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 2D cell recovery medium and 2D culture flask
	<ol>
		<li>Transfer 39 mL of the basal medium into a 50 mL centrifuge tube</li>
		<li>Enrich the basal medium with 20% fetal bovine serum (FBS; 10 mL) and antibiotics (1 mL) (0.5 mL Penicillin-Streptomycin and 0.5 mL Amphotericin B).</li>
		<li>Pre-condition a T75 flask by transferring 6 mL of the cell recovery medium into the flask and incubate it at 37 &#176;C, 5% CO2, and 85% humidity for 45 min (this can be done while performing the cell recovery steps to save time). 
		NOTE: Warm the complete medium to 37 &#176;C before use and store it at 4 &#176;C for a maximum period of 1 week.</li>
	</ol>
	</li>
	<li>2D cell recovery
	<ol>
		<li>Remove a cryovial from the liquid nitrogen storage tank and rapidly defrost the vial in a water bath at 37 &#176;C, until it is completely defrosted.</li>
		<li>Spin down the cryovial using a centrifuge at 1006 x g for 5 min (20 &#176;C) and thereafter remove the supernatant.</li>
		<li>Resuspend the pelleted cells using 1 mL of pre-warmed cell recovery medium.</li>
		<li>Evenly distribute the suspended cells in the pre-conditioned flask (final volume of 7 mL) and incubate at 37 &#176;C, 5% CO2, and 85% humidity.</li>
		<li>After 24 h discard and replace the cell recovery medium.</li>
		<li>Replace the culture medium every 2-3 days until the flask becomes confluent with cells.</li>
	</ol>
	</li>
	<li>Harvest of 2D culture flask in single cell suspension and cell count
	<ol>
		<li>Remove the 2D culture flask from the incubator and discard the culture media into an appropriate waste container.</li>
		<li>Transfer 10 mL of rinse solution into the flask to rinse the cells and remove waste and protein build-up. After that, discard the rinse solution.</li>
		<li>Add 6 mL of detachment solution to the culture flask to detach the cells. Incubate the flask at 37 &#176;C, 5% CO2, and 85% humidity for 2 min. 
		NOTE: Observe the flask under a microscope to confirm that all cells have been detached. If not, gently tap on the flask and incubate for another minute.</li>
		<li>Once all of the cells have been detached, transfer the cell suspension into a 15 mL centrifuge tube and add 1 mL of the cell recovery media (containing 20% FBS) to the tube. This will neutralize the effect of the detachment solution.</li>
		<li>Centrifuge the tube for 5 min at 1711 x g (20 &#176;C).</li>
		<li>Remove the supernatant and resuspend the cells in 1 mL of complete media.</li>
		<li>Remove 10 &#181;L of the cell suspension, place it in a 500 &#181;L microcentrifuge tube and mix it with 10 &#181;L of trypan blue in a 1:1 ratio.</li>
		<li>Place 10 &#181;L of the trypan blue cell solution mix in a cell counting chamber in duplicate.</li>
		<li>Place the cell counting chamber into the automated cell counter. The cell counter will indicate the total number of cells per mL as well as the number of viable and dead cells. Calculate the average viable cell count and the number of viable cells needed to be embedded in the 3D hydrogel (step 2.2.6).</li>
	</ol>
	</li>
</ol>
2. 3D cell culture
<ol>
	<li>Preparing the 3D complete culture medium
	<ol>
		<li>Transfer 44 mL of the basal medium into a 50 mL centrifuge tube.</li>
		<li>Enrich the media with 10% FBS (5 mL) and antibiotics (1 mL) (0.5 mL Penicillin-Streptomycin and 0.5 mL Amphotericin B). 
