Optimize Microplate Assay Protocols for Spheroid Cultures

(See a list of the products featured in this article.)

3D cell models are now increasingly being adopted for cancer, immuno-oncology, neuroscience, and other research areas because their microenvironments more closely resemble the microanatomy of in vivo systems (organized tissues, organs, and tumors), which contain a complex and dynamic set of cell types, chemical gradients, and extracellular matrix (ECM) components. The complexity of 3D cell structures can present a challenge when using cell health assays and protocols originally developed for monolayer (2D) cell cultures. Here we highlight a few basic considerations for adapting and optimizing our microplate-based 2D cell culture viability assays for spheroid cultures, focusing on the optimization of reagent concentration and incubation time.

The optimal reagent concentration for spheroid cultures will provide a high assay-specific signal with the lowest possible nonspecific background, resulting in a high signal-to-noise (S/N) ratio. An assay with a high S/N ratio shows greater sensitivity for detecting changes in cell health due to cell treatments, such as exposure to drugs or test compounds. To demonstrate this effect, we assayed spheroids derived from human lung epithelial cells (A549 cells) using the Invitrogen CyQUANT XTT Cell Viability Assay, a colorimetric microplate assay originally developed to assess 2D cell viability as a function of redox potential (Figure 1). We compared the recommended reagent concentration (1X) with a 2X concentration, in the absence or presence of increasing concentrations of the cytotoxic drug gambogic acid, and found that doubling the reagent concentration increased the S/N ratio, and thus the assay sensitivity. We recommend testing a wide range of reagent concentrations to find the most appropriate one for your 3D assay.

The optimal reagent incubation time for spheroid cultures can be determined by running a time-course experiment and plotting assay signal as a function of incubation time. Figure 2 shows an example of such a time-course experiment using an established microplate viability assay that determines redox potential by measuring the reduction of resazurin to the highly fluorescent (and intensely colored) resorufin. Using the Invitrogen PrestoBlue HS Cell Viability Reagent with various incubation times, we compared the fluorescence generated by A549 cells in a 2D monolayer with that of A549 cell–derived spheroids. The assay protocol recommends incubation times for 2D cell culture of 10 minutes to 3 hours, within the linear range of the time-course curve. Based on our experiments with spheroids, we recommend extending the incubation time—to between 5 and 10 hours—to maximize signal and stay within the linear range of this curve.