Single Cell Effects of Photobiomodulation on Mitochondrial Membrane Potential and Reactive Oxygen Species Production in Human Adipose Mesenchymal Stem Cells

Abstract

Photobiomodulation (PBM) has recently emerged in cellular therapy as a potent alternative in promoting cell proliferation, migration, and differentiation during tissue regeneration. Herein, a single-cell near-infrared (NIR) laser irradiation system (830 nm) and the image-based approaches were proposed for the investigation of the modulatory effects in mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), and vesicle transport in single living human adipose mesenchymal stem cells (hADSCs). The irradiated-hADSCs were then stained with 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) and Rhodamine 123 (Rh123) to represent the ΔΨm and ROS production, respectively, with irradiation in the range of 2.5-10 (J/cm²), where time series of bright-field images were obtained to determine the vesicle transport phenomena. Present results showed that a fluence of 5 J/cm² of PBM significantly enhanced the ΔΨm, ROS, and vesicle transport phenomena compared to the control group (0 J/cm²) after 30 min PBM treatment. These findings demonstrate the efficacy and use of PBM in regulating ΔΨm, ROS, and vesicle transport, which have potential in cell proliferation, migration, and differentiation in cell-based therapy.

Keywords: human adipose-derived mesenchymal stem cell; mitochondrial membrane potential; photobiomodulation; reactive oxygen species; vesicle transport.

Figure 1

Schematic diagram for mechanisms of photobiomodulation. Cytochrome c oxidase (CCO) is a major photoreceptor, located in the mitochondrial respiratory chain at unit IV in the mitochondria, was released by PBM, followed by a functional change in the original mitochondrial respiratory chain to generate ROS and increased mitochondrial membrane potential (ΔΨm) that plays a crucial role in ATP production through oxidative phosphorylation.

Figure 2

Experimental organization for the single-cell NIR laser irradiation system. (A) The proposed experimental setup consists of an 830 nm infrared diode laser, an electrical shutter, laser-focusing optics (condenser), and a specimen holder attaching to the XY-axis motorized stage. The lens pairs, which were used to expand the laser beam eightfold, were combined by two telescopes, including a 1:4 telescope (L1:L2) and 1:2 telescope (L3:L4) with the aim to fill the back aperture of the condenser. The dichroic mirror D1 is located between the visible light source and the condenser to reflect the laser beam into the condenser while allowing visible light to pass for bright field imaging. (B) The experimental setup for the proposed single-cell PBM irradiation system.

Figure 3

Example for showing the determination and selection of the region of interest (ROI) in the irradiated-hADSCs for vesicle transport analysis. The irradiated-hADSCs (Passage 10) were photographed with 100 of kinetic length and 0.1 s of an exposure time by an EMCCD camera before and after 30 min PBM at two fluences, including (A) 0 and (B) 5 J/cm².

Figure 4

A plot of time-lapse Rh123 of mitochondrial membrane potential in PBM. Values demonstrated the ratio of mean intensity before and after 0, 10, 20, 30, 60 min PBM (n = 3). Statistical analysis (one-way ANOVA) for significant differences between after 30 PBM and other time points observed at all fluences, including 0, 2.5, 5, and 10 J/cm² (p ≤ 0.05).

Figure 5

(A) Experimental procedures in investigating the ΔΨm in the irradiated-hADSCs (B) Fluorescence microscopy images of the promotion of the mitochondrial membrane potential in hADSCs (Passage 10) after 30 min PBM at various fluences, including 0, 2.5, 5, and 10 J/cm². These cells were automatically selected and captured after 30 min by EMCCD camera.

Figure 6

Mitochondrial membrane potential in hADSCs (Passage 10) after 30 min PBM at various fluences, including 0, 2.5, 5, and 10 (J/cm²). Values represented as the ratio of mean fluorescence intensity before and after 30 min PBM (S₂/S₁ ± SEM, n = 8). Statistical analysis (one-way ANOVA) for significant differences between 2.5 J/cm²) and two fluences of 5 and 10 J/cm² (* p ≤ 0.05).

Figure 7

(A) Experimental procedures in investigating the ROS production in the irradiated-hADSCs (Passage 10) (B) ROS marker fluorescence microscopy pictures were acquired in the hADSCs after 30 min. These cells were spontaneously picked and captured by an EMCCD camera following various time points of PBM at fluences of 2.5, 5, and 10 J/cm². The scale bar denotes a distance of 10 µm.

Figure 8

Bar charts representing the effects of PBM on intracellular ROS acquired in the hADSCs after 30 min PBM. These cells were spontaneously picked and photographed by an EMCCD camera following PBM at fluences of 2.5 J/cm² (Red), 5 J/cm² (Black), and 10 J/cm² (Purple). The values are the mean intensity ratio (mean ± SEM, n = 6). Statistical analysis (one-way ANOVA) showed significant changes in this study (**** p ≤ 0.0001; ns: p > 0.05).

Figure 9

(A) Experimental procedures in vesicle transport investigation on the irradiated-hADSCs (B) The mergence between bright field and fluorescence images in promoting the vesicle transport in the irradiated-hADSCs (Passage 10) after 30 min treatment at a fluence of 5 (J/cm²) compared to control 0 (J/cm²). The ROI area (64 × 64 pixels) was identified by ImageJ, followed by cutting it and measure the velocity using (Matlab R2021a).

Figure 10

Effects of PBM in vesicle transport on hADSCs. Values were expressed as the velocity’s ratio of vesicle transport before (V₁) and after PBM (V₂) according to time (V₂/V₁ ± SEM). (A) The velocity of the control group (0 J/cm²); (B) treatment group (5 J/cm²) at single-cell level. (C) Vesicle transport in the irradiated-hADSCs (n = 8) after 30 min PBM between control group of 0 J/cm² and PBM group of 5 J/cm². The ensemble average of the velocity ratio (V₂/V₁) for the control and PBM groups were 0.76 and 1.93, respectively (p-value = 0.0117).