		NOTE: Warm the complete media to 37 &#176;C before use and store it at 4 &#176;C for a maximum of one week.</li>
	</ol>
	</li>
	<li>Preparing the hydrogel
	<ol>
		<li>Prepare the fast dextran by centrifuging (1000 x g for 30 s at 20 &#176;C) the fast-dextran vial to concentrate the material. Add 175 &#181;L of water to the fast-dextran vial to achieve a final concentration of 30 mmol/L reactive groups.</li>
		<li>Vortex the tube (medium speed for 30 s) containing the fast-dextran solution until the material is fully dissolved. Incubate the dissolved material on ice for 5 min. Centrifuge the tube, briefly vortex it, and keep it on ice during further use.</li>
		<li>Centrifuge the crosslinker (1000 x g for 30 s at 20 &#176;C) to concentrate the material. Add 188 &#181;L of water to the crosslinker vial to achieve a final concentration of 20 mmol/L of thiol groups.</li>
		<li>Vortex the tube (medium speed for 30 s) containing the crosslinker solution until all material is dissolved. Incubate the dissolved crosslinker at room temperature (RT) for 5 min. Centrifuge the tube, briefly vortex, and keep it at RT during further use.</li>
		<li>Adapt the hydrogel protocol from 30 &#181;L to 10 &#181;L for cost reduction (see Table 1 for specific details).</li>
		<li>Prepare the cell suspension to achieve a seeding density of 2.5 x 103 cells per 10 &#181;L hydrogel.</li>
		<li>Combine the components according to Table 1 to create a master mix. Transfer 9 &#181;L of the master mix to a well in a 96-well strip plate.</li>
		<li>Solidify the gel by adding 1 &#181;L of the degradable crosslinker 3 min after the master mix is transferred.</li>
		<li>Cover the hydrogels with 170 &#181;L pre-warmed complete culture media. Incubate for 1 h. After 1 h, replace the culture medium. 
		NOTE: The hydrogel-embedded cells are ready for further experimentation or analysis within the 3D culture system. Follow additional protocols as needed for downstream applications.</li>
	</ol>
	</li>
</ol>
3. Photobiomodulation exposure
<ol>
	<li>Setting up the laser
	<ol>
		<li>Set up the diode laser system for either green (525 nm) or near-infrared (825 nm) wavelengths. Switch on the lasers and allow them to warm up for the recommended duration.</li>
		<li>Stabilize the power output of the lasers by allowing them to reach a steady state. This ensures consistent and accurate irradiation.</li>
		<li>Measure the power output of the lasers using a laser power meter. Record the power values for each wavelength.</li>
		<li>Use Equation 1 ( Laser irradiation time) to calculate the laser irradiation time. Substitute the values from Table 2 into Equation 1 to determine the appropriate irradiation time for each fluency. In Equation 1, &#34;r&#34; represents the radius of the irradiation zone of the laser. 
		<img alt="Equation 1" src="/files/ftp_upload/66616/66616eq01.jpg" style="margin: auto;" /> 
		<img alt="Equation 2" src="/files/ftp_upload/66616/66616eq02.jpg" style="margin: auto;" /> 
		<img alt="Equation 3" src="/files/ftp_upload/66616/66616eq03.jpg" style="margin: auto;" /> 
		Equation 1</li>
		<li>Refer to Table 2 for the specific parameters needed for the calculation, including fluencies (5 J/cm&#178; or 10 J/cm&#178;) and laser power output.</li>
	</ol>
	</li>
	<li>Irradiating the 3D cell cultures
	<ol>
		<li>After replacing the complete media (step 2.2.9), place the 96-well plate containing hydrogel-embedded cells under the diode laser system. Irradiate the hydrogels with the calculated laser irradiation time for the selected fluency (5 J/cm&#178; or 10 J/cm&#178;) at the chosen wavelength (green or near-infrared). Therefore, the cultures will only receive a single dose.</li>
		<li>Ensure that each experimental group is accompanied by a control (n = 4), consisting of ADSCs embedded in hydrogel without PBM exposure. 
		NOTE: Always wear safety glasses appropriate for the wavelength of light you are working with. The lasers are used outside of a safety cabinet; thus, it is crucially important to disinfect the area before use.</li>
		<li>Incubate the culture plates at 37 &#176;C, 5% CO2, and 85% humidity for the duration of the experiments. Incubate the plates for 24 h for the first analysis or 10 days for the second analysis period, post-irradiation.</li>
	</ol>
	</li>
</ol>
4. Morphology
<ol>
	<li>Inverted light microscopy
	<ol>
		<li>Turn on the inverted light microscope and allow it to warm up for a few minutes. Ensure the microscope is properly aligned and calibrated.</li>
		<li>Prepare the sample for observation on a microscope slide or a specialized cell culture dish, depending on the experimental setup. Calibrate the microscope for optimal imaging conditions.</li>
		<li>Adjust the focus, brightness, and contrast settings as needed. Select the 20x objective lens on the microscope for observing and recording cell morphology.</li>
		<li>Place the sample on the microscope stage and focus on the cells of interest using the eyepiece. Using the eyepiece or camera view, observe and record the cell morphology. Pay attention to cell shape, size, and any distinctive features.</li>
		<li>Attach the camera module to the microscope. Set up the camera for digital imaging. Ensure proper connection and communication between the camera and the microscope.</li>
		<li>Using the 20x objective lens, capture digital pictures of the observed cells. Use the camera controls to adjust exposure, focus, and other relevant settings. 
