Vaezzadeh, M.; Nadort, A.; Igrunkova, A.; Lee, V. S.; Di Ieva, A.; Heng, B.; Guller, A.

Accurate cell counting is essential in tissue engineering and cancer research. The ongoing transition towards advanced 3D in vitro tumour models raises a question about the validity of the standard cell counting protocols, particularly in the systems containing extracellular matrix-based scaffolds. Here, we provide a quantitative analysis of the performance of three popular plate reader-based cell counting/viability assays, such as the Alamar Blue, MTT, CellTiter Glo 3D assays, in 2D monolayer and 3D scaffold-based cultures of U251 human glioblastoma cells, including cell-laden Matrigel plugs, and original tissue engineering constructs based on the decellularised sheep brain scaffolds. We quantitatively characterized the assays linearity, precision, biological and technical reproducibility, proportionality, and inter-assay agreement. The study revealed that assays performance is highly platform-dependent, with 2D cultures allowing significantly more precise and reliable measurements than in 3D ECM scaffold-based cultures. The numerical results provided in this study can help researchers make informed decisions when working with 3D scaffold-based in vitro tumour models and for other tissue engineering purposes where precise cell counting is essential.