Photobiomodulation as an antioxidant substitute in post-thawing trauma of human stem cells from the apical papilla

Cell culture and cryopreservation

SCAP at a passage of 3–5 were used in this experiment. SCAP were isolated from a human tooth. We obtained Institutional Review Board approval at Seoul National University Hospital (Seoul, South Korea; IRB number CRI05004). Informed consent was obtained from all participants. All experiments were performed in accordance with the Declaration of Helsinki. The manuscript does not contain information or images to identify study participants. Therefore, patient consent for the publication of our experimental data does not seem necessary. Basal media is composed of Alpha-MEM, FBS, and an antibiotic (all of them from Welgene, Korea) at a ratio of 100:10:1. This was used as a complete media. The cells were cultured in an incubator (371 Steri Cycle CO₂ Incubator, Thermofisher, USA) at a temperature of 37 °C and 5% carbon dioxide. For cryopreservation, EDTA-trypsinized cells were suspended in freezing media of basal media, FBS, and dimethyl sulfoxide (DMSO) (D8418-250ML, Sigma, USA) in a ratio of 6:3:1. A mixture of cells and freezing media was placed in a freezing vial and placed in isopropanol at − 70 °C for 12 h. After 12 h, freezing vials were moved to the liquid nitrogen tank for more than 24 h. Frozen cells were used in experiments after thawing in a 37 °C-waterbath for 2 min.

PBM system and chemical treatments

The 950 nm NIR device was made with 44 20-mm-spaced LEDs with a wavelength of 950 nm (PV810-3C6W-EDISAA, KAOS, Korea). Well plates were positioned 50 mm above the LEDs. Cells were uniformly irradiated with 950 nm NIR through dispersers from the backlight of an LED TV produced by Samsung Co., Ltd. (Fig. 1A). The 8-bit microcontroller-based (UM_MC95FG308_V3.20_EN, Korea) device operated in CW mode and PW modes with frequencies of 3, 30, 300, and 3000 Hz. The duty cycle was adjusted to 10, 20, 30, 40, and 80% in PW mode. In CW mode, the NIR-irradiated power density was 5.856 (± 0.534) mW/cm² as measured by the power meter (PM-USB-100, Thorlabs, USA). The irradiation flux was 175.68 mJ/cm² in total. When duty cycles were 10, 20, 30, 40, and 80%, the irradiation time was adjusted to 300, 150, 100, 75, and 37.5 s, respectively. In CW mode, the irradiation time was adjusted to 30 s. For experiments, cells contained in a well plate were placed on a transparent acrylic plate in the 37 °C incubator (MyCO₂, Hanil Science Medical, Korea).

NAC treatment

The NAC group was created to compare the effects on post-thawing trauma treatment with the NAC of an antioxidant. In NAC groups, the processing conditions for the reagent NAC used to reduce ROS are as follows. NAC (A7250, Sigma, USA) was dissolved in deionized water to 10 mM and was mixed with media to a 0.1 mM final concentration. After thawing, media mixed with NAC was added to cells instead of irradiating. Those cells were incubated at 37 °C for 30 min and washed twice with phosphate buffered saline (PBS). In this state, we incubated cells for 6 h with complete media to measure apoptosis or started other measurements immediately.

Hydrogen peroxide treatment

The following are conditions under which the cells were treated with hydrogen peroxide to increase ROS. A 3% hydrogen peroxide solution (Sigma) was mixed with media to a solution of 0.25 mM. The cells were seeded on the well plate and incubated for 24 h. After 24 h, the media was suctioned out and replaced with media containing hydrogen peroxide. We cultured this for 1 h at 37 °C and washed it twice with PBS. The complete media was added after washing. After hydrogen peroxide, we irradiated cells with 950 nm NIR or treated cells with NAC.

Delayed luminescence

For DL measurements, a 24-well plate was set on the acrylic plate (Fig. 1A). A mixture of PBS of 0.5 mL and 1.0 × 10⁴ cells per well was loaded into the well. Here, PBS was used in the culture media due to the fluorescence from FBS. A probe from the power meter was fixed 5 mm above the surface of the mixture. The irradiation followed the procedure above. The intensity of NIR was measured by calculating decay time. Delayed luminescence follows Eq. (1) which is the hyperbolic function⁵¹. The curves of NIR intensity were fitted in Matlab.

Here, I₀ is the initial intensity, β is the delayed luminescence index factor, τ is the delayed luminescence characteristic, and T is the decay time. Decay time was calculated by Eq. (2).

