Exploring Mitochondria! Energy Metabolism of Single 3D Microtissue Spheroids using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate. Extracellular flux (XF) analyzers have been used to explore mitochondria! function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondria! energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers, DNA content and type. The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

Automated summary

This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using the XFe96 XF analyzer and spheroid microplates. XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of different sizes, volumes, cell numbers, DNA content, and type. The optimal concentrations of mitochondrial effector compounds are used to probe specific parameters of mitochondrial energy metabolism. The paper also discusses methods to normalize data obtained from spheroids and addresses considerations when exploring spheroid metabolism using XF technology.