Introduction
Regenerative medicine is a multidisciplinary field that leverages cellular therapies to restore or establish normal function of damaged tissues and organs [1]. Recently, there has been a surge of interest in the therapeutic potential of adipose‐derived stem cells (ADSCs) due to their accessibility, ease of isolation, ability to differentiate into various cell types and potent regenerative capabilities [2]. ADSCs are a valuable source of multipotent cells that can be readily obtained from the patient's own adipose tissue, thereby reducing the risk of immune rejection and ethical concerns [3].
Photobiomodulation (PBM), a form of low‐level light therapy, has been shown to significantly enhance the proliferation and differentiation of stem cells, thus making it a promising approach for various regenerative applications [4, 5]. PBM uses light in the visible spectrum or in the near‐infrared range to modulate cellular functions including proliferation, modulation of inflammation, and tissue repair [6]. The cellular mechanisms of PBM have been intensively studied in numerous biochemical applications and clinical trials; however, a comprehensive understanding of all PBM‐mediated pathways remains elusive. For instance, PBM efficacy depends critically on the therapeutic window chosen and on parameters such as wavelength, fluence, mode of application, duration of exposure, as well as the cellular environment and the specific cell type involved [5, 7, 8].
PBM fluence plays a significant role and in many studies researchers have explored the use of various fluences of PBM when treating ADSCs, in two‐dimensional (2D) cell culture, fluences around 5 J/cm2 has been identified as ideal [9, 10]. In three‐dimensional (3D) cell culture environments when using scaffolds such as hydrogel or Matrigel fluences ranging from 2 to 10 J/cm2 have been explored with focus mainly on differentiation when using PBM [11, 12], thus highlighting the significance of fluence calibration when shifting from 2D to 3D cell culture models.
Near‐infrared (NIR) light, especially at wavelengths that is around 825 nm, has demonstrated notably promising effects on stimulating cellular metabolism and enhancing tissue regeneration processes within the body [13]. Significant work in the application of PBM at 825 nm has been conducted primarily in 2D environments, which inherently limits the ability to translate findings effectively from in vitro laboratory studies to actual in vivo situations [14, 15].
The in vitro culture of ADSCs represents a vital and critical step in the application within various fields of regenerative medicine [16]. The choice made between 2D, and 3D cell culture models can significantly influence not only cell behavior but also their overall functionality and efficacy in therapeutic applications. These variations in culture methods can alter the cells' biochemical and mechanical environment, which ultimately impacts their growth, differentiation, and potential to repair damaged tissues [17, 18]. Traditional 2D cell culture methods, where cells are grown in a monolayer on flat surfaces, have been widely used in research. However, these methods often fail to replicate the complex interactions and microenvironments found in vivo. The 3D microenvironment more closely mimics the in vivo conditions, potentially providing a more accurate representation of the cellular responses to PBM [19, 20, 21]. One of the commonly used 3D models in 3D cell culture systems is the spheroid model. Spheroids are among the simplest and most effective 3D models, as cells aggregate to form compact cellular clusters with enhanced cell‐to‐cell interactions, resulting in a spherical formation. To date, spheroids have been widely used in various studies, including disease modeling, drug development, and testing [22, 23, 24].
Recent studies increasingly point to the synergistic potential of integrating PBM with 3D cell culture systems to enhance cell survival, viability, and functional maturation under physiologically relevant conditions. This dual application is rapidly gaining interest as researchers explore how precise light parameters within the optical therapeutic window can be tailored to optimize tissue‐engineered constructs and overcome common limitations of 3D cell culture, such as poor nutrient diffusion and reduced cell viability [12]. This study investigates the effects of PBM at 825 nm on ADSCs cultured in both 2D and 3D environments, focusing on cellular proliferation, cytotoxicity, and morphological changes. By comparing these two culture systems, we aim to elucidate the optimal conditions for the use of PBM in regenerative medicine.