Cell viability

The Wst-1 assay kit (EZ-3000, Dogen, Korea) was used to estimate cell viability. After thawing, 1 × 10⁴ cells were seeded in a well of a 96-well plate and treated according to group. The 950 nm group was irradiated by 950 nm NIR, and the NAC-group was treated with 0.1 mM NAC. All wst-1 assays were conducted 24 h after treatments. For the assay, media was removed, and cells were washed with PBS. Wst-1 solution and growth media were mixed at a ratio of 1:10. The working solution was 110 ul per well. The cells were incubated for 1 h in the 37 °C incubator. Formazan dye formed was transported (100 ml each) to a new 96-well plate, and absorbance was measured at 450 nm by ELISA.

Apoptosis

After post-thawing treatment, 1.0 × 10⁵ cells were seeded in a 24-well plate for each group and incubated for 6 h. After that, cells were harvested and placed in 1.5 ml EP tubes. The harvested cells were washed with PBS. Then, 0.5 ml cold 1× binding buffer and 1.25 µl Annexin V-FITC were added to the cells, according to the manufacturer’s recommendations (EzWay Annexin V-FITC Apoptosis Detection Kit, k29100, Komabiotech, Korea). After being suspended, the sample was incubated for 15 min at room temperature in the dark. After staining, supernatant from centrifugation at 3000 RPM for 5 min was removed from the cells. Then, 0.5 ml cold 1× binding buffer and 10 ul PI were added to the cells. The cells were resuspended in PBS and analyzed immediately via flow cytometry (FACSVerse, Becton Dickinson, USA).

Intercellular ROS

To measure intercellular ROS, each group of harvested cells was dyed with CM-H2DCFDA (C6827, Invitrogen, USA) immediately after treatment. The cells were suspended in pre-warmed PBS containing dye with a final concentration of 10 μM. The cells were incubated for 15 min at 37 °C, centrifuged for 5 min at 3000 rpm, and the supernatant was removed. The cells were resuspended in culture media and incubated for 20 min at 37 °C. After incubating and centrifuging, the cells with removed supernatant were resuspended in pre-warmed PBS and analyzed via FACS as described above.

Mitochondrial ROS

MitoSOX (Invitrogen) was used for measuring mitochondrial ROS. According to the protocol, 50 μg of MitoSOX was dissolved in 13 μl of DMSO to prepare a 5 mM MitoSOX stock solution. The stock solution was diluted to a final concentration of 5 μM in Hanks’ balanced salt solution (HBSS). Then, 500 ul of working solution was added to each sample. The cells were incubated for 10 min and washed three times with warm HBSS. After washing, samples were mounted in warm buffer, and their MFIs were immediately measured using FACS.

Real-time polymerase chain reaction

SCAP cells (5.0 × 10⁵) were seeded in a 4-well plate and harvested 6 h after PBM treatment. The mRNA was extracted (RNeasy Mini kit, Qiagen, Germany), and the cDNA was synthesized using commercial kits (Rever Tra Ace qPCR Master Mix, Toyobo, Japan). mRNA was quantified with a plate reader (Take 3 at Synergy HT, Biotek, USA), reverse transcription was conducted using a thermal cycler (Qiagen, Germany), and real-time PCR was performed using a CFX96 Real-Time System (BioRad, USA). For real-time PCR, Hardshell 96-Well PCR Plates (BioRad, USA), Microseal ‘B’ seals (BioRad, USA), and SYBR green master mix (BioRad, USA) were used. All the processes followed a protocol provided by the manufacturers. The sequence of primers used in quantitative real-time PCR is given in Table 2.

Mitochondrial mass

For the mitochondrial mass assay, 1.0 × 10⁵ SCAP cells were used for each sample and were analyzed with Mito-tracker deep red (M22426, Invitrogen, USA). Briefly, a 1 mM stock solution was prepared with 50 μg of Mito-tracker deep red and 92 µl DMSO. The stock solution was diluted with PBS to form a 200 nM working solution. The harvested cells were washed with pre-warmed PBS and centrifuged for 3 min at 3000×g. The pre-warmed working solution was added to the cells (with supernatant removed) and incubated for 30 min at 37 °C. After that, the cells were washed with pre-warmed PBS and centrifuged for 3 min at 3000×g. MFI was immediately measured with FACS. Then, the cells were resuspended in 20 µl PBS and were put on a confocal dish in 10 µl (101350, SPL, Korea). Images of dyed cells were produced at a rate of 64× using a confocal microscope (LSM800, ZEISS, Germany).

