Life Or Death Decisions: How To Choose A Cytotoxicity Assay

There are many things that can make these cultures sick—an imbalance in the salt, temperature, or pH of its growth medium, chemical toxins, secreted waste products, signals transmitted from other cells (by contact or secreted messengers), mutations, contamination, radiation, etc. Thus a culture’s health can be used as a barometer of the conditions under which the cells thrive, and of those that inhibit their vitality.

One of the big pushes in cytotoxicity testing is in the area of drug development, and in the cosmetics and detergent industries, notes Frank Barile, associate professor of pharmacology at St. John’s University in Jamaica, New York. “By performing these assays, the impetus in the field seems to be moving away from more expensive animal-based toxicity testing… It’s taken a long time for industry to realize the potential of using in vitro assays as pre-clinical testing.” The academic world, on the other hand, has been developing and using in vitro assays for long time, Barile says, mostly for supplementing or replacing animal testing.

Cytotoxicity testing can also yield information about the functions of different genes, for example, or the effect of a variety of treatments—ranging from radiation to a seemingly benign dye or peptide, to industrial waste products introduced into the environment—that may affect the health of a culture. Such determinations can be as simple as viewing the cultures under a microscope, or as complex as monitoring radioactive metabolic by-products chromatographically separated from culture supernatant.

A typical kit might measure the presence of intracellular enzymes such as lactate dehydrogenase (LDH), adenylate kinase (AK), or glucose 6-phosphate dehydrogenase (G6PD) in the culture supernatant. When cell membranes are compromised they become porous and allow these stable macromolecules to leak out and be quantitated using a variety of fluorescent, luminescent, and colorimetric assays. Similar assays pre-load cells with a radioactive (51Cr) or non-radioactive substance (usually an ester that is cleaved to a non-membrane-permeable product), and then measure the amount released into the supernatant upon loss of membrane integrity (such assays are often used in cell-mediated cytotoxicity assays). Other viability assays being used to measure cytotoxicity rely on the fact that adherent cells generally let go of their plastic substrate when they die—dead cells are washed away, and the remaining cells are counted or otherwise quantitated.

Roche Applied Science’s Apoptosis, Cell Death and Cell Proliferation, 3rd edition, guides researchers interested in cytotoxicity through a flow chart to DNA fragmentation kits—which measure apoptosis—and proliferation assays, along with an LDH detection kit. This kind of advice is not atypical, nor is it necessarily off base. Terry Riss, senior product manager for cellular analysis at Promega estimates that more than half of those using proliferation assays are actually using them to detect cytotoxicity. “Typically, they’ll start with a relatively large number of cells … treat with a compound, and try to determine after a 24 or 28 hour exposure period how many of those cells are left.” Compare that to a no-treatment control and a positive control (using a known toxin), he continues, and they can see the relative effect of their different compounds.

For researchers interested in looking at inhibited function of viable cells rather than death, though, there are several options.

Similarly, cancer cell lines—from any origin—are frequently used because of their ease of manipulation in culture, he continues. They retain at least some of their metabolic activity and differentiated features, and are “very useful in identifying different properties of the cells without the annoying problem of aging.”

Other cell lines’ properties may make them useful for some tests, but unsuitable for others. An immunologist might look at the ability of a cytotoxic T cell to kill other cells in response to antigen, for example, while a neurobiologist can measure a neuron’s ability to propagate an action potential. And in general, depending upon the receptors a particular cell lineage may display on their surface, a culture’s response to its environment may vary greatly. Thus while certain assays making use of linage-specific factors may be a useful indication of that culture’s health, they often cannot be translated into a general cellular assay. Other, more ubiquitous ways to assess the effects of compounds and environmental changes should be employed.