Methods
Cell Culture
Immortalized adipose‐derived stem cells (ASC52telo hTERT, ATCC SCRC‐4000) were used in this study. Briefly, cells were cultured as monolayers in a T75 tissue culture flask, containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum, 1% penicillin–streptomycin, and 1% amphotericin B. Thereafter, cells were incubated in a 85% humidified incubator at 37°C with 5% carbon dioxide (CO2) and media was changed every 24 h. Upon confluency of 90%, cells were seeded in a 96‐well plate at a concentration of 9 × 103 in 200 μL in complete media as stated above. For 3D cell culture, cells were seeded in U‐bottom ultra‐low attachment 96‐well plate to allow for spheroid formation. The cells were incubated for 24 h prior to irradiation.
Photobiomodulation
Following 24‐h incubation, cells were irradiated using an 825 nm near‐infrared laser diode at a room temperature of 37°C in the dark. The laser beam (3.4 cm diameter) was positioned centrally to irradiate four wells simultaneously, with the beam midpoint aligned at the intersection of the four quadrants. Each spheroid was carefully placed at the center of it's respective well to ensure even energy distribution across all samples. Laser output power (mW) was verified before each session using a FieldMate Laser Power Meter (Coherent Inc., USA) to confirm consistent fluence delivery. All laser parameters, including energy, fluence, and exposure time, are provided in Table 1.
| Average power output (Mw) | Light source | Irradiance (Mw/cm)2 | Irradiation time (s) | Fluence (J/cm)2 |
|---|---|---|---|---|
| 180.58 ± 8.77 | Diode | 19.9 | 251.26 | 5 |
| Diode | 19.9 | 502.5 | 10 | |
| Diode | 19.9 | 753.75 | 15 |
Morphological Analysis
Morphological changes in 2D cell culture were visualized 24‐ and 72‐h post‐irradiation (hpi) using an inverted light microscope (Olympus CKX41, C5060‐ADUS) attached to a digital camera. Morphological changes and size diameter were observed and analyzed 24 and 72 hpi using Leica Mica confocal microscope (Leica Microsystems, Germany) under bright field for 3D cell culture.
Biochemical Assays
Trypan Blue Exclusion Cell Concentration
Cell concentration on viable cells in 2D cell culture was determined using the Trypan Blue dye exclusion assay. The quantitative viability assays were performed using the entire well to measure the concentration of viable cells. Briefly, a 1:1 ratio of an equal volume of Trypan Blue Stain (0.4%) was mixed with the cell suspension. The homogeneous mixture was then loaded onto a Countess Cell Counting Chamber Slide and analyzed using a Countess II Automated Cell Counter (ThermoFisher, AMQAX1000) to calculate the cell concentration of viable cells.
Live/Dead Cell Viability
Cellular viability was assessed in the control and experimental groups. Briefly cells and spheroids were washed thrice with 1X ice‐cold phosphate‐buffered saline (PBS) to remove any residual medium. The cells and spheroids were then dual stained with 1 μg/mL of acridine orange (AO) and ethidium bromide (EtBr) in PBS for 5 min at room temperature. AO stains viable cells by intercalating into DNA and emitting green fluorescence, while EtBr penetrates only compromised membranes, labeling non‐viable cells with red fluorescence [25, 26]. After staining, cells were rinsed thrice with 1X PBS to remove excess dye. Fluorescence imaging was performed using a Leica Mica confocal microscope (Leica Microsystems, Germany), utilizing AO and EtBr channels for visualization and analysis.
Membrane Cytotoxicity Assay (LDH)
Cell membrane integrity was evaluated using the CytoTox 96 Non‐Radioactive Cytotoxicity Assay (Promega, G179A, Madison, WI, USA), which measures the amount of lactate dehydrogenase (LDH) released from damaged cells. Absorbance values were colorimetrically recorded at 490 nm using the VICTOR3 Multilabel Plate Reader (Model 1420‐014, PerkinElmer Life and Analytical Sciences, Waltham, MA, USA).
Cell Proliferation (ATP) Assay
Cell proliferation was assessed using the CellTiter‐Glo 3D Cell Viability Assay (Promega, G968A, Madison, WI, USA), which quantifies ATP, through a luminescent reaction to detect metabolically active cell. Following the manufacturer's instructions, the luminescent signal was measured using the VICTOR3 Multilabel Plate Reader (Model 1420‐014, PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) and expressed in relative light units (RLUs).
