Choosing the Best Cell-Based Assay for Your GLP Lab Study
Selecting an appropriate analytical methodology is a fundamental aspect of GLP-compliant studies. Assay selection influences regulatory acceptance, data quality, and program success rates. Cell-based assays are robust analytical tools for evaluating drug efficacy, safety, and mechanisms of drug action. These assay systems have diverse applications, including cell proliferation assays for growth inhibition studies, cell viability assays for cytotoxicity assessments, and specialized cell-based screening assays and cell-based functional assays for mechanistic evaluations.
A strategic approach to assay selection in GLP-compliant studies ensures reliable data generation and regulatory submissions for large and Small Molecule Bioanalysis. This article discusses aspects of selecting an optimal cell-based assay based on regulatory requirements, analytical capabilities, and study objectives.
GLP validation for cell-based assays
A robust assay validation instills confidence in the accuracy and reliability of the generated data. A comprehensive validation approach for cell-based assays ensures data integrity and regulatory compliance in a GLP environment. The essential validation parameters for cell proliferation assays and cell viability assays include precision, specificity, accuracy, stability, and linearity. However, validating Cell-based Assays has unique challenges compared to conventional methods, such as matrix effects and biological variability. Hence, robust GLP-compliant documentation is necessary, including standard operating procedures, quality control specifications, and validation protocols.
Nonetheless, reliable assay performance requires establishing reference standards, qualified cell lines, and control materials. Adequate considerations are necessary for validation and regulatory submissions of small and large molecule bioanalysis. In conclusion, a rigorous validation approach ensures reliability, accuracy, and regulatory acceptance for cell-based assays.
Choosing cell-based assays
Cell-based assays help researchers study effects on more than just protein secretion or gene expression. It enables understanding complex events at a whole-cell level, such as apoptosis and cell division. Cell-based Assays can be performed on a wide range of cell types, including immortalized cell lines, primary cells, transfected or edited cells, stem cell-derived cells, and patient-derived cells. The underlying principle in all these assay formats is that they are conducted in vitro.
Live cell counting is one of the simplest cell-based assays. The primary goal of these assays is to measure cell viability. However, it is often beneficial to determine the cell count at the end of a study. Researchers may need to know the total number of live or dead cells. Understanding the way a particular cell died may be another goal of the experiment. All this information is critical to understanding different elements of cell-based assays.
Membrane integrity is a key indicator of cell viability. When a particular cell dies, its membrane leaks the internal components into the culture medium, for example, the enzyme lactate dehydrogenase. Treating lactate dehydrogenase with a specific substrate generates a quantifiable product and helps determine the number of non-viable cells in the population.
Often, studies focus on more than just live or dead cells. Experiments may be designed to understand effects on cell proliferation, apoptosis, or necrosis, or a cell’s ability to inhibit or activate a particular signaling pathway. Many of these assays are conducted through cultured cells as opposed to lysed cells. For instance, researchers add annexin V at the beginning with propidium iodide to determine necrosing and apoptosing cells. On the other hand, homogenous assay versions allow real-time monitoring without wash steps. Although researchers perform some assays sequentially or together, it is crucial to understand the underlying chemistries involved, including the readouts, to ensure compatibility.
Before choosing a cell-based assay, researchers often focus on the appropriateness of the assay systems to accurately measure the intended target. The primary aim should be to consider the end goal of an experiment. A study focused on cell viability may not necessarily measure cytotoxicity or proliferation, or the mechanism of drug action. Researchers should consider aspects of multiplexing, which may initially cost more but could provide faster results. On the other hand, considering the need for another assay may help understand the need for studying interferences.
Must Read: Cell Proliferation Assay in Drug Screening and Toxicity Testing
Some other considerations in selecting a cell-based assay include readout and optimization. Identifying the readout type early on has multiple advantages. However, the selection depends on the intended output of the experiment. Imaging generally requires extensive equipment, setup, and analysis, but can generate relatively more data. On the other hand, researchers may be more focused on speed. Here, one may choose the speed and sensitivity of luminescence detection, the frugality of the colorimetric technique, or the multiplexing and localization ability of the fluorescence method. Importantly, some experiments may need the minimization of assay steps. In this case, scientists can opt for a homogeneous assay.
Hence, choosing a cell-based assay has several considerations. Researchers may use the in vitro cell-based assays section of the National Library of Medicine’s Assay Guidance Manual for learning more about the planning and conduct of cell-based assays.
Conclusion
An informed assay selection is critical for successful toxicological investigations and optimizing GLP laboratory operations. However, validated cell-based assay platforms are essential for supporting drug development decisions and regulatory submissions. A comprehensive understanding of cell proliferation assays and cell viability assays enhances data quality and experimental design. Hence, understanding the basics of assay selection offers competitive advantages in regulatory approvals and accelerates drug development timelines.