| Summary: | 3D cell cultures are becoming the new standard for cell-based in vitro research, due to their higher transferrability toward in vivo biology. The lack of established techniques for the non-destructive quantification of relevant variables, however, constitutes a major barrier to the adoption of these technologies, as it increases the resources needed for the experimentation and reduces its accuracy. In this review, we aim at addressing this limitation by providing an overview of different non-destructive approaches for the evaluation of biological features commonly quantified in a number of studies and applications. In this regard, we will cover cell viability, gene expression, population distribution, cell morphology and interactions between the cells and the environment. This analysis is expected to promote the use of the showcased technologies, together with the further development of these and other monitoring methods for 3D cell cultures. Overall, an extensive technology shift is required, in order for monolayer cultures to be superseded, but the potential benefit derived from an increased accuracy of in vitro studies, justifies the effort and the investment.
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