Statistical Analysis
Data was expressed as mean ± standard error of the mean (SEM) from four biological replicates. Statistical comparisons between control and experimental groups were performed using two‐way ANOVA together with a Tukey post hoc test. All graphical analyses were conducted using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). Statistical significance was set at p < 0.05, with significance levels indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Results
2D Morphological Analysis and Cell Concentration
Morphological analysis in the 2D cell culture environment (Figure 1A), showed minor changes in both control and PBM‐treated groups. However, PBM treatment did not induce significant alterations in cell structure at either 24‐ or 72‐hpi when compared to the control. Cells retained their typical spindle‐like morphological characteristic of fibroblast‐like cells. However, there was an increase in the number of cells there were observed in all PBM treated groups both at 24 and 72 hpi, significantly at 5 J/cm2 by 72 hpi when compared to the control. Analysis of viable cell concentration (Figure 1B) showed a significant decrease 24 hpi following PBM treatment with 5 J/cm2 (p < 0.0001) and 10 J/cm2 (p < 0.0001), while the 15 J/cm2 group displayed a comparable concentration to the control. Over time, however, by 72 hpi, the concentration of viable cells was significantly increased across all treatment groups, particularly at 10 (p < 0.0001) and 15 J/cm2 (p < 0.0001).
Effect of PBM on cell morphology and viability in 2D cell culture. (A) Representative phase‐contrast microscopy images of cells at 24 and 72 hpi with PBM at doses of 5, 10, and 15 J/cm, compared to untreated control. Across all time points and treatment conditions, cells retained normal spindle‐shaped morphology, with no significant morphological alterations observed in PBM‐treated groups relative to controls. (B) Quantification of viable cell concentration at 24 and 72 hpi using a viability assay. At 24 hpi, treatment with 5 and 10 J/cmsignificantly decreased the concentration of viable cells compared to the control. At 72 hpi, an increase in cell concentration was observed in all PBM‐treated groups. Data are presented as mean ± SEM (= 4). Statistical analysis was performed using two‐way ANOVA followed by Tukey's post hoc test. *< 0.05; ****< 0.0001. 2 2 n p p
Spheroid Morphology and Size
Morphological characteristics (Figure 2A) of the spheroid was detected using bright field, it was observed that the spheroids maintained a uniform compact spherical shape in all PBM treated groups both 24 and 72 hpi when compared to the control. Spheroid size (Figure 2B) was measured and observed to have decreased in size in the 5 J/cm2 (p < 0.01) and 15 J/cm2 (p < 0.01) when compared to the control. Over time, by 72 hpi spheroid size was maintained at levels similar to that of the control in all PBM treated groups.
Effect of PBM on spheroid morphology and size in 3D cell culture. (A) Representative phase‐contrast microscopy images of spheroids at 24 and 72 hpi with PBM at doses of 5, 10, and 15 J/cm, compared to untreated control. (B) growth kinetics of spheroids in size 24 and 96 hpi. Initially 24 hpi there was a decrease in spheroid size at 5 and 15 J/cmwhen compared to the control. 2 2
Cell Viability
In a 2D cell culture environment, cellular viability analysis, Figure 3A shows a decreased cell viability was noted in cells exposed to PBM at 15 J/cm2, there was an increase in cell death at 5 and 10 J/cm2 over time at 72 hpi, but at 15 J/cm2 showed cell recovery (Figure 3B). In spheroid analysis, Figure 3C an increase in viable spheroid was observed in all PBM treatment groups. Over time, by 72 hpi, an increase in cell death was seen significantly at 15 J/cm2 treatment groups compared to the control (Figure 3D).
