June 15, 2014 (Vol. 34, No. 12)

Reagent Penetration Key to Interrogating Cells in the Center of Spheroids

There is growing use of three-dimensional (3D) cell culture models to attempt to reproduce cell-to-cell and cell-to-matrix interactions occurring in vivo. The goal is to create model systems for experimentation that more closely predict what happens in living organisms. It has been appreciated for decades that growing cells in 3D arrangements enables expression of differentiated phenotypes not possible in cultures of cells grown on a plastic surface.

Although creating cultures of cells in 3D arrangements is more complex, the desire to use physiologically relevant model systems to better predict in vivo biology justifies the increased investment of using more complex systems. There is a wide variety of approaches that have been developed to support creation of 3D cultures of cells. The approaches can be generally classified as those that provide a scaffold to support the 3D arrangement of cells and scaffold-free approaches. Each approach can offer specific advantages or present disadvantages depending on the particular application.

One feature in common with all 3D culture models is the increase in thickness of cell layers compared to cells grown as a single layer on a plastic surface. Most commercial cell-based assays were developed using two-dimensional (2D) monolayers of cells and have not been validated for use on various 3D cell culture models. A major problem is effective lysis of all cells in large 3D spheroids.

There is an unmet need for assays validated for use with 3D culture models. To address that need, the first approach we took for validating assays was to combine the hanging drop method to generate 3D spheroids and the ATP assay to measure cell viability. The hanging drop method forms 3D spheroids of cells at the liquid-air interface of a suspended drop of cell culture medium.

Spheroids can be generated using a 96- or 384-well arrayed format. Their size can be controlled by the number of cells added and is very reproducible well-to-well. The reproducible size of multiple replicates of spheroids was important to our work to determine how size affects the performance of different assays.

Our initial experiments included a direct comparison of ATP assay reagent from two commercial sources. To visualize the lytic process, we combined a DNA binding dye (CellTox™ Green Cytotoxicity Assay) with the ATP detection reagent. The DNA binding dye is not permeable to live cells, but becomes fluorescent when it binds to DNA in cells that have lost membrane integrity. We could view spheroids treated with that combination of reagents to see which cells became stained after membrane disruption. As expected, we observed a substantial difference in the lytic capacity of different commercial reagents and, even though the CellTiter-Glo® Assay was superior, the results suggested the need to further characterize and improve assays applied to 3D culture models.

Additional experimental work led to development of a new reagent formulation (CellTiter-Glo 3D Assay) that more effectively lyses cells in large 3D spheroids and extracts ATP. The new formulation contains a higher detergent concentration, which was enabled by the properties of a recombinant form of luciferase that is stable to harsh reagent conditions. The protocol incorporates better mixing and longer exposure to the lytic reagent.

The Table illustrates a comparison of the recovery of ATP from different sizes of 3D spheroids using different reagents. Acid extraction of samples and comparison to ATP standards was used to measure the efficiency of ATP extraction. We also have confirmed improved performance of the new CellTiter-Glo 3D Assay with other models that use hydrogel and inert scaffolds.

HCT116 cells were cultured in the InSphero GravityPLUS™ 3D Cell Culture system for four days to form a range of spheroid sizes. Spheroids were assayed by adding an equal volume of the indicated reagent, shaking for five minutes, and recording luminescence after 30 minutes. The amount of ATP per spheroid was calculated by comparing the luminescence to ATP standard curves.

Protocol Modification as an Alternative to Reformulating Reagents

Reformulating reagents to contain more detergent will not work for all cell-based assays. In some cases, simply modifying the assay protocol can accomplish the desired result. If the marker being measured is not stable to harsh detergent conditions, an alternate approach must be used to extract the marker from the inner cell layers of spheroids. For example, although the luminogenic caspase-3 assay reagent (Caspase-Glo® 3/7 Assay) contains the stable form of luciferase, increasing detergent concentration results in limited recovery of caspase-3 enzymatic activity.

If the marker is unstable, the logical approach to improve assay performance is to apply a form of physical disruption of the spheroid or to increase the incubation time to aid penetration of lytic reagents. Figure 1 shows improved lysis of cells by extending the period of mixing the lytic reagent using a plate shaker.

Figure 1. HCT116 cells were cultured in the InSphero GravityPLUS 3D Cell Culture system for four days to form ~330 µm diameter spheroids. Caspase-Glo® 3/7 Assay reagent containing CellTox™ Green DNA dye to indicate lysis was added and the samples shaken for 5 (left) or 30 (right) minutes. Samples were imaged using confocal laser microscopy.

Path Forward

We are continuing efforts to validate the use of other assay chemistries with 3D spheroids, including RNA extraction, glutathione content, reactive oxygen species, and expression of luciferase reporter genes (Figure 2). For example, expression of a constitutive luciferase reporter follows the pattern of ATP content over a range of spheroid sizes whereas expression of an HIF-1 driven luciferase reporter shows an increase in larger spheroids, suggesting hypoxia-induced expression in the larger structures where oxygen diffusion is limited.

Figure 2. HCT116 cells expressing NanoLuc® luciferase under a constitutive promoter (A) or an HIF-1 promoter (B) were cultured in the InSphero GravityPLUS 3D Cell Culture system for four days to form ~200–700 µm diameter spheroids. An equal volume of Nano-Glo® Reagent or CellTiter-Glo® 3D Reagent was added to each well; the plate was shaken for 10 minutes; and the luminescence was recorded after a total of 30 minutes of incubation.

Figure 2


The expanded use of 3D cell culture model systems has outpaced the development of assay methods to measure the biology in large spheroids of cells. There is a significant risk involved with assuming commercially available products developed using monolayers of cells will work efficiently with 3D cultures. There is a clear unmet need for validated assays for interrogating markers in large 3D culture models. We recommend validating assays with each 3D model system and each cell type used. The challenges of interrogating cells in the center of 3D spheroids may require reformulating reagents to have increased lytic capacity.

In situations where the desired marker cannot withstand increased detergent concentrations to achieve lysis, altering assay protocols to incorporate more vigorous physical disruption steps and/or increasing incubation time in the presence of lytic reagents are logical first steps to achieve release of markers from large 3D spheroids.

Terry Riss ([email protected]) is senior product specialist, Mike P. Valley is senior research scientist, Kevin R. Kupcho is research scientist, Chad A. Zimprich is research scientist, and Dan Lazar is senior research scientist, all at Promega. Wolfgang Moritz is head of R&D at InSphero.

Previous articleSartorius Stedim Biotech Introduces Novel Single-Use Bag Family
Next articleTop 30 Grant-Giving Disease Foundations: 2014 Edition