Cell viability measures the proportion of live, healthy cells within a population, reflecting their ability to remain functional. This measurement is fundamental in biological research because cell health directly influences the reliability of experimental results. Determining whether cells are alive, dying, or dead is the initial step in countless studies, from routine cell culture maintenance to complex drug development. A viable cell is physically intact, metabolically active, and capable of future division. Scientists employ various techniques to assess these different facets of a cell’s overall health.
Checking Cell Membrane Integrity
The most direct assessment of cell viability focuses on the integrity of the plasma membrane, which acts as the cell’s physical barrier. A healthy cell maintains a tightly regulated, impermeable membrane that controls the passage of substances. When a cell dies, this selective barrier breaks down, becoming porous and allowing large molecules to pass freely.
The Trypan Blue dye exclusion test is a standard method that exploits this principle to distinguish between live and dead cells. Trypan Blue is a negatively charged dye normally excluded from the cytoplasm of viable cells with intact membranes. Non-viable cells, having compromised membranes, allow the dye to enter, staining the cell body a distinctive blue color.
Researchers mix a cell suspension with the dye and manually count the cells using a hemocytometer under a microscope. Cells that remain clear are counted as viable, while the blue-stained cells are counted as non-viable. This provides a quick percentage of live cells, offering an immediate snapshot of the population’s physical health.
Assessing Cellular Metabolism
While membrane integrity provides a quick physical check, assessing cell health requires measuring internal functional activity, or metabolism. Metabolic assays gauge processes like enzyme activity or respiration, which are ongoing signs of life. A common approach involves colorimetric assays that rely on metabolically active cells to chemically transform a colorless substrate into a colored product.
Tetrazolium salts, such as MTT and XTT, are frequently used in these tests. Viable cells contain active mitochondrial dehydrogenase enzymes, which are part of the cell’s energy-producing machinery. These enzymes interact with the tetrazolium compound and reduce it.
In the MTT assay, the yellow tetrazolium salt is reduced inside the cell to formazan, an insoluble purple crystal product. The crystals must be chemically dissolved using a solvent before the intensity of the purple color can be measured using a spectrophotometer. The resulting color intensity is directly proportional to the number of metabolically active cells.
The XTT assay operates on a similar principle but is often preferred for convenience. The XTT salt is reduced by active cells to an orange-colored, water-soluble formazan. This product can be measured directly without a separate solubilization step, simplifying the procedure and making it suitable for high-throughput screening.
Monitoring Cell Growth and Division
Cell viability involves not only immediate survival but also the ability to thrive over time, reflected in the cell’s potential to proliferate. A cell population that cannot grow and divide is considered compromised, even if individual cells are currently alive. Monitoring cell growth and division provides an indication of long-term health and reproductive fitness.
The simplest method involves direct cell counting over several days to track changes in population number. This can be done manually using a hemocytometer, or with automated cell counters that often incorporate a dye exclusion test to count only viable cells. The resulting growth curve reveals the population doubling time and its responsiveness to the culture environment.
More sophisticated techniques measure DNA synthesis, a required step before a cell can divide. Assays using molecules like Bromodeoxyuridine (BrdU) incorporate this synthetic nucleoside analog into the newly synthesized DNA of dividing cells. The incorporated BrdU is then detected using specific antibodies, marking the cells that are actively preparing for division. This approach measures the cell’s potential to proliferate.
Why Viability Testing is Essential
Viability testing provides necessary context for virtually all cell-based research and applications. Without knowing the percentage of healthy, functioning cells, it is impossible to interpret experimental results accurately.
In the pharmaceutical industry, viability assays are routinely used in drug discovery and toxicology screening. Researchers screen potential new medicines to determine their cytotoxicity, ensuring that a compound kills diseased cells, such as cancer cells, without excessive harm to healthy tissues.
Viability testing also serves as a quality control measure in biotechnology, ensuring the health and potency of cells used in cell-based therapies and regenerative medicine. In fundamental biological research, these measurements are necessary to optimize culture conditions and understand how cells respond to environmental changes or specific stimuli.