Cell Viability Assays for 3D Cell Culture

One of the most common assays used with in vitro models is an assessment of total ATP which is a broad method for evaluation how a compound or large molecule treatment effect cells’ metabolic pathway. ATP is the molecule that provides cells with energy and its quantity within cells is directly correlated with the overall metabolic health of cells. Therefore, measuring ATP with a direct luminescence assay is a common approach for quantifying the overall health or cells within an in vitro assay.

At Visikol we routinely assess 2D and 3D cell culture models for ATP content after dosing with compounds and antibodies of interest by using Celltiter-Glo in our cell viability assays. The application of  Celltiter-Glo reagents to cell culture models lyse the cells and subsequently creates a luminescent signal that is proportional to the amount of ATP present. This signal can be detected rapidly using a plate reader and then a standard curve can be used to quantitatively determine the amount of ATP present. A total ATP assay helps quantify the metabolic functionality of cells within a culture. While a total ATP assay is rapid and inexpensive, it only provides a single data point from a well and does not provide spatial or contextual information in more complex 3D cell culture models (e.g. organoids, microtissues, spheroids) such as which cells are metabolically healthy (i.e. stromal vs tumoral) or where those cells are located (i.e. exterior vs interior). Therefore, the use of this assay depends on the type of data that a researcher wants to get from a cell culture model and if a single end point is acceptable or if complex high content confocal imaging is required.

An alternative assay Visikol provides is a Realtime-Glo assay which targets a cell’s reduction potential. Realtime-Glo will only be integrated into those cells with a reduction potential, indicating which cells are viable. This assay is nonlytic and can allow for multiplexing with other cell-based assays which allows for substantially more information to be collected from each model including spatial and contextual information. Viability can be monitored using luminescence which is directly proportional to the number of living cells. When applying this assay for use with 3D cell culture models, the models are fixed and labeled using immunofluorescence followed by processing with Visikol Histo-MTM tissue clearing which allows for the entire model to be characterized in 3D. These assays help expedite drug discovery in the early stages and provide a useful method to quantify and visualize the efficacy of a particular compound based on a specific target such are ATP or reduction potential. If you are interested in discussing these assays in more detail as well as our cell culture models to understand which assays best meet your cost, throughput and end-point needs, reach out today.

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