels of the mitochondrial enzyme cytochrome c oxidase relative to a nuclear encoded mitochondrial protein frataxin using our Biogenesis assay. The increase in COX/frataxin can be as much as 200%. Whether this is seen in the viability experiments is a function of concentration range (hence the need for an IC₅₀) and the strength of opposing effects that cause drug induced cell death. As shown, a range of compounds induce increased OXPHOS biogenesis at low concentrations, or before the number of cell divisions is sufficient for direct inhibition of biogenesis takes effect.

Figure 2. Increase in mitochondrial biogenesis after 1 population doubling in the presence of several compounds at 40µM. The up-regulation seen is maximal at this time for most compounds, the exceptions include DNP and nefazadone where the increase continues for 2 doublings (maximum up-regulation in parentheses).

Q3. Are not most cells used in drug screening transformed cells, and does this not alter cellular metabolism?
A3. Yes, this is true and it does lead to potential problems. The most significant issue is that transformed/cancer cells use glycolysis for energy rather than oxidative phophorylation. Even primary cell lines prefer glycolysis over OXPHOS when glucose is plentiful. As a result, compounds that act to inhibit one or more of the complexes of oxidative phosphorylation do not have the profound effect they would be expected to have. It is possible to overcome this problem by changing the substrate for cell growth. When cells are grown in galactose to inhibit glucose uptake, and glutamine to provide electrons from the Krebs cycle, cells grow using OXPHOS and not glycolysis. As shown in Figure 3 (below), those drugs with a direct effect on one or more of the OXPHOS complexes affect cell viability dramatically when the assay is carried out in this way but have limited effect when glucose is used as substate. Therefore it is advantageous to perform the viability assay under both substrate conditions when direct effects on OXPHOS are predicted, or have been determined by our OXPHOS enzyme assays.

Figure 3. Drugs that inhibit one or more of the OXPHOS complexes (e.g. nefazodone, which is a strong inhibitor of Complex I) are much more toxic in galactose+glutamine.