2.1.

Cell Culture

Cell lines were obtained from the American Type Tissue Culture Collection (ATCC, Rockville, Maryland): A549 adenocarcinoma human alveolar epithelial cells, PtK2 rat kangaroo renal epithelial cells, and U2OS human osteosarcoma cells. Cells were grown in T-75 flasks (Corning, Fisher, Pittsburgh, Pennsylvania) in a 37°C, 5% ${\mathrm{CO}}_2/95\%$ air incubator using Dulbecco’s Modified Eagle’s Medium (DMEM) for A549 and U2OS cells and Modified Eagle’s Medium (MEM) for PtK2 cells. Both media types were supplemented with 10% fetal bovine serum (Mediatach, Manassas, Virginia). Cells were trypsinized with TrypLE (Invitrogen Life Technologies, Carsbad, California), seeded into 6-well or 96-well plates (Corning, Fisher, Pittsburgh, Pennsylvania) and grown to confluency.

2.2.

Photoirradiation and Devices

The two-light sources used were (1) a red (652 nm) and near-infrared (806 nm) emitting laser (Biolitec AG, Jena, Germany), and (2) an LED array with wavelengths at 637 and 901 nm (Biophotas Inc., Tustin, California). The LLLT phototherapy was delivered using the parameters shown in Table 1 and spectra given in Fig. 1(a)–1(d). The emission spectra from each light source were measured and the maximum intensity value was normalized to one. The full bandwidth was determined by measuring the bandwidth at half of the maximum intensity. Each of the four wavelengths was administered to a confluent 6-well dish. The light from the laser was coupled to an optical fiber terminating in a front distributing lens with a full angle of divergence of 34.7 deg and a beam diameter of 0.6 mm at 0 mm from the lens (FD, Medlight, Ecublens, Switzerland). The samples were exposed to a circular beam of light with the dimensions and dosimetry listed in Table 1. The LED array was placed 1.3 cm (measured from diode to cells) from the bottom of the sample dish. Each dish (well) was suspended above the LED array by a transparent stage. Laser and LED powers were measured using a FieldMate laser power meter (Coherent, Santa Clara, California). Output from either the optical fiber or LED was measured 1.3 cm from the detector, which has an area of $1{\mathrm{cm}}^2$. Dosimetry values were calculated based on a 30-min light exposure. Samples were kept either in the dark or under low-level light for all other processing steps.

Table 1

Dimensions and dosimetry of light delivery.

Fig. 1

Emission spectra of all four light sources. The emission spectra from either the laser (a and b) or LED (c and d) was measured and plotted. The maximum intensity value was normalized to one for all curves plotted and the full bandwidth was determined as the bandwidth at half maximum intensity (black line). The full bandwidth and maximum emission wavelength is indicated for all curves. Arbitrary units (a.u.).

2.3.

Heat Measurement

A RAYNGER MX4 (Raytek, Santa Cruz, California) infrared-detecting thermometer was used to measure the temperature of the cell culture sample before and after LLLT. The laser temperature gun measures the selected area within the well with a relationship of $60:1$ between the distance and spot size at the focal point. The aiming laser beam of the gun was aimed at multiple positions within each sample and for multiple culture dishes. Final temperature measurements were normalized to the initial (before any light treatment was administered) temperature.

2.4.

Scratch Wound Closure Assay

The scratch wound closure assay method has been previously described.¹¹ Briefly 600 to 700-μm wide single-line scratches were mechanically generated in a cell monolayer by a 200-μl plastic tip. The area of the open wound was recorded for 24 h and quantified using a custom MATLAB^® script. Cells from both edges of the wound migrated into the open wound area until closure was complete. The wound closure rate was determined by plotting changes in the open wound area as a function of time.

2.5.

