Advantages and limitations of using cell viability assays for 3D bioprinted constructs.

Bioprinting shows promise for bioengineered scaffolds and 3D disease models, but assessing the viability of embedded cells is challenging. Conventional assays are limited by the technical problems that derive from using multi-layered bioink matrices dispersing cells in three dimensions. 
In this study, we tested bioprinted osteogenic bioinks as a model system. Alginate- or gelatin-based bioinks were loaded with/without ceramic microparticles and osteogenic cells (bone tumour cells, with or without normal bone cells). Despite demonstrating 80-90% viability through manual counting and live/dead staining, this was time-consuming and operator-dependent. Moreover, for the alginate-bioprinted scaffold, cell spheroids could not be distinguished from single cells. The indirect assay (alamarBlue), was faster but less accurate than live/dead staining due to dependence on hydrogel permeability. Automated confocal microscope acquisition and cell counting of live/dead staining was more reproducible, reliable, faster, efficient, and avoided overestimates compared to manual cell counting by optical microscopy. Finally, for 1.2 mm-thick 3D bioprints, dual-photon confocal scanning with vital staining greatly improved the precision of the evaluation of cell distribution and viability and cell-cell interactions through the z-axis. 
In summary, automated confocal microscopy and cell counting provided superior accuracy for the assessment of cell viability and interactions in 3D bioprinted models compared to most commonly and currently used techniques&#xD.

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