Tailoring metabolic activity assays for tumour-engineered 3D models
Julien Clegg1, Rodrigo Curvello1, Anastasiia Gabrielyan2
1Department of Chemical and Biological Engineering, Faculty of Engineering, Monash University, Victoria, Australia.
Resazurin-based assays like PrestoBlue and AlamarBlue can monitor cell behavior in 3D hydrogel models. Optimal performance for these metabolic assays in pancreatic cancer cell cultures requires specific reagent concentrations and incubation times.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Drug Discovery
Background:
- Monitoring cell behavior in 3D hydrogel models is crucial for drug development and understanding treatment responses.
- Resazurin-based assays (PrestoBlue, AlamarBlue) are common for metabolic activity but lack defined protocols for 3D hydrogels.
- Assay performance is influenced by cell type, culture conditions, and measurement sensitivity.
Purpose of the Study:
- To evaluate the performance of PrestoBlue and AlamarBlue assays in hydrogel-based 3D pancreatic cancer models.
- To determine optimal reagent concentrations and incubation times for these metabolic assays in 3D cultures.
- To compare metabolic assays with direct cell counting (CyQuant) for assessing drug treatment responses in complex 3D environments.
Main Methods:
- Pancreatic cancer cells were cultured in gelatin methacryloyl and collagen hydrogels.
- Metabolic activity was assessed using varying concentrations (4%, 10%) and incubation times (45 min, 2 h, 4 h) of PrestoBlue and AlamarBlue.
- Multicellular 3D cultures (cancer cells, fibroblasts, PBMCs) were treated with gemcitabine and nab-paclitaxel, with responses compared to CyQuant assays.
Main Results:
- In monocultures, increased reagent concentration and incubation time enhanced fluorescent intensity.
- A 10% reagent concentration and 2-hour incubation were found suitable for both hydrogels and cell lines.
- In multicellular cultures, metabolic assays detected drug responses but overestimated cell growth compared to CyQuant.
Conclusions:
- Optimized conditions (10% reagent, 2h incubation) enable effective use of PrestoBlue and AlamarBlue in 3D hydrogel models for cancer cell monocultures.
- For multicellular 3D cultures, metabolic assays should be complemented with direct cell viability assays like CyQuant to accurately assess drug effects.
- Combined approaches are recommended for reliable evaluation of cell viability and growth in complex 3D cancer models.
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