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 HLHA23X task ALC-R007; the University Research Council, grant number 2022URC00513; the Department of Science and Technology&#39;s South African Research Chairs Initiative (DST-NRF/SARChI), grant number 98337. The funding bodies played no role in the design of the study, collection, analysis, interpretation of the data or writing the manuscript. The authors thank the University of Johannesburg (UJ) and Laser Research Centre (LRC) for their use of the facilities and resources.

NOTE: See the Table of Materials for details related to all materials, reagents, and software used in this protocol. The protocol has been graphically summarized in Figure 1.
1. Two- dimensional (2D) cell culture
NOTE: Immortalised ADSCs (1 x106 cells) are stored at -195.8 &#176;C in liquid nitrogen in a cryopreservation vial containing 1 mL of cell freezing media.
<ol>
	<li>Preparing the 2...

Photobiomodulation Growth Augmentation of Hydrogel Embedded Stem Cells 

<ol>
	<li>Zuk, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+cells+from+human+adipose+tissue:+Implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: Implications for cell-based therapies.</a> Tissue Eng. 7 (2), 211-228 (2001).</li><li>Yuan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+improving+adipose-derived+stem+cells+for+tissue+regeneration.">Strategies for improving adipose-derived stem cells for tissue regeneration.</a> Burns Trauma. 10, (2022).</li><li>Nilforoushzadeh, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+spheroids+embedded+in+an+injectable+thermosensitive+hydrogel:+An+in+situ+drug+formation+platform+for+accelerated+wound+healing.">Mesenchymal stem cell spheroids embedded in an injectable thermosensitive hydrogel: An in situ drug formation platform for accelerated wound healing.</a> ACS Biomater Sci Eng. 6 (9), 5096-5109 (2020).</li><li>Yang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermosensitive+injectable+chitosan/collagen/&#946;-glycerophosphate+composite+hydrogels+for+enhancing+wound+healing+by+encapsulating+mesenchymal+stem+cell+spheroids.">Thermosensitive injectable chitosan/collagen/&#946;-glycerophosphate composite hydrogels for enhancing wound healing by encapsulating mesenchymal stem cell spheroids.</a> ACS Omega. 5 (33), 21015-21023 (2020).</li><li>Chimenti, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+roles+of+direct+regeneration+versus+paracrine+effects+of+human+cardiosphere-derived+cells+transplanted+into+infarcted+mice.">Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice.</a> Circ Res. 106 (5), 971-980 (2010).</li><li>Hassan, W., Dong, Y., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+and+3d+culture+of+human+adipose-derived+stem+cells+in+an+in-situ+crosslinked+hybrid+hydrogel+composed+of+peg-based+hyperbranched+copolymer+and+hyaluronic+acid.">Encapsulation and 3d culture of human adipose-derived stem cells in an in-situ crosslinked hybrid hydrogel composed of peg-based hyperbranched copolymer and hyaluronic acid.</a> Stem Cell Res Ther. 4 (2), 32 (2013).</li><li>Wu, K. H., Mo, X. M., Han, Z. C., Zhou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+engraftment+and+survival+in+the+ischemic+heart.">Stem cell engraftment and survival in the ischemic heart.</a> The Ann Thorac Surg. 92 (5), 1917-1925 (2011).</li><li>Lee, K., Silva, E. A., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factor+delivery-based+tissue+engineering:+General+approaches+and+a+review+of+recent+developments.">Growth factor delivery-based tissue engineering: General approaches and a review of recent developments.</a> J R Soc Interface. 8 (55), 153-170 (2011).</li><li>Koivunotko, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+potential+of+human+adipose-derived+mesenchymal+stromal+cells+in+nanofibrillated+cellulose+hydrogel.">Angiogenic potential of human adipose-derived mesenchymal stromal cells in nanofibrillated cellulose hydrogel.</a> Biomedicines. 10 (10), 2584 (2022).</li><li>Dong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+and+tunable+gelatin+hydrogels+enhance+stem+cell+retention+and+improve+cutaneous+wound+healing.">Injectable and tunable gelatin hydrogels enhance stem cell retention and improve cutaneous wound healing.</a> Adv Funct Mater. 27 (24), 1606619 (2017).</li><li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+extracellular+matrix+mimics+for+3d+cell+culture.">Hydrogels as extracellular matrix mimics for 3d cell culture.</a> Biotechnol Bioeng. 103 (4), 655-663 (2009).</li><li>Mantha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart+hydrogels+in+tissue+engineering+and+regenerative+medicine.">Smart hydrogels in tissue engineering and regenerative medicine.</a> Materials. 12 (20), 3323 (2019).</li><li>Sung, T. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+culturing+of+human+adipose-derived+stem+cells+enhances+their+pluripotency+and+differentiation+abilities.">3D culturing of human adipose-derived stem cells enhances their pluripotency and differentiation abilities.</a> J Mater Sci Technol. 63, 9-17 (2021).</li><li>Garg, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+force+seeding+of+hydrogels+for+adipose-derived+stem+cell+delivery+in+wounds.">Capillary force seeding of hydrogels for adipose-derived stem cell delivery in wounds.</a> Stem Cells Transl Med. 3 (9), 1079-1089 (2014).</li><li>Kim, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cell-containing+hyaluronic+acid/alginate+hydrogel+improves+vocal+fold+wound+healing.">Adipose-derived stem cell-containing hyaluronic acid/alginate hydrogel improves vocal fold wound healing.</a> Laryngoscope. 124 (3), E64-E72 (2014).