When Dr Simone Sleep first entered the world of photobiomodulation, the science seemed full of possibilities. Yet the results, scattered and uneven, rarely agreed.
“At Griffith University, our focus has been on understanding how PBM influences mitochondrial function in human dental stem cells. However, one of the primary challenges in the field is inconsistency. Different laboratories report varying outcomes, much of which can be attributed to differences in model systems.” - Dr Sleep
Working within the School of Medicine and Dentistry at Griffith University, Simone, together with Professor Roy George, Professor Deanne Hryciw, and Professor Praveen Arany, sought to address a fundamental translational challenge:
How do you standardise PBM dose–response in a system that actually reflects native dental tissue?
Most high-throughput screening platforms are designed for 2D cultures. They offer convenience, but the world they create is flat, missing oxygen gradients, the resistance of real tissue, and the slow movement of molecules through a living matrix.
“For tissue regeneration, including dentin and bone, diffusion and oxygen tension are critical. We required a three-dimensional (3D) model that more accurately represented native tissue while remaining compatible with our Seahorse XFe96 metabolic platform.” - Dr Sleep
Translating a 2D workflow into a 3D system was not straightforward. New complications emerged, each persistent.
The search was for a matrix with the right balance: soft enough to mimic living tissue, strong enough to maintain its shape, and capable of meeting the demands of high-throughput assays.
Progress came through careful pairing of a biological model and steady control of the matrix. Using human dental pulp stem cells cultured under hypoxic (~5% O₂), xeno-free conditions, the team developed a high-throughput 3D spheroid workflow inside LunaGel™. Using a hydrogel was not enough. What mattered was shaping it and tuning its properties until it responded as needed.
“With LunaGel™ and the LunaCrosslinker™, we could consistently tune stiffness to around ~3.5 kPa,” Dr Sleep explains. “That balance was critical - firm enough to maintain spheroid integrity, but permissive enough for small-molecule diffusion during Seahorse assays.”
With control over stiffness and careful timing during the mitochondrial stress test, responses that had once been hidden began to surface. The effects of PBM, previously blurred by diffusion limits, became clearer.
Once optimised, the workflow adhered to a defined structure:
The team observed clear, dose- and time-dependent PBM effects on both basal and maximal respiration, which were not reproducible under 2D conditions.
Matrix optimisation and diffusion testing demonstrated that stiffness directly influenced modulator entry. Softer matrices facilitated more efficient diffusion, whereas stiffer matrices slowed metabolic response curves. These insights are critical for the accurate interpretation of mitochondrial data.
“The combination of stiffness tuning and Seahorse timing adjustments was decisive,” Dr Sleep notes. “It allowed us to reveal PBM-dependent mitochondrial changes in 3D that simply weren’t visible in 2D systems.”
Beyond the scientific findings, the workflow enhanced experimental efficiency.
The system:
“Ease of use was important. We saved setup time, and once optimised, the workflow was robust and reproducible.” - Dr Sleep
For translational research, reproducibility is essential. A stable 3D readout enables rational PBM dose selection and speeds the development of clinically relevant protocols.
Dr Sleep emphasises that successful 3D optimisation requires careful planning.
“Most high-throughput instruments were designed for 2D applications. It is necessary to plan a focused 3D optimisation phase.”
Her recommendations:
“Once optimised, the system is highly effective. Combining LunaGel™ with real-time mitochondrial stress testing provides a level of insight into PBM bioenergetics that was previously unattainable.”
Dr Simone Sleep’s work shows that the transition from 2D to 3D is not just an increase in complexity, but an exercise in achieving precise control.
By standardising matrix stiffness, diffusion timing, hypoxic conditions, and reporting parameters, her team established a reproducible 3D metabolic workflow capable of generating clinically relevant PBM insights.
For researchers at the intersection of regenerative medicine and metabolic modulation, structured 3D systems have become foundational rather than optional.