Microscopy

Phase contrast images were captured through a $10\times$ magnification Ph1 NA 0.25 objective (Zeiss, Jena, Germany). Fluorescent images were captured through a $63\times \mathrm{Ph}3$ NA 1.4 objective (Zeiss, Jena, Germany). An inverted microscope (Axiovert 135, Zeiss, Jena, Germany) with a charge-coupled device camera ($\mathrm{ORCA}\text{-}{\mathrm{R}}^2$ Hamamatsu, Bridgewater, New Jersey) was used and images were acquired using custom Robolase II software developed previously.¹² The microscope stage was modified to accommodate a multiwell format for high-throughput analyses.

2.6.

Transwell Migration Assay

A standard transwell migration assay method was used as previously described.¹¹ Briefly, cells were serum-starved overnight prior to being seeded ($5\times {10}^4\mathrm{cells}$ in 100 μl of DMEM or MEM/0.1% FBS) into the top chamber of a 24-well transwell plate with an 8.0-μm pore polycarbonate membrane (Corning, Fisher). MEM or DMEM/10% FBS was placed in the bottom chamber. Cells were allowed to adhere to the membrane for 24 h and then were exposed to light for 30 min. After an additional 24 h, the cells were fixed in 70% cold ethanol. A cotton swab was used to remove cells remaining on top of the membrane. Cell migration through the membrane was measured using the cell nuclei stain bisbenzimide H 33342 trihydrochloride (Hoechst, Invitrogen Life Technologies, Grand Island, New York). Cell number was quantified using the ImageJ (Wayne Rasband, NIH, Bethesda, Maryland) cell counter plug-in.

2.7.

MTS Cell Proliferation

PtK2 and U2OS cells were seeded at $2\times {10}^4$ per well, and A549 cells were seeded at $1\times {10}^4$ per well in a 96-well plate format. Cells were incubated overnight to permit cell attachment. The cells were then exposed to light at all four wavelengths for 30 min (Table 1). At 24 h the level of cell proliferation was determined using a MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) cell proliferation assay (CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, Wisconsin) according to the manufacturer’s instructions. The MTS dye was added directly to cell culture media and incubated with the cells for 2 h at 37°C. Dye absorbance at 490 nm was measured and plotted for both untreated (no-light) and light-treated cells.

2.8.

Statistical Analysis

Data are presented as $\text{mean}\pm \text{standard error}$ of the mean (SEM). Student’s $t$-tests were used for experiments that contained only two conditions, and one-way analysis of variance followed by Bonferroni’s post-test was used for experiments containing three or more conditions. A $p$ value $<0.05$ was considered to be statistically significant.

3.1.

LLLT Enhances Wound Closure

LLLT with either the laser or LED light source significantly enhanced in vitro wound closure in all three cell types at all four wavelengths tested. The initial wound size was evaluated and then compared with the wound 24-h postlight exposure [Figs. 2(a) and 2(b) and Figs. 3(a) and 3(b)]. All treatment conditions demonstrated statistically significant increases in wound closure rates when compared with the untreated controls.

Fig. 2

Low-level light therapy (LLLT) increases in vitro wound healing in A549, PtK2 and U2OS using 637 and 652 nm. (a and b) Mechanical scratch wounds were generated in a monolayer of the respective cell type, and treated with LLLT 637 or 652 nm. Twenty-four hours later, the area of the wound was evaluated by a computer-based system as described in Sec. 2 and compared with the untreated control. The normalized average wound area was determined by dividing the final wound area by the initial wound area and averaging replicates. The green area marks the wound, bar, 100 μm. Data represent the $\text{mean}\pm \text{standard error}$ of the mean (SEM) of at least three separate experiments with $N=12$; ****$p<0.0001$, **$p<0.01$ compared with control or for the indicated comparison.

Fig. 3

LLLT increases in vitro wound healing in A549, PtK2, and U2OS using wavelengths 806 and 901 nm. (a and b) Cells were treated and evaluated as in (Fig. 2), but treated with LLLT 806 or 901 nm and wound healing was measured after 24 h. The green area marks the wound, bar, 100 μm. Data represent the $\text{mean}\pm \mathrm{SEM}$ of at least three separate experiments with $N=12$; ****$p<0.0001$, *$p<0.05$ compared with control or for the indicated comparison.

