Non-destructive monitoring of 3D cell cultures: new technologies and applications

Abstract

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 Overall, an extensive technology shift is required, in order for monolayer cultures to be superseded, but the potential benefit derived from an increased accuracy of in vitro studies, justifies the effort and the investment.

Keywords: 3D cell culture; Non-destructive technology; in-vitro quantitative analysis.

Figure 1. Summary of 3D cell culture models.

In (A) the scaffold-based (SB) setups are presented, while (B) and (C) illustrate scaffold free (SF) and hybrid methods, respectively.

Figure 2. Non-destructive methods for viability quantification.

(A) Working principle of the LDH assay. (B) Representative image of a 3D cell culture obtained with light-sheet microscopy. Image reproduced from Alladin et al. (2020). (C) Example of 2D conductivity map reconstructed from EIT measurements. Image extracted from Wu et al. (2018).

Figure 3. Image exemplifying the combination of fluorescent and colorimetric readout methods.

Image extracted from Ursache et al. (2018). © 2017 The Authors The Plant Journal © 2017 John Wiley and Sons Ltd.

Figure 4. 3D Raman map of a cell adhered to a scaffold.

Image extracted from Baldock et al. (2019).

Figure 5. Schematic representation of the phantoms and results presented in Cortesi et al. (2021b), Lovecchio et al. (2022).

Figure 6. Schematic representation of the techniques presented within this work (coloured ovals) and of their use to quantify different biological variables (text fields).