</li><li>Dong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformable+hyaluronic+acid+hydrogel+delivers+adipose-derived+stem+cells+and+promotes+regeneration+of+burn+injury.">Conformable hyaluronic acid hydrogel delivers adipose-derived stem cells and promotes regeneration of burn injury.</a> Acta Biomater. 108, 56-66 (2020).</li><li>Truegel3d hydrogel for 3d cell culture. Merck Available from: <a target="_blank" href="https://www.sigmaaldrich.com/ZA/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/3d-cell-culture/truegel3d">https://www.sigmaaldrich.com/ZA/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/3d-cell-culture/truegel3d</a> (2024)</li><li>Braccini, S., Tacchini, C., Chiellini, F., Puppi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+hydrogels+for+in+vitro+3d+ovarian+cancer+modeling.">Polymeric hydrogels for in vitro 3d ovarian cancer modeling.</a> Int J Mol Sci. 23 (6), 3265 (2022).</li><li>Mashinchian, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+transcriptomic+profiling+using+cell+encapsulation+identifies+effector+pathways+of+systemic+aging.">In vivo transcriptomic profiling using cell encapsulation identifies effector pathways of systemic aging.</a> eLife. 11, e57393 (2022).</li><li>Matsushige, C., Xu, X., Miyagi, M., Zuo, Y. Y., Yamazaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rgd-modified+dextran+hydrogel+promotes+follicle+growth+in+three-dimensional+ovarian+tissue+culture+in+mice.">Rgd-modified dextran hydrogel promotes follicle growth in three-dimensional ovarian tissue culture in mice.</a> Theriogenology. 183, 120-131 (2022).</li><li>Marx, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+some+labs+put+more+bio+into+biomaterials.">How some labs put more bio into biomaterials.</a> Nat Methods. 16 (5), 365-368 (2019).</li><li>Marques, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobiomodulation+therapy+weaknesses.">Photobiomodulation therapy weaknesses.</a> Laser Dent Sci. 6 (3), 131-132 (2022).</li><li>Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+mitochondrial+redox+signaling+in+photobiomodulation.">Mechanisms and mitochondrial redox signaling in photobiomodulation.</a> Photochem Photobiol. 94 (2), 199-212 (2018).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-level+controllable+blue+LEDs+irradiation+enhances+human+dental+pulp+stem+cells+osteogenic+differentiation+via+transient+receptor+potential+vanilloid+1.">Low-level controllable blue LEDs irradiation enhances human dental pulp stem cells osteogenic differentiation via transient receptor potential vanilloid 1.</a> J Photochem Photobiol B. 233, 112472 (2022).</li><li>Chang, S. -. Y., Carpena, N. T., Kang, B. J., Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+photobiomodulation+on+stem+cells+important+for+regenerative+medicine.">Effects of photobiomodulation on stem cells important for regenerative medicine.</a> Med Lasers. 9 (2), 134-141 (2020).</li><li>Bikmulina, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+2d:+Effects+of+photobiomodulation+in+3d+tissue-like+systems.">Beyond 2d: Effects of photobiomodulation in 3d tissue-like systems.</a> J Biomed Opt. 25 (4), 048001 (2020).</li><li>Ahmadi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+photobiomodulation-preconditioned+diabetic+stem+cells+accelerates+ischemic+wound+healing+in+diabetic+rats.">Transplantation of photobiomodulation-preconditioned diabetic stem cells accelerates ischemic wound healing in diabetic rats.</a> Stem Cell Res Ther. 11 (1), 494 (2020).</li><li>Mao, A. S., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine:+Current+therapies+and+future+directions.">Regenerative medicine: Current therapies and future directions.</a> Proc Natl Acad Sci U S A. 112 (47), 14452-14459 (2015).</li><li>De Andrade, A. L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+photobiomodulation+on+the+behaviour+of+mesenchymal+stem+cells+in+three-dimensional+cultures.">Effect of photobiomodulation on the behaviour of mesenchymal stem cells in three-dimensional cultures.</a> Lasers Med Sci. 38 (1), 221 (2023).</li><li>Diniz, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobiomodulation+of+mesenchymal+stem+cells+encapsulated+in+an+injectable+rhbmp4-loaded+hydrogel+directs+hard+tissue+bioengineering.">Photobiomodulation of mesenchymal stem cells encapsulated in an injectable rhbmp4-loaded hydrogel directs hard tissue bioengineering.</a> J Cell Physiol. 233 (6), 4907-4918 (2018).</li><li>Carter, M., Shieh, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to Research Techniques in Neuroscience. , (2015).</li><li>Lutolf, M. 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P., Sutherland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shedding+of+mitotic+cells+from+the+surface+of+multicell+spheroids+during+growth.">Shedding of mitotic cells from the surface of multicell spheroids during growth.</a> J Cell Physiol. 106 (1), 23-32 (1981).</li><li>Bogacheva, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+pluripotent+stem+cells+into+definitive+endoderm+cells+in+various+flexible+three-dimensional+cell+culture+systems:+Possibilities+and+limitations.">Differentiation of human pluripotent stem cells into definitive endoderm cells in various flexible three-dimensional cell culture systems: Possibilities and limitations.</a> Front Cell Dev Biol. 9, 726499 (2021).</li><li>Chen, X., Thibeault, S. 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ADSCs are an ideal cell type to use for regenerative medicine as they stimulate various processes to aid in wound healing3,4. However, there are several challenges that need to be circumvented, e.g., poor survival rates and ineffective engraftment of the cells in an injury site9. Immortalized cells were used as a commercially available cell line, as they can be passaged for more generations compared to primary cells, they do not need to be...

