Cell viability refers to the proportion of living cells within a given population. This measurement indicates whether cells are alive and capable of performing their normal functions. Assessing cell viability is fundamental across scientific disciplines, providing insights into cellular health and how cells respond to different environments or treatments.
Why Measuring Cell Viability Is Important
Understanding cell viability plays a significant role in biological research and development. In drug discovery, it evaluates the potential toxicity of new compounds and determines effective drug dosages, especially for identifying treatments that target diseased cells while sparing healthy ones. Cell viability testing is also integrated into toxicology screening, where it helps assess the effects of chemicals or environmental contaminants on living cells and establish safe exposure levels.
Beyond drug development, measuring cell viability is important in cell therapy to ensure the quality and functionality of therapeutic cells before use. It is also relevant in basic biological studies and disease research, providing a measure of cellular health influenced by factors like culture conditions, pharmacological agents, or disease states.
Knowing cell culture viability is necessary for reliable experimental outcomes, as non-viable cells can significantly alter results.
Assessing Cell Membrane Integrity
Methods focusing on cell membrane integrity determine viability based on the principle that live cells maintain intact membranes, which act as barriers to certain substances. Dead or dying cells possess compromised membranes that allow these substances to enter freely. This distinction enables researchers to differentiate between viable and non-viable cells.
The Trypan Blue exclusion assay uses a dye that cannot pass through the intact membranes of living cells. Cells with damaged membranes take up the dye, appearing blue under a microscope. Researchers count the number of blue (non-viable) and colorless (viable) cells using a hemocytometer, calculating the percentage of viable cells. This visual method is a rapid way to assess cell viability.
Propidium Iodide (PI) staining utilizes a fluorescent dye excluded from live cells with intact membranes. When a cell’s membrane is compromised, PI enters and binds to DNA, emitting red fluorescence. This method is frequently used with flow cytometry, which quantifies fluorescent (dead or dying) versus non-fluorescent (viable) cells. PI staining offers a quantitative measure of viability and can also provide information about the cell cycle and DNA content.
Assays Based on Cellular Activity
Cell viability can be inferred by measuring a cell’s metabolic activity or enzyme function, as only metabolically active cells perform these processes. These assays often involve a color change or light emission proportional to the number of living cells.
The MTT assay is a colorimetric method where metabolically active cells convert a yellow tetrazolium salt, MTT, into purple formazan crystals. The insoluble formazan crystals are then dissolved, and the resulting purple color intensity is measured spectrophotometrically, with a stronger color indicating more viable cells.
Similar to MTT, the XTT assay uses a yellow tetrazolium salt that metabolically active cells reduce into an orange formazan product. An advantage of the XTT assay is that the formazan product is water-soluble, eliminating the need for a separate solubilization step. The intensity of the orange color, measured spectrophotometrically, directly correlates with the number of metabolically active cells.
The AlamarBlue assay uses a cell-permeable, non-toxic dye called resazurin. In living cells, resazurin (blue and weakly fluorescent) is reduced to resorufin (pink and highly fluorescent). This color change and increase in fluorescence intensity can be measured by absorbance or fluorescence, providing a quantitative measure of cell viability. The non-toxic nature of AlamarBlue allows for continuous monitoring of cells over time.
ATP assays measure the amount of adenosine triphosphate (ATP) present in cells. ATP is a molecule that provides energy for cellular processes and is found only in metabolically active cells. When cells lose viability, their ATP levels rapidly decrease. These assays use the enzyme luciferase, which reacts with ATP to produce light (bioluminescence); the intensity of the emitted light, measured with a luminometer, is directly proportional to the amount of ATP and the number of viable cells. ATP assays are known for their high sensitivity and rapid results.
Interpreting and Applying Viability Data
Interpreting cell viability data involves understanding the context of the experiment and the specific characteristics of the cells. The percentage of viable cells or signal readings provide a snapshot of cellular health, but various factors can influence these measurements. Cell type, for instance, can affect how cells respond to experimental conditions or specific assay reagents.
Environmental factors and experimental conditions, such as temperature, pH, nutrient availability, and exposure to compounds, can significantly impact cell viability. High viability readings indicate a healthy cell population, while low readings suggest cell stress, damage, or death. Researchers establish control groups to provide a baseline for comparison, helping to interpret the effects of specific treatments.
Choosing the appropriate viability assay depends on practical considerations. Factors like throughput, cost, and sensitivity play a role in method selection. Some assays are better suited for high-throughput screening, while others offer greater sensitivity for detecting subtle changes in cell health. Understanding each method’s strengths and limitations allows for accurate and reliable assessment of cell viability in diverse research and industrial applications.