Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Area of Science:

• Cell Biology
• Biotechnology
• Biochemistry

Background:

• Three-dimensional (3D) spheroids mimic in vivo cellular environments, offering advantages over 2D cultures for physiological relevance.
• Spheroid models are crucial in oncology, diabetes, stem cell biology, and tissue engineering.
• Assessing metabolic function in 3D spheroids is vital but lacks standardized protocols for mitochondrial energy metabolism.

Purpose of the Study:

• To provide detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using XFe96 XF analyzer.
• To optimize and validate the use of Extracellular Flux (XF) technology for spheroid metabolic analysis.
• To establish a reproducible method for studying spheroid metabolism.

Main Methods:

• Utilized XFe96 spheroid microplates with an XF analyzer for metabolic flux analysis.
• Employed specific mitochondrial effector compounds (oligomycin, BAM15, rotenone, antimycin A) at optimal concentrations.
• Applied normalization methods for data obtained from 3D spheroids of varying characteristics.

Main Results:

• Demonstrated XF technology's capability to differentiate cellular respiration in 3D spheroids.
• Showcased distinctions based on spheroid size, volume, cell number, and DNA content.
• Identified optimal effector compound concentrations for specific mitochondrial energy metabolism parameters.

Conclusions:

• The developed protocols enable detailed analysis of mitochondrial energy metabolism in 3D spheroids.
• This methodology supports advanced in vitro spheroid models for various research fields.
• The findings facilitate a deeper understanding of spheroid metabolic function and its applications.