Representative micrographs showing cell viability (live/dead staining) in ADSCs following PBM treatment. (A) PBM treated groups and the control 24 h post‐irradiation (hpi) in 2D cell culture; (B) PBM‐treated group and the control, 72 hpi in 2D cell culture; (C) PBM‐treated groups and the control, 24 hpi in 3D cell culture; and (D) PBM‐treated groups and the control, 72 hpi in 3D cell culture. Live cells are stained green (Acridine Orange), and dead cells are stained red (Ethidium bromide). In 2D 15 J/cmat 72 hpi showed cellular recovery with increase in number of viable cells. In spheroids 15 J/cmat 72 hpi showed increased cell death compared to the control. 2 2
Membrane Integrity
Cellular membrane damage was assessed using the lactate dehydrogenase (LDH) assay by measuring the amount of LDH leakage into the cell culture media. An increase in LDH release is observed at 24 hpi in 2D, and it was observed to have increased significantly at 5 J/cm2 in 2D cell cultures compared to 3D cell cultures (Figure 4A). When comparing LDH release to the control, it was observed in the 5 J/cm2 (p < 0.001) and 15 J/cm2 (p < 0.001) in 2D cell culture. In spheroid analysis, when compared to the control, no significant difference was observed.
At 72 hpi, increased LDH release was significantly increased in 2D cell culture for both the control and PBM treated groups (Figure 4B). When comparing LDH release in PBM treated groups and the control, a significant increase was recorded at 5 (p < 0.05) and 15 J/cm2 (p < 0.01) while 10 J/cm2 (p < 0.01) showed a significant decrease. In spheroid analysis a significant decrease in LDH was observed in 10 (p < 0.001) and 15 J/cm2 (p < 0.0001) PBM‐treated groups. Despite the increase in LDH release, the levels were significantly lower than those observed in the positive control, which represented 100% cell death with complete membrane damage.
Comparison of cell membrane integrity between 2D monolayer and 3D spheroid following PBM treatment. Cell membrane integrity was assessed using the LDH release assay at two time points: (A) 24 and (B) 72 hpi. Data represent mean ± SEM from= 4 independent experiments. Statistical analysis was performed using two‐way ANOVA followed by Tukey's post hoc test to compare 2D versus spheroid groups at each time point. Statistical significance is indicated as *< 0.05, **< 0.01, ***< 0.001, and ****< 0.0001. n p p p p
Effects of PBM on Proliferation
Cellular proliferation was evaluated, 24 hpi (Figure 5A) in 2D cell culture, and ATP production statistically remained constant compared to the control. While in spheroids, a significant increase was observed in all PBM treated groups with 10 J/cm2 (p < 0.0001), showing the highest proliferation. Over time (Figure 5B), by 72 hpi in 2D cell culture, a significant increase in ATP production was observed at 5 J/cm2 (p < 0.0001) while it also significantly decreased at 15 J/cm2 (p < 0.0001). In spheroids, ATP production levels in all treatment groups were similar, with the 10 and 15 J/cm2 groups showing slightly higher ATP levels compared to the control.
Comparison of cellular proliferation between 2D monolayer and 3D spheroid following PBM treatment. Cellular proliferation was assessed using ATP assay at two time points: (A) 24 and (B) 72 hpi. Data represent mean ± SEM from= 4 independent experiments. Statistical analysis was performed using two‐way ANOVA followed by Tukey's post hoc test to compare 2D versus spheroid groups at each time point. Statistical significance is indicated as ***< 0.001 and ****< 0.0001. n p p
Discussion
The study elucidates the differential responses of ADSCs to PBM in 2D and 3D cell culture environments using key metrics such as morphological analysis, live/dead assays, LDH release, and ATP production. The application of PBM has increased interest in the application of regenerative medicine owing to its ability to improve ADSCs' proliferation without changing the cell properties [27]. The application of PBM in regenerative applications depends on the light parameters, which need to be optimized within the potential therapeutic window [28, 29]. While much work has been applied in the 2D cell culture environment which fail to represent the in vivo microenvironment.
The results confirm that PBM enhances ADSC proliferation and viability while minimizing cytotoxic effects when appropriately dosed, aligning with prior findings that highlight PBM's capacity to stimulate mitochondrial activity and promote cellular regeneration [12, 27, 28]. Our observations revealed that in 2D cell cultures, PBM at 5 J/cm2 enhanced proliferation and ATP synthesis, whereas higher fluences (10 and 15 J/cm2) induced morphological alterations such as cell shrinkage and detachment, indicating increased stress and possible phototoxic effects. This agrees with previous studies suggesting that PBM effects are biphasic, beneficial at lower doses but inhibitory at higher exposures [18, 22].