ADSCs are mesenchymal multipotent progenitor cells with the capacity to self-renew and differentiate into several cell lineages. These cells can be harvested from the stromal vascular fraction (SVF) of adipose tissue during a lipoaspiration procedure1. ADSCs have emerged as an ideal stem cell type to use in regenerative medicine because these cells are abundant, minimally invasive to harvest, easily accessible, and well characterized2. Stem cell therapy offers a possible avenue for wound healing by stimulating cell migration, proliferation, neovascularization, and reducing inflammation within wounds3,4. Roughly 80% of the regenerative capacity of ADSCs is attributable to paracrine signaling via their secretome5. Previously, it was suggested a direct local injection of stem cells or growth factors into damaged tissue could illicit sufficient in vivo repair mechanisms6,7,8. However, this approach faced several challenges, such as poor survival and reduced stem cell engraftment within damaged tissues as a result of the inflammatory environment 9. Furthermore, one of the reasons cited was a lack of an extracellular matrix to support the survival and functionality of the transplanted cells10. To overcome these challenges, emphasis is now being placed on the development of biomaterial carriers to support stem cell viability and function.
Three-dimensional (3D) cell culture enhances cell-to-cell and cell-to-matrix interaction in vitro to provide an environment that better resembles the in vivo environment11. Hydrogels have been extensively studied as a class of biomaterial carriers that provide a 3D environment for stem cell culture. These structures are made of water and crosslinked polymers12. Encapsulation of ADSCs in hydrogel has virtually no cytotoxic effect on the cells during culture while maintaining the viability of the cells6. Stem cells cultured in 3D demonstrate enhanced retention of their stemness and improved differentiation capacity13. Similarly, hydrogel-seeded ADSCs demonstrated increased viability and accelerated wound closure in animal models14. Furthermore, hydrogel encapsulation significantly increases the engraftment and retention of ADSCs in wounds15,16. TrueGel3D is made of a polymer, either polyvinyl alcohol or dextran, solidified by a crosslinker, either cyclodextrin or polyethylene glycol17. The gel is a synthetic hydrogel that does not contain any animal products that may interfere with the experiments or trigger an immune reaction during the transplantation of the gel into a patient while effectively mimicking an extracellular matrix18. The gel is fully customizable by altering the composition and individual components. It can house different stem cells and support the differentiation of several cell types by adjusting the stiffness of the gel19. Attachment sites can be created through the addition of peptides20. The gel is degradable by the secretion of metalloproteases, allowing for cell migration21. Lastly, it is clear and allows for imaging techniques.
PBM is a minimally invasive and easily performed form of low-level laser therapy used to stimulate intracellular chromophores. Different wavelengths elicit different effects on cells22. Light in the red to near-infrared range stimulates increased adenosine triphosphate (ATP) and reactive oxygen species (ROS) production by enhancing flux through the electron transport chain23. Light in the blue and green ranges stimulates light-gated ion channels, allowing for the non-specific influx of cations, such as calcium and magnesium, into cells, which is known to enhance differentiation24. The net effect is the generation of secondary messengers that stimulate the transcription of factors triggering downstream cellular processes such as migration, proliferation, and differentiation25. PBM can be used to pre-condition cells to proliferate or differentiate before transplanting the cells into an adverse environment, e.g., damaged tissue26. Pre- and post-transplant PBM (630 nm and 810 nm) exposure of ADSCs significantly enhanced the viability and function of these cells in vivo in a diabetic rat model27. Regenerative medicine requires an adequate number of cells for effective repair of tissues28. In 3D cell culture, ADSCs have been associated with slower proliferation rates compared to two-dimensional cell culture6. However, PBM can be used to augment the 3D cell culture process of ADSCs by enhancing viability, proliferation, migration, and differentiation29,30.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>525 nm diode laser</td>
