What is a Cell Toxicity Assay?
A cell toxicity assay is an in vitro bioanalytical test that assesses the adverse effects of a substance or treatment on cells. In this test, cells are exposed to the substance of interest, and after incubation, the number of viable cells is determined. Cell cytotoxicity assays help develop therapies and rely on biological parameters such as cell viability, proliferation, and function. In drug discovery, cell toxicity assay is a key tool that determines the fate of potential therapeutic candidates.
Importance of Cell Toxicity Assay in Cancer
In cancer research, Cell Toxicity Assays are employed for cytotoxicity screening of chemotherapeutic agents and determining their effectiveness in killing cancer cells. Screening potential anti-cancer molecules is a crucial path toward discovering new anti-cancer therapeutics. It helps assess drug dosage and safety. These assays also help evaluate drug resistance in cancer cells and understand why and how some cancer cells develop resistance to anti-cancer therapies. This understanding is essential in developing strategies to overcome this resistance when developing anti-cancer therapeutics. Thus, cell cytotoxicity assays play a key role in developing targeted cancer therapies that target cancer cells with high specificity, causing minimal damage to normal cells.Â
Based on the method employed and biological endpoints, cell toxicity assays may be of different types, including enzymatic cytotoxicity assays, viability assays, apoptosis assays, autophagy assays, and cell proliferation assays. Enzymatic cytotoxicity assays include LDH release assay and glutamate dehydrogenase release assay. These assays help to assess membrane integrity and damage. Viability assays include MTT/MTS/XTT assay and trypan blue exclusion test. They help to determine the viability of cells and the loss of membrane integrity. Apoptosis assays include the caspase activity assay, the annexin V assay, and the TUNEL assay. These assays help assess the cell apoptosis based on different apoptosis markers. Autophagy assays include the LC3-II conversion assay and the P62/SQSTM1 accumulation assay. They help determine the autophagic activity of the cells. Lastly, cell proliferation assays include BrdU Incorporation and Ki-67 staining assays that help evaluate the growth and division of cells, a key marker enabling distinction between normal and pathological states. The researcher needs to select the appropriate cytotoxicity assay based on the specificity and sensitivity of the assay, mechanism of detection, properties of the test molecule, and availability of associated techniques in the laboratory.
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The cell toxicity assays are routinely used in cancer research as part of preclinical trials. As opposed to animal models, determining cell toxicity using in vitro cell culture has been a preferred choice among researchers. The high specificity and sensitivity of cell toxicity assay are some of the key reasons. These assays employ diverse readout techniques, including colorimetry and fluorimetry.
Advancements in Cell Toxicity Assay
Cell toxicity assays are crucial to drug development, including cancer research. Classical assays, frequently based on labeling of live/dead cells, were time-consuming and required the expertise of highly skilled professionals. Modern trends in assay technology highlight the transformation in screening capacity, accuracy, sensitivity at a single-cell resolution, and automation to minimize user intervention. Microfabrication and surface engineering technologies have fostered a paradigm shift in assay technology, including cell cytotoxicity assays.Â
The latest advancements in this area include high-throughput screening technologies, multiplex assays, 3D cell culture models, and new assays. High-throughput technologies allow researchers to determine the cytotoxic effect of several molecules of interest on different cell lines. This technology not only screens several target molecules rapidly but also has high accuracy and efficiency. Some assays incorporating high-throughput technology are the MTT assay, cell titer glow assay, plate reader assay, and flow cytometry assay. Multiplexing capabilities allow the simultaneous detection of multiple aspects of cell viability and toxicity within the same environment. This ability eliminates the need to run several separate assays, thus minimizing time and cost. Multiplexed cell cytotoxicity assays are comprehensive and efficient, have reduced sample requirements, and have improved data interpretation. Some Multiplex Assay Services available include the MultiTox-Glo Multiplex Cytotoxicity assay, the MultiTox-Fluor-Multiplex Cytotoxicity assay, and the Live Cell Multiplex assay. Next, the inclusion of 3D cell culture models helps better mimic the physiological conditions of the cells within the body and thus helps to generate more biologically relevant data, which in turn offers an improved prediction of drug efficacy and cell toxicity in the body. Developing new assays with high sensitivity, such as real-time assays, flow cytometry, LDH assay, and trypan blue exclusion assay, provides real-time insight into cellular processes and information at a single cell resolution, which was previously difficult to obtain.Â
Conclusion
The cornerstone of cancer research involves the development of anti-cancer therapeutics that can eliminate cancerous cells, causing minimal damage to healthy cells and tissues. Cell cytotoxicity assays help to determine the capability of potential anti-cancer drug molecules to kill cancer cells and the damage they cause to the healthy cell population. Cell toxicity assays are of different types, with each offering unique insight into different cell processes. With technological advancements, cell cytotoxicity assays and associated techniques have come a long way, providing real-time information at an unparalleled resolution.
