Photobiomodulation of human dermal fibroblasts in vitro: decisive role of cell culture conditions and treatment protocols on experimental outcome

Abstract

Photobiomodulation-based (LLLT) therapies show tantalizing promise for treatment of skin diseases. Confidence in this approach is blighted however by lamentable inconsistency in published experimental designs, and so complicates interpretation. Here we interrogate the appropriateness of a range of previously-reported treatment parameters, including light wavelength, irradiance and radiant exposure, as well as cell culture conditions (e.g., serum concentration, cell confluency, medium refreshment, direct/indirect treatment, oxygen concentration, etc.), in primary cultures of normal human dermal fibroblasts exposed to visible and near infra-red (NIR) light. Apart from irradiance, all study parameters impacted significantly on fibroblast metabolic activity. Moreover, when cells were grown at atmospheric O2 levels (i.e. 20%) short wavelength light inhibited cell metabolism, while negligible effects were seen with long visible and NIR wavelength. By contrast, NIR stimulated cells when exposed to dermal tissue oxygen levels (approx. 2%). The impact of culture conditions was further seen when inhibitory effects of short wavelength light were reduced with increasing serum concentration and cell confluency. We conclude that a significant source of problematic interpretations in photobiomodulation reports derives from poor optimization of study design. Further development of this field using in vitro/ex vivo models should embrace significant standardization of study design, ideally within a design-of-experiment setting.

Differential response of human dermal fibroblast subpopulations to visible and near‐infrared light: Potential of photobiomodulation for addressing cutaneous conditions

“…Therefore, the observation of photorejuvenation, wound healing or hair regrowth effects cannot yet be associated with a stimulation/inhibition of a particular cell type, for example, dermal fibroblasts. Some of our results concurred with previous research studies . In particular, it was clear that three consecutive exposures to short visible wavelengths (⇐530 nm), exerted inhibitory effects on DFs while long visible and NIR wavelengths (>=530 nm) had a neutral impact at standard environmental oxygen concentration.…”

Does blue light restore human epidermal barrier function via activation of Opsin during cutaneous wound healing?

“…Reduced proliferation and metabolic activity has also been observed in primary cultures of human dermal fibroblasts after irradiation with blue light . Recently, Mignon et al demonstrated a stimulatory effect with a single treatment of 450 nm light at 2 J/cm 2 on metabolic activity of dermal fibroblasts, with suppressive action following additional irradiations . In a scratch‐wound assay, high‐dose blue light (55 J/cm 2 ) inhibited fibroblast migration, while lower doses (2, 5, and 10 J/cm 2 ) had no effect .…”

Effects of blue light on inflammation and skin barrier recovery following acute perturbation. Pilot study results in healthy human subjects

“…Intriguingly, however, the same dose of blue light was shown to have differential effects on distinct lineages of human dermal fibroblasts . In addition to differential biological responses elicited by different cell lineages and irradiation parameters, identifying effective therapeutic parameters for blue light‐based therapies is further complicated by the still incomplete knowledge of the photoacceptors in the skin . Besides nitrosated proteins, the effects of blue light could be mediated by photoacceptors in mitochondria, including cytochrome c oxidase, but also by cryptochromes and other flavoproteins, and all could lead to the generation of NO and reactive oxygen species (ROS) or to the activation of G‐protein‐coupled receptors .…”