			<td>National Laser Centre of South Africa</td>
			<td>EN 60825-1:2007</td>
			<td></td>
		</tr>
		<tr>
			<td>825 nm diode laser</td>
			<td>National Laser Centre of South Africa</td>
			<td>SN 101080908ADR-1800</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Strip Plates</td>
			<td>Sigma-Aldrich</td>
			<td>BR782301</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td>Antibiotic (0.5%; 0.5 mL)</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell Viability Assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td>ATP reagent, Proliferation assay Kit</td>
		</tr>
		<tr>
			<td>Corning 2 mL External Threaded Polypropylene Cryogenic Vial</td>
			<td>Corning</td>
			<td>430659</td>
			<td>cryovial</td>
		</tr>
		<tr>
			<td>CryoSOfree</td>
			<td>Sigma-Aldrich</td>
			<td>C9249</td>
			<td>Cell freezing media</td>
		</tr>
		<tr>
			<td>CytoTox96 Non-Radioactive Cytotoxicity Assay</td>
			<td>Promega</td>
			<td>G1780</td>
			<td>Cytotoxicity reagent</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Media</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td>Basal medium (39 mL/44 mL)</td>
		</tr>
		<tr>
			<td>FieldMate Laser Power Meter</td>
			<td>Coherent</td>
			<td>1098297</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-bottomed Corning 96 well clear polystyrene plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3370</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>Biochrom</td>
			<td>S0615</td>
			<td>Culture medium enrichment (5 mL; 10% / 10 mL; 20%)</td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Sigma-Aldrich</td>
			<td>H9394</td>
			<td>Rinse solution</td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>51026280</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus Labofuge 400</td>
			<td>Thermo Scientific</td>
			<td>75008371</td>
			<td>Plate spinner for 96 well plates</td>
		</tr>
		<tr>
			<td>Heraeus Megafuge 16R centrifuge</td>
			<td>ThermoFisher</td>
			<td>75004270</td>
			<td></td>
		</tr>
		<tr>
			<td>Immortalized ADSCs</td>
			<td>ATCC</td>
			<td>ASC52Telo hTERT, ATCC SCRC-4000</td>
			<td>Passage 37</td>
		</tr>
		<tr>
			<td>Invitrogen Countess 3</td>
			<td>Invitrogen</td>
			<td>AMQAX2000</td>
			<td>Automated cell counter for Trypan Blue</td>
		</tr>
		<tr>
			<td>Julabo TW20 waterbath</td>
			<td>Sigma-Aldrich</td>
			<td>Z615501</td>
			<td>Waterbath used to warm media to 37 &#176;C</td>
		</tr>
		<tr>
			<td>Olympus CellSens Entry</td>
			<td>Olympus</td>
			<td>Version 3.2 (23706)&#160;</td>
			<td>Imaging software: digital image acquisition</td>
		</tr>
		<tr>
			<td>Olympus CKX41</td>
			<td>Olympus</td>
			<td>SN9B02019</td>
			<td>Inverted light microscope</td>
		</tr>
		<tr>
			<td>Olympus SC30 camera</td>
			<td>Olympus</td>
			<td>SN57000530</td>
			<td>Camera attached to inverted light microscope</td>
		</tr>
		<tr>
			<td>Opaque-walled Corning 96 well solid polystyrene microplates</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3912</td>
			<td>Opaque well used for ATP luminescence</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td>Antibiotic (0.5%; 0.5 mL)</td>
		</tr>
		<tr>
			<td>SigmaPlot 12.0</td>
			<td>Systat Software Incorporated</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrueGel3D &#8211; True3</td>
			<td>Sigma-Aldrich</td>
			<td>TRUE3-1KT</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>TrueGel3D Enzymatic Cell Recovery Solution</td>
			<td>Sigma-Aldrich</td>
			<td>TRUEENZ</td>
			<td>01:20</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Thermo Fisher - Invitrogen</td>
			<td>T10282</td>
			<td>0.4% solution</td>
		</tr>
		<tr>
			<td>TrypLE Select Enzyme (1x)</td>
			<td>Gibco</td>
			<td>12563029</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Victor Nivo Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>HH3522019094</td>
			<td>Spectrophotometric plate reader</td>
		</tr>
	</tbody>
</table>