Mitochondrial membrane potential

MMP was measured using a JC1-mitochondrial membrane potential assay kit (ab113850, Abcam, USA). Harvested cells were washed with PBS. JC-1 diluted with 1× dilution buffer was added to cells to a final concentration of 10 μM. The cells were washed twice with the 1xX dilution buffer after incubating for 30 min. Then, 50 µl of 2.0 × 10⁵ cells was transported to each well of the 96-Well Black Polystyrene Microplate (CLS3603, Corning, USA) followed by 50 µl of the buffer. The relative fluorescence (in RFUs) was measured using a Spark™ 10 M multimode microplate reader. JC1 exists in two forms, aggregate and monomer, depending on the mitochondrial membrane potential, and it emits different fluorescence depending on type. In intact cells, the membrane of the mitochondria is well permeated and exists in the state of JC1-aggregate, producing a 590 nm wavelength fluorescence. In damaged cells, mitochondria remain in the cytoplasm and presents a 529 nm fluorescence in the JC-1 monomer. Thus, the state of the MMP is inferred from the ratio between two intensities of fluorescence, which is called the JC1 ratio and is obtained as JC1 ratio = RFU(Aggregate)/RFU(Monomer)²⁷.

CCO assay

In the CCO activity measurement, there were two groups, the 950 nm group and the control group. Here, 1.0 × 10⁶ cells were used in the measurement. A Triton solution with 1% Triton X-100 (X100, Sigma, USA) was added to the harvested cells and incubated in ice for 10 min. After centrifugation, the cells were diluted in assay buffer and measured with a multimode reader (Spark 10 M, Tecan, Switzerland) at 570 nm. The amount of protein was calculated with a linear regression equation obtained from the standard curve. The slope of the absorbance data in kinetic mode presents a variation of absorbance over time. The slope was divided by the amount of protein, and the result was divided by the molar extinction coefficient provided from Abcam. The final result of the calculation was CCO activity, and the units were units/protein. A definition of “unit” is the amount that oxidizes 1 μmol cytochrome c per minute at 25 °C and pH 7.2.

ATP assay

The ATP assay employed the ATP assay kit (Abcam, ab83355) to compare the 950 nm group and the control. A standard curve was obtained and used. First, 1.0 × 10⁶ cells were washed with cold PBS. After washing, the 100 µl ATP assay buffer included in the kit was added to lysate cells. After lysis, cells were deproteinized with cold perchloric acid (PCA) and KOH. The hydrogen ion concentration was kept in the range of pH 6.5–8. The ATP working solution was prepared according to the given protocol and added to the cells. They were incubated in a dark room for 30 min at room temperature, and absorbance was measured at 570 nm.

Alkaline phosphatase (ALP) assay

ALP activity is the main biomarker of osteogenic differentiation. Alkaline Phosphatase Yellow (pNPP) Liquid Substrate (P7998-100ML, Sigma, USA) was used. The ALP assay was implemented on and days 3 and 5 after osteogenic induction. A mixture of the OM ingredient and the complete media in a 1:50 ratio was used for osteogenic induction media. The OM ingredient was prepared with 100 ml deionized water, 10.8 g β-glycerophosphate, 88.1 mg ascorbic acid, and 196.2 μg dexamethasone. Then, 3.0 × 10³ cells per well were seeded in a 96-well plate and incubated for 24 h at 37 °C. After that, they were washed with PBS and incubated with osteogenic induction media. At 3 and 5 days after osteogenic induction, ALP activity was measured. The cells were washed twice with a diluted assay buffer (10× Assay Buffer, ThermoFisher) with deionized water in a 1:10 ratio. After washing, 70 µl of the mixture of TritonX-100 (X100, Sigma, USA) and the diluted assay buffer in a ratio of 1:500 was added. The cells were placed in a 4 °C refrigerator for 10 min, and 50 ul diluted TritonX-100 was transferred to a new well plate. Then, 50 µl pNPP solution per well was added to the new well plate with diluted Triton X-100, incubated at 37 °C for 1 h and 15 min, and absorbance was measured by ELISA at 405 nm. Then, 5 µl of the remaining diluted TritonX-100 solution was transferred to a new well plate, and 200 µl of BCA solution (Pierce BCA protein assay reagent A, B, Thermo, USA) was added per well. After incubating at 37 °C for 30 min, the absorbance was measured at 570 nm with ELISA. To determine how much protein was present per well, absorbance data were calibrated using a BCA standard. The blank value was subtracted from the absorbance measured at 405 nm, and the result was divided by the amount of protein.

Statistical analysis

All experimental data were expressed as mean ± standard deviation (SD). Statistical significance between the experimental groups and control was determined using one-way ANOVA with R-3.6.3 software. Different letters at the tops of columns indicate significant difference at P < 0.05 according to Duncan’s multiple range test, performed using R-3.6.3.

Institutional review board statement

Human stem cells from the apical papilla were isolated from a tooth and we obtained Institutional Review Board approval at Seoul National University Hospital (Seoul, South Korea; IRB number CRI05004).

Informed consent statement

The manuscript does not contain information or images that could lead to identification of a study participant. Therefore, the consent of patients for publication of our experimental data is not necessary.