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bioRxiv · 10.64898/2026.04.27.720021

What Can We Count On? Performance of Microplate Cell Counting Assays in 2D Monolayer and 3D ECM-based In Vitro Tumour Models

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

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. ToC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/720021v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16018d9org.highwire.dtl.DTLVardef@1ff7d6dorg.highwire.dtl.DTLVardef@838021org.highwire.dtl.DTLVardef@1510d5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Vaezzadeh, M., Nadort, A., Igrunkova, A., Lee, V. S., Di Ieva, A., Heng, B., Guller, A.. 2026-04-30. What Can We Count On? Performance of Microplate Cell Counting Assays in 2D Monolayer and 3D ECM-based In Vitro Tumour Models. https://doi.org/10.64898/2026.04.27.720021

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