Nadort, A. | Search bioRxiv

bioRxiv2026

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

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

bioengineering↗

bioRxiv2020

Tissue engineered model of hepatic breast cancer micrometastasis shows host-dependent colonization patterns and drug responses

Early stages of colonization of distant organs by metastatic cancer cells (micrometastasis) remain almost inaccessible to study due to lack of relevant experimental approaches. Here, we show the first 3D tissue engineered model of hepatic micrometastasis of triple negative breast cancer (TNBC). It reproduces characteristic histopathological features of the disease and reveals that metastatic TNBC cells colonize liver parenchymal and stromal extracellular matrix with different speed and by different strategies. These engineered tumors induce the angiogenic switch when grafted in vivo, confirming their metastatic-specific behaviour. Furthermore, we proved feasibility and biological relevance of our model for drug and nanoparticle testing and found a down-regulatory effect of the liver microenvironment of the sensitivity of TNBC cells to chemotherapeutic drug doxorubicin in free and nanoformulated forms. The convenient and affordable methodology established here can be translated to other types of metastatic tumors for basic cancer biology research and adapted for high-throughput assays.

bioengineering↗