Fig. 4

Effect of culture matrix on MCTS cell viability. (A) Representative cell viability images of the CRC MCTS. Cells were stained with Cell Tracker Green CMFDA (1 µM) and seeded at 3,000 cells/well in U-bottom 96-well cell repellent plates. The concentrations of the extracellular matrices were 2.5 mg/mL methylcellulose, 2.5% Matrigel, and 25 µg/mL collagen type I. After 72 h, the MCTS were stained with DAPI to label dead cells, and pictures were taken using a Leica DM6000B microscope with 4X or 10X. (B) Cell viability quantification of the MCTS cultured in different matrices. Cells were stained at 72 h with Zombie Violet (BioLegend), and cell death was analysed by flow cytometry. Statistical analyses were done independently for every cell line, using one-way ANOVA with culture condition as explanatory variable followed by Tukey’s HSD tests for pairwise comparisons (n = 3 independent replicates per group of 12 spheroids each). No statistical significant differences were found between cell death rate in absence of matrix vs. any of the tested matrices, or between culturing the cells in 2D vs. 3D.