Conversely, in 3D spheroid cultures, PBM at 10 J/cm2 produced the most favorable response, characterized by increased metabolic activity and sustained viability over time. The 3D cell culture environment provided superior structural stability and resilience, likely due to enhanced cell–cell and cell–extracellular matrix (ECM) interactions, which buffer against PBM‐induced stress [20, 21]. These findings corroborate reports by Wu et al., who demonstrated that 3D spheroids exhibit greater resistance to photothermal and oxidative stress due to ECM‐mediated signaling and diffusion gradients that better mimic in vivo conditions [30]. This stability in 3D cell cultures is likely a reflection of the in vivo‐like conditions that the ECM provides, reducing the direct stress impact of PBM [12].
Live/dead staining assays showed increased cell death at 15 J/cm2 in the 2D environment over time. In the 3D environment, however, higher survival rates were observed, suggesting the protective role of the 3D ECM against PBM‐induced cytotoxicity [26]. LDH release, a marker of cellular membrane damage, was higher in 2D cell cultures, particularly at 5 J/cm2, compared to 3D cell cultures. This indicates greater membrane integrity disruption in the 2D environment. However, even in 2D cell cultures, the LDH release levels were significantly lower than the positive control, representing 100% cell death, highlighting PBM induced sublethal stress rather than extensive cytotoxicity [31].
ATP production analysis further underscored these differences. In 2D cell cultures, ATP levels were generally higher, reflecting elevated metabolic activity due to the easier access to nutrients and oxygen [32]. However, at 15 J/cm2, ATP production significantly dropped at 24 hpi, indicating initial metabolic stress. By 72 hpi, ATP production at 5 J/cm2 increased significantly, suggesting an adaptive metabolic response, while 15 J/cm2 maintained the lowest ATP production, indicating sustained metabolic inhibition [33]. In 3D cell cultures, ATP production peaked at 10 J/cm2 24 hpi, suggesting an optimal PBM dose for metabolic stimulation. Over time, ATP production levels across all treatment groups became similar, with the highest levels at 10 and 15 J/cm2, reflecting effective cellular adaptation and enhanced metabolic activity in the 3D environment [5, 12].
In a 2D environment, 5 J/cm2 was noted as the most optimal fluence for PBM, as it promoted ATP production, minimized cytotoxicity and membrane damage, and maintained cellular morphology and viability better than 10 or 15 J/cm2. This fluence offered a balance between stimulating cellular functions and avoiding excessive stress or damage. In a 3D environment, ATP the 10 J/cm2 fluence has been shown to optimally balance the metabolic stimulation and cell viability, promoting cellular health and function with minimal cytotoxic effects. This fluence level enhanced ATP production, maintained membrane integrity, and supported overall cellular resilience, making it the most effective PBM dose among the tested fluences. Over time, ATP levels converged across groups, with sustained high production at 10 and 15 J/cm2, indicating effective adaptation and metabolic resilience in 3D spheroids. Cevik et al. demonstrated that PBM enhances mitochondrial function and metabolic activity in 3D‐cultured stem cells, further supporting our findings [29].
This work emphasizes how crucial 3D spheroid culture environments are for giving a more accurate representation of in vivo circumstances, which serves as a link to translate in vitro results for clinical use. Optimizing PBM procedures and boosting the effectiveness of regenerative medicine treatments requires an understanding of the different cellular responses between 2D and 3D cell cultures. Consequently, even though 2D cell culture techniques have proven useful for several tests and investigations, it's critical to acknowledge their limitations in accurately simulating the intricacy of in vivo tissue settings. Cellular homeostasis and other physiological processes may be misunderstood or overestimated if 2D cell culture models are used excessively without taking into account the variations in cellular responses between 2D and 3D cell cultures [29, 34, 35]. All things considered, the findings demonstrate how crucial the culture environment is in determining how cells react to PBM. The 3D cell culture technique offers superior protection and fosters cellular resistance against PBM‐induced stress because of its in vivo‐like conditions. These results highlight how crucial it is to optimize PBM protocols in regenerative medicine utilizing 3D cell culture models in order to maximize therapeutic efficacy and minimize cellular harm.
