Simple strategies to increase the biological significance of a cytotoxic test based on 3D cell cultures

Objectives: Three-dimensional (3D) models have greatly improved our understanding of tumor pathophysiology. Scale-up of these models is needed to obtain broader data reproducibility. Our goal was to demonstrate that spheroids heterogeneous in size and shape may respond in different ways to chemical and physical treatments. Finally, we validated a viability test capable of providing rigorous data in large spheroids (≤ 650 μm). Methods: A549 and MRC-5 spheroids were obtained using a rotatory cell culture system (RCCS) and a pellet culture system, respectively. The open-source AnaSP and ReViSP software tools were used to monitor morphological 2D and 3D parameters. For live imaging, spheroids of MRC-5 cell line were stained for DNA with Hoechst 33342 and imaged using a Lightsheet Z.1 microscope. The viability was evaluated with CellTiter-Glo® 3D Assay. Results: We selected 30 spheroids of similar volumes (0.112 ± 0.013 mm3) but of different shapes, (15 spherical (SI ≥ 0.90) and 15 non-spherical (SI < 0.90)) to analyze how different shapes influence the metabolic state of spheroids. The data obtained from CellTiter-Glo® 3D showed a significantly reduced viability of spherical spheroids with respect to the irregular-shaped group (P = 0.045). To validate the data obtained, we evaluated the viability of spheroids belonging to 5 different volumetric categories. A linear increase in the bioluminescence signal moving from spheroids of 0.025 to 0.150 mm3 in volume