Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

Abstract

Along with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures. Diffusion rates for nutrients and oxygen may limit metabolic activity which is often measured as a marker for cell viability. For assays that lyse cells, the penetration of reagents to achieve uniform cell lysis must be considered. For live cell fluorescent imaging assays, the diffusion of fluorescent probes and penetration of photons of light for probe excitation and fluorescent emission must be considered. This review will provide an overview of factors to consider when implementing assays to interrogate three dimensional cell culture models.

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“…Within the spectrum of available 3D models, one should then consider the complexity, costs, throughput and availability of imaging methods. 59 Yet, depending on the research question, one could still consider 3D models as simplified representations of reality. While it is certainly true that these models can bring crucial insights into the relationship between cellular complexity, space‐geometry, and EV biology, they are commonly not vascularized and/or do not contain stroma as found in tissues within in a living organism.…”