Cell Viability Assay: Essential Methods for Measuring Living Cell Populations
Understanding Cell Viability Fundamentals
Cell viability is defined as the percentage of live, healthy cells within a cell population. This critical parameter is used in biological research to evaluate cellular population, its functionality, and response to treatments.
Principles and Mechanisms of MTT Cell Viability Assays
Metabolic Activity
Cell viability assays are based on the metabolic activity of living cells and detect specific enzymes that are only functional in living cells. These enzymes convert specific non-toxic substrates into detectable products. Various readout methods are used to measure cell viability:
- Colorimetric assay (e.g., MTT cell viability assay) assesses cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes reduce the yellow tetrazolium dye MTT to its insoluble formazan, which has a purple color and is measured by absorbance. The intensity of the color correlates with the number of living cells.
- Fluorescence-based assays use non-fluorescent substrates that enzymes can convert into fluorescent products, which can then be detected using a fluorescence plate reader or microscope. For example, resazurin is reduced to the bright red-fluorescent compound resorufin in viable cells.
- Luminescence-based assays measure ATP levels by using an enzymatic process to convert substrates into products that emit light.
ATP Production
Adenosine triphosphate (ATP) is a nucleoside triphosphate that provides energy to drive and supports many processes in living cells. ATP can be produced only in living cells.
ATP-based cell viability assays rely on luciferase enzymes that react with ATP to produce light. The intensity of the converted light is directly proportional to the number of viable cells in the sample. Luminescence-based cell viability assays provide a rapid, highly sensitive, and quantitative assessment of cell viability and metabolic activity in a wide range of biological applications, such as drug screening and cytotoxicity testing, high-throughput cell viability analysis in multi-well formats.
Membrane Integrity
Membrane integrity is a key indicator of cell viability. In viable cells, the plasma membrane is intact and does not allow cell viability dyes to enter. When cells die, their plasma membranes become permeable, allowing dyes to enter the cell. Trypan blue, a non-fluorescent dye, selectively enters cells with compromised membranes and is excluded by intact cell membranes. Dead cells appear blue under a microscope. Similarly, the fluorescent dye propidium iodide (PI) cannot cross intact membranes. However, in dead or dying cells with compromised membranes, PI enters through the damaged membrane and binds to DNA, emitting red fluorescence.
DNA Synthesis
DNA synthesis is an indicator of cell proliferation and S-phase activity. BrdU (Bromodeoxyuridine) and EdU (5-ethynyl-2′-deoxyuridine) assays are widely used in DNA synthesis assays to monitor the incorporation of synthetic nucleoside analogs into newly replicating DNA.
BrdU is a thymidine analog that incorporates into newly synthesizing DNA during the S-phase. Incorporated BrdU can be detected to identify cell proliferation. Detection of incorporated BrdU requires DNA denaturation to expose the BrdU epitopes for anti-BrdU antibody binding. The BrdU assay is well-established, extensively validated and is compatible with co-staining for other markers. A disadvantage of this method is that the harsh denaturation can damage cellular structures and affect other epitopes.
EdU is a thymidine analogue that incorporates into the newly synthesized DNA of dividing cells during active DNA replication. It can be detected via a copper-catalyzed click chemistry reaction using fluorescent azide. Unlike the BrdU assay, DNA denaturation is not required for the EdU assay. This preserves cell structure, reduces the harshness for cells and saves assay time.
Applications of Cell Viability Assay
Cell Viability Assay in Pharmaceutical Research and Drug Discovery
Cell Viability Assay are widely used to screen potential drug candidates, determine their cytotoxicity and optimal dosage, and identify drugs that effectively inhibit diseased cells (e.g. cancer cells) while minimizing harm to healthy cells.
Cell Viability Assay in Toxicology and Safety Assessment
Cell viability testing is an important assay to evaluate the toxic effects of chemicals and environmental contaminants on living cells, as well as the concentrations at which these effects occur. These assays are used in toxicology and safety assessments to evaluate the toxic effects of chemicals or environmental contaminants, determine safe exposure levels, support regulatory assessments and risk evaluations, and screen for potential therapeutic or hazardous components to protect public health.
Cell Viability Assay in Cell-Based Therapies and Medicine
In stem cell research and the development of cellular therapies, cell viability assays ensure the safety, potency and functionality of the therapeutic cells. Cell viability assays are used for quality control and to confirm the health and functionality of therapeutic cells prior to their use and to ensure they exert their therapeutic effects after transplantation. These assays make stem cell and cellular therapies and regenerative medicine more reliable, effective, and safer.
Future Directions in Cell Viability Assay
Emerging Technologies and Novel Biomarkers
Innovative approaches such as microfluidic devices and 3D cell culture models provide more accurate and precise tools for assessing cell viability in response to drugs, toxins, and environmental conditions. Microfluidic devices are platforms that enable precise control of the cellular microenvironment, including nutrient flow, oxygen gradients and shear stress, in dynamic, in vivo-like conditions. 3D cell culture models mimic the architecture and function of tissues. These techniques provide more realistic data on cell viability, cell survival, drug responses, and toxicity compared to flat 2D cultures.
These innovative approaches improve cell viability evaluation in drug development, toxicology, and regenerative medicine.
Integration with Other Cell Analysis Assays
Cell viability assays have been integrated into multimodal approaches, combining with additional cellular measurements such as apoptosis, proliferation, metabolic activity and gene expression. These approaches offer a more comprehensive understanding of cell health and function. They allow cytotoxic and cytostatic effects to be differentiated and early stress responses to be detected. Combining cell viability assays with additional cellular measurements provides a more complex interpretation of cellular responses in drug screening, toxicology and the development of cell-based therapies.
Modern powerful tools like AI and machine learning (ML) offer the automation of cell viability analysis and enhance data interpretation. These technologies can:
- Rapidly process large, complex datasets.
- Identify patterns and correlations across multiple cellular parameters
- Reduce human error and variability.
- Create predictive models of drug responses and toxicity outcomes.
AI and ML are transforming cell viability testing into a more efficient and insightful process for research and drug development by increasing speed, consistency, and analytical depth.
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