However, the present study presents several limitations. First, the use of immortalized ADSCs, while advantageous for reproducibility, abundance, and low inter‐experimental variability, high proliferative rate, low apoptosis, and multipotent differentiation potential, may not fully represent the physiological behavior of primary ADSCs derived from donor tissue [14]. Second, spheroid size and morphology were monitored, more advanced analyses (e.g., oxygen and nutrient diffusion profiling or transcriptomic assessment) could provide deeper mechanistic insights into PBM effects in 3D systems. Third, the study focused primarily on viability and metabolic activity without assessing differentiation potential or functional outcomes. Future studies should therefore investigate PBM‐induced differentiation, ECM remodeling, and signaling pathway activation in 3D ADSC constructs, ideally using patient‐derived primary cells. Integration of PBM with biofabrication techniques such as hydrogel scaffolds or bioprinted constructs may further enhance translational relevance and optimize therapeutic applications in regenerative medicine.
Conclusion
This study demonstrates that ADSCs responses to PBM are highly dependent on the cell culture dimensionality and fluence applied. In 2D cell cultures, a fluence of 5 J/cm2 was optimal for enhancing metabolic activity and maintaining cellular integrity, while in 3D spheroids, 10 J/cm2 provided the most balanced response, enhancing viability and ATP production with minimal cytotoxic effects. Importantly, this work reinforces the need to contextualize PBM parameters within physiologically relevant 3D systems to avoid overestimation of in vitro effects. Although 2D models remain useful for initial mechanistic studies, 3D cell cultures provide a more predictive platform for translating PBM‐enhanced stem cell therapies into clinical practice. Future investigations incorporating primary ADSCs, differentiation assays, and long‐term functionality studies will be essential to fully elucidate PBM's regenerative potential and optimize its integration into tissue engineering and clinical regenerative protocols.
Author Contributions
Precious Earldom Mulaudzi, Anine Crous: conceptualization. Precious Earldom Mulaudzi, Anine Crous: data curation. Precious Earldom Mulaudzi, Anine Crous: formal analysis. Anine Crous and Heidi Abrahamse: funding acquisition. Precious Earldom Mulaudzi, Anine Crous: investigation. Precious Earldom Mulaudzi, Anine Crous: methodology. Heidi Abrahamse, Anine Crous: project administration. Heidi Abrahamse, Anine Crous: resources. Precious Earldom Mulaudzi: visualization. Precious Earldom Mulaudzi and Anine Crous: writing – original draft. Precious Earldom Mulaudzi, Heidi Abrahamse, and Anine Crous: writing – review and editing. All authors read and approved the final manuscript.
Funding
This research was funded by the National Research Foundation of South Africa Thuthuka Instrument, grant number TTK2205035996; the Department of Science and Innovation (DSI) funded African Laser Centre (ALC), grant number HLHA26X task ALC‐R003; the University Research Council, grant number 2024URC00813; the National Research Foundation Doctoral grant, grant number PMDS22070532778; and the Department of Science and Innovation South African Research Chairs Initiative (DSI‐NRF/SARChI), grant number 98337.
Ethics Statement
The study was reviewed and approved by the University of Johannesburg, Higher Degree Committee (HDC) and Research Ethics Committee (REC) clearance certificates (HDC‐01‐31‐2023 and REC‐1972‐2023).
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgments
The authors thank the University of Johannesburg (UJ), the Laser Research Centre (LRC) for use of their facilities and resources and the National Laser Centre for their laser equipment used in this study. We acknowledge Promolab Pty Ltd. T/A Separations for their partnership with the Laser Research Centre, providing the Leica Mica Microhub in support of the microscopy images featured in this article. The South African Medical Research Council (SAMRC), through its Division of Research Capacity Development under the Research Capacity Development Initiative from funding received from the South African National Treasury. The content and findings reported/illustrated are the sole deduction, view and responsibility of the researcher and do not reflect the official position and sentiments of the SAMRals.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
References
Associated Data
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.