three&#45;dimensional cell culture of adipose-derived stem cells in a hydrogel with photobiomodulation augmentation

Adipose-derived stem cells (ADSCs), possessing multipotent mesenchymal characteristics akin to stem cells, are frequently employed in regenerative medicine due to their capacity for a diverse range of cell differentiation and their ability to enhance migration, proliferation, and mitigate inflammation. However, ADSCs often face challenges in survival and engraftment within wounds, primarily due to unfavorable inflammatory conditions. To address this issue, hydrogels have been developed to sustain ADSC viability in wounds and expedite the wound healing process. Here, we aimed to assess the synergistic impact of photobiomodulation (PBM) on ADSC proliferation and cytotoxicity within a 3D cell culture framework. Immortalized ADSCs were seeded into 10 &#181;L hydrogels at a density of 2.5 x 103 cells and subjected to irradiation using 525 nm and 825 nm diodes at fluencies of 5 J/cm2 and 10 J/cm2. Morphological changes, cytotoxicity, and proliferation were evaluated at 24 h and 10 days post-PBM exposure. The ADSCs exhibited a rounded morphology and were dispersed throughout the gel as individual cells or spheroid aggregates. Importantly, both PBM and 3D culture framework displayed no cytotoxic effects on the cells, while PBM significantly enhanced the proliferation rates of ADSCs. In conclusion, this study demonstrates the use of hydrogel as a suitable 3D environment for ADSC culture and introduces PBM as a significant augmentation strategy, particularly addressing the slow proliferation rates associated with 3D cell culture.

Author Spotlight: Advancements in Stem Cell Regenerative Therapy Through Photobiomodulation

Three&#45;Dimensional Cell Culture of Adipose-Derived Stem Cells in a Hydrogel with Photobiomodulation Augmentation

The Laser Research Centre are leading efforts to investigate phototherapy or laser radiation of biological tissue, focusing on stem cell photobiomodulation for regenerative therapy. In this study, we investigated stem cell photobiomodulation in adipose-derived stem cells using three-dimensional cell culture. Recent research has been focusing on combining photobiomodulation and adipose-derived stem cells for stem cell regenerative therapy.Studies have indicated that photobiomodulation enhances stem cell viability, proliferation, and differentiation potential. 3D cell culture techniques are an advancement of photobiomodulation research. As 3D cell culture supplies us with a physiologically relevant environment to study cell behavior.The current study demonstrated that hydrogel has no cytotoxic effect on cells over a 10-day culture period. And that photobiomodulation has significant positive augmentative potential and can significantly up-regulate the proliferation rates of adipose-derived mesenchymal stem cells despite being in a three-dimensional cell culture. The results of the current study will pave the way for the development of standardized photobiomodulation parameters and culture conditions for the differentiation of adipose-derived mesenchymal stem cells into various specialized types of cells, especially tenocytes.

To assess the morphology and visually inspect the cell density of the hydrogels, inverse microscopy was used (Figure 2). The ADSCs retained a rounded morphology 24 h after seeding and PBM exposure. The cells were scattered throughout the gel as single cells or in grape-like clusters. The morphology was unchanged after 10 days in 3D culture. No definitive difference in morphology was noted between the experimental groups and controls or between the different experimental groups.
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Watch this Scientific Journal Video about Advancements in Stem Cell Regenerative Therapy Through Photobiomodulation at JoVE.com

Here, we present a protocol demonstrating the use of hydrogel as a three-dimensional (3D) cell culture framework for adipose-derived stem cell (ADSC) culture and introducing photobiomodulation (PBM) to enhance the proliferation of ADSCs within the 3D culture setting.

Adipose-derived stem cells (ADSCs), possessing multipotent mesenchymal characteristics akin to stem cells, are frequently employed in regenerative ...

threedimensional-cell-culture-adipose-derived-stem-cells-hydrogel