A 3D naive biological model for high-throughput live-cell analysis of mitochondrial oxygen consumption in response to photobiomodulation treatment

Abstract Purpose Understanding the photobiomodulation (PBM) dose response for mitochondrial activity and tissue regeneration is challenging due to inconsistent results and the absence of advanced, high-throughput 3D in vitro techniques to accurately assess PBM effects. This study introduces a 3D native biological model optimized for high-throughput live-cell analysis, enabling precise measurement of mitochondrial oxygen consumption in response to two PBM treatment time points. Methodology This model is specifically designed for human dental pulp stem cells in vitro, incorporating hypoxic conditions, replacing fetal bovine serum with antibody A and B male human serum, and utilizing the advanced LunaGel™ technology to facilitate spheroid formation. This model ensures experimental reproducibility by adhering to the core principles of metabolic biochemistry. Here we describe protocols to optimise this complex model to suit the Seahorse XFe96 extracellular flux analyser using mitochondrial stress modulators. The process involves setting up the laser and related equipment to establish a reliable publication standard for future PBM experiments. Results LunaGel™ optimized at 3.5 kPa stiffness, effectively supported hDPSC culture and Seahorse XFe96 analysis under hypoxic conditions with human serum. Oxygen consumption rates (OCR) varied with hydrogel stiffness, with higher stiffness showing increased OCR but no significant differences overall. Post-PBM treatment, basal respiration significantly increased at 5 J/cm² after 24 h, while maximal respiration was elevated immediately at 5 J/cm² and reduced at 15 J/cm². Cells exhibited healthy growth and morphology under hypoxia environment. Conclusion We demonstrate an improved accuracy in evaluating bioenergetic responses with this approach and its effects post-PBM.