Investigating the effects of low intensity visible light on human keratinocytes using a customized LED exposure system

Abstract

Photobiomodulation (PBM) refers to the use of light to modulate cellular processes, and has demonstrated utility in improving wound healing outcomes, and reducing pain and inflammation. Despite the potential benefits of PBM, the precise molecular mechanisms through which it influences cell behavior are not yet well understood. Inconsistent reporting of key light parameters has created uncertainty around optimal exposure profiles. In addition, very low intensities of light, < 0.1 J/cm2, have not been thoroughly examined for their use in PBM. Here, we present a custom-made compact, and modular LED-based exposure system for studying the effects of very low-intensity visible light (cell proliferation, migration, ROS production, and mitochondrial membrane potential) of three different wavelengths in a parallel manner. The device allows for six repeats of three different exposure conditions plus a non-irradiated control on a single 24-well plate. The immortalised human keratinocyte cell line, HaCaT, was selected as a major cellular component of the skin epidermal barrier. Furthermore, an in vitro wound model was developed by allowing the HaCaT to form a confluent monolayer, then scratching the cells with a pipette tip to form a wound. Cells were exposed to yellow (585 nm, 0.09 mW, ~ 3.7 mJ/cm2), orange (610 nm, 0.8 mW, ~ 31 mJ/cm2), and red (660 nm, 0.8 mW, ~ 31 mJ/cm2) light for 10 min. 48 h post-irradiation, immunohistochemistry was performed to evaluate cell viability, proliferation, ROS production, and mitochondrial membrane potential. The results demonstrate increased proliferation and decreased scratch area for all exposure conditions, however only red light increased the mitochondrial activity. Oxidative stress levels did not increase for any of the exposures. The present exposure system provides opportunities to better understand the complex cellular mechanisms driven by the irradiation of skin cells with visible light.

Photobiomodulation and Wound Healing: Low-Level Laser Therapy at 661 nm in a Scratch Assay Keratinocyte Model

“…As the scratch assay experiments were performed in reduced FBS (1%) compared, it can be concluded that the scratch wound model's closure is influenced by another factor, possibly keratinocyte migration. This hypothesis is further supported by the work of Sutterby [ 24 ] and Sperandio [ 25 ], suggesting the role of red light on cell migration. Although the two experiments, proliferation and scratch assay, were performed with different FBS supplementations and can not be directly compared, we can assume that in a real wound, low-power laser light will contribute to wound closure by both proliferation and migration.…”

Clinical Applicability of Visible Light‐Mediated Cross‐linking for Structural Soft Tissue Reconstruction

“…This stimulation may be caused by exposure to low‐intensity visible light, which has been shown to stimulate cell metabolism in a range of cell types. [ 49,50 ] Nevertheless, the relatively stable metabolic activity observed across culture in most groups was expected as adipose tissue largely contains terminally‐differentiated adipocytes, which undergo minimal proliferation in this state. [ 51,52 ] Despite this, the DNA‐content of the collagen‐encapsulated controls increased over culture; while the low photocross‐linking intensities slowly reduced with large decreases observed at high cross‐linking intensities (Figure 3b).…”

Effects of Combined Blue light and 5-ALA on cell death in B16F1 melanoma and HaCaT cells

“…Notably, blue light alone decreased ROS levels in HaCaT cells, suggesting a potential cell-protective effect of blue light in certain cell types. Sutterby et al demonstrated that monochromatic visible light (yellow, orange, and red light) induces proliferation and wound healing in HaCaT cells [29]. The effect of blue light was not assessed, thus this question requires further investigation.…”