Three-dimensional (3D) cell cultures mimic the in vivo microenvironment more closely than traditional 2D cultures. 3D spheroids, self-assembled aggregates of cells, contain cell-cell contacts and have nutrient, drug, and oxygen mass-transfer gradients. They also have proliferating cells at the periphery and quiescent cells within. Cells at the core of larger spheroids become necrotic, much like some tumors. These features distinguish 3D cell cultures from dispersed cell monolayers. They also impact how basic parameters, like cell viability and cytotoxicity, can be assessed. This application note presents two approaches to measuring these variables in 3D spheroids.Materials & Methods
HEK293 cells and HCT116 cells were seeded at different cell densities in 96-well Perfecta3D® Hanging Drop Plates and cultured for seven and five days, respectively. Cell viability was assessed using Cayman's Perfecta3D® Cell Viability Kit by transferring spheroids to a round-bottomed plate (included), adding reagents, incubating 3.5 hours at 37°C, and reading absorbance at 450 nm. The cytotoxicity of staurosporine (stauro) was evaluated using Cayman's Perfecta3D® LDH Cytotoxicity Assay Kit: after exposing spheroids to stauro (FC = 4 or 40 µM), 10 µl of media from each well was mixed with reaction mixture in a 96-well plate, incubated for 30 minutes at room temperature, and absorbance was read at 490 nm.Results & Discussion
HEK293 formed uniformly round 3D spheroids when grown in Perfecta3D® Hanging Drop Plates (Figure 1A). Spheroids ranged in size with smaller ones appearing translucent, while the core region of larger spheroids was opaque. This suggests a technical issue confronting spheroid analysis: probes that must be internalized by cells cannot be accurately assessed due to variability in light transmission. The Perfecta3D® Cell Viability Kit uses the cell-impermeable indicator WST-1, which is reduced to a yellow formazan product at a rate proportional to the number of viable cells. While WST-1 formazan absorbance increased with initial seeding density, the relationship in seven day spheroids was not linear (Figure 1B). Given that WST-1 is also an indicator of cell proliferation, we conclude that spheroids seeded at lower densities proliferated faster than those seeded at higher densities.
The Perfecta3D® LDH Cytotoxicity Assay Kit measures lactate dehydrogenase (LDH) released by cells that have membranes damaged due to necrosis or apoptosis. This sensitive assay requires only 10 µl of media, which can be taken from the Perfecta3D® Hanging Drop Plate without disturbing the spheroids. Stauro dose-dependently induced cytotoxicity in spheroids of HCT116 cells (Figure 1C).Conclusion
Two new kits from Cayman Chemical (www.caymanchemical.com) help researchers assess cell viability and cytotoxicity in 3D spheroids rapidly and accurately. Each kit includes a Perfecta3D® Hanging Drop Plate plus all necessary reagents.