CellTiter-Glo® 3D: A Sensitive, Accurate Viability Assay for 3D Cell Cultures

Two-dimensional cell culture has long served as an invaluable tool for in vitro experiments. However, results derived from studying flat monolayers of cells adhered to plastic often do not properly reflect the in vivo situation. Various characteristics of actual tissue, such as morphology, cell-cell communication, gene expression, and biological responsiveness are much better represented by microtissues produced under 3D cell culture conditions that replicate these biological functions. Much effort has gone into the development of the variety of 3D cell culture models available, but little effort has gone into developing the reagents to effectively assay these models. There is a significant risk to data quality in assuming that assays designed for 2D monolayers will work equally well for 3D microtissues, especially considering the increased need for reagent penetration in microtissue spheroids. The CellTiter-Glo® 3D Assay is a new, single-component, liquid reagent for measuring cell viability. The assay has increased lytic capacity and is designed specifically for use with microtissues produced through 3D cell culture.

Initial studies with the classic CellTiter-Glo® Assay demonstrated effective lytic capacity with small microtissues. However, as the microtissues became larger the classic reagent under-reported the total amount of ATP present—as judged by comparison to results from a laborious trichloroacetic acid extraction procedure. Therefore, we optimized the CellTiter-Glo® Assay formulation to increase lytic capacity and generate a new, 3D-optimized bioluminescent cell viability assay. As shown in Table 1, both the classic CellTiter-Glo® and the CellTiter-Glo® 3D Assay reagents recover a similar amount of ATP from small HCT116 colon cancer spheroids (~200 µm diameter), but as the spheroids get larger (> 350 µm diameter), the CellTiter-Glo® 3D Reagent recovers 20–40% more ATP than the classic reagent. We have also observed similar increases in recovery for other cell types, including HEK293 embryonic kidney cells and HepG2 liver carcinoma cells (data not shown).

As with the classic CellTiter-Glo® Reagent, the CellTiter-Glo® 3D Reagent generates a luminescent signal that is proportional to the amount of ATP present using pure ATP (Figure 1, Panel A), and the amount of ATP is directly proportional to the number of cells present in 2D cell culture (Figure 1, Panel B). 

However, for microtissues in 3D culture, the correlation between cells seeded and luminescent signal is often not linear. For example, with hanging-drop spheroids produced using the InSphero GravityPLUS™ 3D Culture and Assay Platform (Figure 2, Panel A), effects such as contact inhibition on cell proliferation and reduced metabolic activity in the central region of large spheroids cause the relationship between seeded cell number and luminescent signal to be curvilinear (Figure 2, Panel B). However, by including CellTox™ Green Dye, a cell membrane-impermeant fluorogenic DNA-binding dye, in our lytic assay we can show that as microtissue size increases, the amount of ATP detected (luminescence, RLU) by the CellTiter-Glo® 3D Assay is proportional to the amount of DNA detected (fluorescence, RFU; Figure 2, Panel C). This confirms that the CellTiter-Glo® 3D Assay is effectively lysing cells, resulting in release of ATP, and can accurately report the number of viable cells in microtissues produced through 3D cell culture.

The CellTiter-Glo® 3D Assay is significantly brighter than a competing bioluminescent ATP detection assay for measuring cell viability (Figure 3, Panel A). As shown in Table 2, this difference is due to the recovery of considerably more ATP from both small and large microtissues by the CellTiter-Glo® 3D Reagent. This is likely due (in part) to greater preservation of the ATP because of the composition of the CellTiter-Glo® 3D Reagent, but also to a striking difference in lytic effectiveness (Figure 3, Panel B). When CellTox™ Green Dye is added to each reagent prior to addition to the spheroid samples, confocal laser fluorescent microscopy of ~300 µm HCT116 spheroids illustrates that the CellTiter-Glo® 3D Reagent penetrates almost completely through the entire spheroid thickness. In contrast, the competitor reagent only penetrates the outer third of the same-sized spheroids.

In common with many bioluminescent assays, the CellTiter-Glo® 3D Assay exhibits a signal that is orders of magnitude above background (Figure 4). In contrast, other non-lytic viability assays that measure changes in fluorescence (e.g., alamarBlue®) or absorbance (e.g., MTT) generate signals that are only modestly higher than their no-cell control signals. In addition to this enhanced sensitivity, the CellTiter-Glo® 3D Assay has a much shorter assay time. The luminescent assay can be measured in 30 minutes or less, whereas alamarBlue® and MTT assays typically require multiple-hour incubations.