Although this is a logical and essential step in study design and execution, some companies do not follow this study tier regimen. They may assume that if a compound has not demonstrated toxicity in a certain model, it will not demonstrate such reaction in any model. However, different cell lines and models exhibit various sensitivity ranges and any compound, at sufficiently high concentration, may compromise cell viability. The concentration threshold at which the compound significantly affects viability is termed “cytotoxic concentration.” This means that from a certain exposure and higher, the cell will enter a mode that will significantly compromise its functionality and will eventually lead to its elimination. Dead cells do not express biomarkers; therefore, exposure to cytotoxic levels without knowing that they compromise viability will ultimately compromise study results as well.
This preliminary assay of cellular viability is termed “the range finder experiment” as it is simply utilized to establish the range of concentration at which the cells maintain their functionality and are capable of metabolism, division and proliferation. Different cell types, cultures and co-culture models exhibit various sensitivities to exposure; i.e., a viability range that has been established in a certain cell type. For example, human keratinocytes cannot be used when a different model is utilized; such as 3T3 mouse-derived fibroblasts. The implication is that prior to any study that is conducted in a model never before tested; the tested composition should be re-tested for cytotoxicity to determine the appropriate range finder to the model of interest.
All Models Have Limits
Utilizing cell cultures to study safety end-points in vitro means that the active compound tested, if soluble in the culture media, comes in close contact with the cell’s membrane. This close interaction may not happen when the compound is applied to the skin. When applied to healthy intact skin, the compound of interest will only interact with viable cells if partitioning into and through the stratum corneum, where it will be exposed to a variety of cells, mediators and conditions, and not just to one or two cell types.
Therefore, every cell culture or related model must be considered as a limited and not as an accurate clinically predictive study and should be viewed as such. Still, one can learn a great deal from cell culture studies and can attempt to analyze data obtained to form a hypothesis on the compound or formulation activity.
To establish cell viability data; scientists assume that a viable cell will express the following signs of livelihood and functionality:
- It will metabolize;
- It will divide and proliferate; and
- It will maintain its boundaries from the surrounding; therefore, its membrane integrity will be intact.
Probably the most popular assay to establish the concentration range of viability is the “MTT assay.” MTT is the abbreviation for the chemical name of the dye used in this study 3-(4,5- dimethylthiazol-2-yl) -2,5-diphenylphenyl tetrazolium bromide. This is a colorimetic assay, meaning it utilizes a method of determining the concentration of a compound that reacts to generate color with an aid of an algorithm that correlates the color strength to the compound concentration. The assay is an indication for cell metabolism and it is an objective quantitative measurement. In brief, it is based on the assumption that under certain conditions the activity of a cellular enzyme, NAD(P)H-dependent oxidoreductase, may reflect the number of viable cells present in a culture. This family of enzymes is capable of reducing the MTT dye to its insoluble purple color compound, formazan. After the formazan is formed, it is dissolved into a colored solution by adding a solubilizer such as sodium dodecyl sulfate. The absorbance of this solution at a certain wavelength (usually between 500 and 600nm) is determined spectrophotometrically and is correlated to cell viability.
An illustration on the next page outlines the basic steps of an MTT assay.
Another common way to determine cellular viability is by testing the cell’s membrane integrity. The cell membrane is a vital organelle that protects it from its environment and allows it to function at its intracellular specific conditions. In addition, it is the bridge and connection between the cell to its surrounding, a path to balanced nutrition and controlled waste management. When the cell membrane integrity is breached beyond its ability to repair, the internal cellular environment is no longer protected and the cell loses its ability to function. Compounds that will impart membrane integrity violation may therefore be cytotoxic. When the cell membrane is compromised to the extent that the cell viably is in danger, it will release to the cell media an enzyme called lactate dehydrogenase (LDH). The LDH assay is a means of measuring either the number of cells via total cytoplasmic lactate dehydrogenase (LDH) or membrane integrity as a function of the amount of cytoplasmic LDH released into the medium. This is a relatively simple and accurate method that reportedly yields reproducible results. The assay is based on the reduction of NAD by LDH. Similar to the MTT assay, the resulting reduced NAD (NADH) is converted to a tetrazolium dye. This later colored compound is measured spectrophotometrically. If the cell membrane is compromised, it will be lysed prior to assaying the medium and an increase or decrease in cell numbers results in a concomitant change in the amount of substrate converted.
Indications of Proliferation
An additional endpoint to assess cell viability is a direct measure of cell number in a culture over a time period. This kinetics of growth or decline in number of cells is a direct indication to cell proliferation or death, respectively. Live healthy cells in a culture go through mitosis and their number is elevated until they reach 100% confluence which means that the seeded surface is completely covered by the cells, no space is left for additional cells to grow as a monolayer. When observed under the microscope, scientists can compare the cell population density at the beginning of the experiment to that of later time-points. However, in order to achieve accurate quantitative measurement, the cells should be counted. This can be difficult and time consuming if conducted manually although such practice is possible. Cells are often stained with trypan blue or another dye to ease cell count and observation under the microscope.
Another means to ease the burden of cell counting is “The Countess” automated cell counter. This instrument also requires prior staining with trypan blue. The Countess reportedly produces accurate cell and viability counts and completes all calculations in 30 seconds, using as little as 5 µL of a sample. A cell viability percentage value is calculated automatically. Basically, if the cell number in the culture declines over time, it is most likely associated with cell death and if it elevated it is associated with mitosis and viability.
Cell viability is a standalone assay; as well as the basis of assessment for more complicated toxicological endpoints. For example, it is used to evaluate lethal dose in an in vitro assay. The rationale for this lies in the fact that an essential organ failure is the cause of lethality. As such, if a threshold cytotoxic concentration of compound exposure is established to cells of vital internal organs, it may be correlated to the lethal dose. Additional example in which cell viability is used to determine other endpoints are skin and eye irritation. When skin or eye tissue is irritated, it is manifested biologically as an accelerated cell death. Therefore there is a correlation between irritation and cytotoxicity, which will be detailed in future columns.
In summary, determination of cell viability is a key measurement in any cell culture study and should be conducted prior to the actual study. If the intention of the actual study is to quantify the expression of specific biological markers, it is essential that the cells are alive and functioning; otherwise no markers will be detected and as such, if no prior knowledge is established it can present a misleading or confusing alteration in data or observation to the study and be interpreted as an actual effect and not associated to cell death.
There are varieties of methods to detect cell viability. It is recommended to run at least two methods to establish the non-cytotoxic concentration range before the study of interest is conducted. Ideally, the cell viability assay is conducted, concentration range that allows maintenance of cell functionality is established and the study of choice is conducted within this range.
Dr. Nava Dayan LLC
Nava Dayan Ph.D. is the owner of Dr. Nava Dayan L.L.C, a skin science and research consultancy and serving the pharmaceutical, cosmetic, and personal care industries. She has 25 years of experience in the skin care segment, and more than 150 publication credits.