In some cases (U2OS at 637 nm, U2OS at 901 nm, and PtK2 at 901 nm) the LED treatment produced a statistically greater closure rate than the laser. However, it should be noted that there is a small amount of heat given off by the LED device (3°C for 637 and 2°C for 901 nm), whereas there is no measurable temperature change during treatment with the laser source. Heat alone from the LED circuitry was tested for all assays run in this study, and it was found to have no effect in most cases. The U2OS cells showed no change in wound closure after heat treatment alone. This suggests that the differences observed between LED and laser treatments for U2OS cells are attributed to wavelength differences. In contrast PtK2 cells showed a significant increase in wound healing rate after heat treatment alone ($p<0.05$ compared with the untreated control), which suggests that for this nontransformed marsupial cell type, at least some of the difference observed between the laser and LED, is likely due to the heat given off by the LED array. However, the marsupial cells did respond with increased wound healing when treated with the laser, which did not generate any heat.

3.2.

LLLT Improves Cell Migration

Using the transwell assay LLLT was shown to significantly stimulate in vitro cell migration in all three cell types A549, PtK2, and U2OS [Figs. 4(a) and 4(b) and Figs. 5(a) and 5(b)]. Cells were serum starved prior to seeding onto a transwell insert and treated with laser 652 and 806 nm, and LED 637 and 901 nm. It should be noted that serum starvation was only used for the transwell assay, as it was the only assay carried out in this work requiring this pretreatment step. A549 and PtK2 cells demonstrated similar efficacy at all four wavelengths tested. U2OS cells, however, showed a statistically better response for the two LED wavelengths (637 and 901 nm) compared with the two laser wavelengths (652 and 806 nm). In U2OS cells the LED and laser sources exhibited no difference in cell migration after heat treatment this was also true for A549 and PtK2 cells.

Fig. 4

LLLT 637 and 652 nm increase in vitro cell migration. (a and b) A549, PtK2, or U2OS cells were seeded onto a polycarbonate membrane and allowed to adhere to the transwell insert followed by treatment with either LLLT 637 or 652 nm. Transwell migration was visualized using Hoechst 33342 (blue) and quantified using the cell counter ImageJ plugin at 24-h posttreatment as described in Sec. 2. Bar 20 μm. Data represent the $\text{mean}\pm \mathrm{SEM}$ of at least three separate experiments with $N=3$ fields of view; ****$p<0.0001$ compared with control or for the indicated comparison.

Fig. 5

LLLT 806 and 901 nm increase in vitro cell migration. (a and b) A549, PtK2, or U2OS cells were treated and quantified as in (Fig. 4), but using LLLT 806 or 901 nm. Bar 20 μm. Data represent the $\text{mean}\pm \mathrm{SEM}$ of at least three separate experiments with $N=3$ fields of view; ****$p<0.0001$, ***$p<0.001$, **$p<0.01$ compared with control or for the indicated comparison.

3.3.

LLLT and Cell Proliferation

For three wavelengths (652, 637, and 901 nm), there was no measureable difference in proliferation in A549, PtK2, and U2OS cells compared to unexposed control cells. However, at 901 nm the A549 and U2OS cells exhibited no increase in cell proliferation compared with the controls, but the PtK2 cells did [Figs. 6(a) and 6(b)]. No difference was detected from heat treatment alone in all three cell types.

Fig. 6

LLLT does not increase in vitro cellular proliferation for most conditions. (a and b) A549, PtK2, or U2OS cells were treated with LLLT as indicated, and cell proliferation was measured 24-h posttreatment as described in Sec. 2. Data represent the $\text{mean}\pm \mathrm{SEM}$ of at least three separate experiments with $n=8$ **$p<0.01$ compared with control.