Bioprinting shows promise for bioengineered scaffolds and three-dimensional (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 tumor 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.

中文翻译：

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使用细胞活力测定进行 3D 生物打印结构的优点和局限性

生物打印显示出生物工程支架和三维 (3D) 疾病模型的前景，但评估嵌入细胞的活力具有挑战性。传统的检测方法受到使用多层生物墨水基质在三个维度上分散细胞所产生的技术问题的限制。在这项研究中，我们测试了生物打印的成骨生物墨水作为模型系统。基于藻酸盐或明胶的生物墨水负载有/没有陶瓷微粒和成骨细胞（骨肿瘤细胞，有或没有正常骨细胞）。尽管通过手动计数和活/死染色证明了 80%–90% 的活力，但这非常耗时且依赖于操作员。此外，对于藻酸盐生物打印支架，无法将细胞球体与单个细胞区分开。由于依赖于水凝胶渗透性，间接测定（alamarBlue）比活/死染色更快，但准确性较低。与光学显微镜手动细胞计数相比，自动共焦显微镜采集和活/死染色细胞计数更具重复性、可靠、更快、高效，并且避免了高估。最后，对于 1.2 毫米厚的 3D 生物打印，采用活体染色的双光子共聚焦扫描通过z-轴。总之，与最常用和当前使用的技术相比，自动共焦显微镜和细胞计数为 3D 生物打印模型中的细胞活力和相互作用的评估提供了更高的准确性。