		NOTE: Ensure that the captured images are of high quality and provide a representative view of the cell morphology.</li>
		<li>Launch the imaging software on the computer. Capture the image and use the analysis tools within the software to analyze cell morphology.</li>
		<li>Measure cell dimensions, count cells, or perform any relevant analysis based on the experimental objectives.</li>
		<li>Add an appropriate scale bar to the images using the software. Use the known magnification of the microscope objective to accurately represent the scale of the images.</li>
	</ol>
	</li>
</ol>
5. Biochemical assays
<ol>
	<li>Cell recovery 
	NOTE: The cells need to be recovered from the hydrogel in a single-cell suspension for further downstream biochemical assays.
	<ol>
		<li>Prepare the enzymatic cell recovery solution. In a sterile environment, prepare a working solution by diluting the enzymatic cell recovery solution with phosphate-buffered saline (PBS) at a ratio of 1:20 (Solution: PBS). If recovering cells from a specific number of gels or wells is required, calculate the total volume required based on 30 &#181;L of recovery solution per gel.</li>
		<li>Add 30 &#181;L of the working solution to each gel or well containing the cells needing recovery. Ensure thorough and even distribution of the recovery solution by gently rocking or swirling the plate. Incubate the plate with the recovery solution according to the recommended incubation time. This information can typically be found in the product datasheet or protocol provided by the supplier.</li>
		<li>After the incubation period, carefully remove the plate from the incubator. Centrifuge the plates at 1409 x g for 5 min (20 &#176;C) to pellet the cells. Refer to the product datasheet or protocol for recommended centrifugation conditions. Once centrifugation is complete, carefully discard the supernatant, being cautious not to disturb the cell pellet.</li>
		<li>Gently resuspend the pelleted cells in 220 &#181;L of sterile PBS. Ensure thorough mixing to achieve a homogeneous single-cell suspension. The cells are now ready for downstream biochemical assays. Follow the specific protocols for the intended assays, considering the nature of the recovered cells and the requirements of the experimental setup.</li>
	</ol>
	</li>
	<li>Lactate dehydrogenase (LDH) cytotoxicity assay
	<ol>
		<li>For each well, carefully remove 50 &#181;L of complete culture media, ensuring minimal disturbance to the cell layer.</li>
		<li>Add 50 &#181;L of the cytotoxicity reagent directly to each well containing the remaining 50 &#181;L of culture media. Mix gently by pipetting up and down to ensure thorough mixing of the sample with the reagent.</li>
		<li>Prepare a positive control. Set up triplicate wells with 5 &#181;L of 10x lysis solution per 50 &#181;L of positive control cells included in the kit.</li>
		<li>Incubate the plate at the recommended temperature and duration. This step allows the cytotoxicity reagent to lyse the cells and release LDH into the culture media.</li>
		<li>After the incubation period, add 50 &#181;L of the stop solution directly to each well containing the cytotoxicity reagent and cell lysate. Mix gently by pipetting up and down to ensure proper stopping of the reaction. 
		NOTE: The stop solution halts further LDH release and ensures the stability of the color development.</li>
		<li>Set up the spectrophotometric plate reader according to the manufacturer&#39;s instructions. Record the absorbance of each well at 490 nm. This wavelength is specific to the colorimetric detection of the formazan product generated by the LDH reaction.</li>
		<li>Subtract background absorbance readings from both treatment groups and control wells. Obtain background readings from wells containing media and cytotoxicity reagents without cells.</li>
		<li>Analyze the corrected absorbance values for each sample to determine the cytotoxicity level. Higher absorbance values indicate increased LDH release and, consequently, higher cytotoxicity.</li>
		<li>Ensure that each experimental group and control consisted of four repeats (step 3.2.2) for statistical accuracy. Calculate and import the average absorbance of each group and control into an appropriate statistical program.</li>
		<li>Compare the results between treatment groups and appropriate controls to assess the cytotoxic effects accurately and record all absorbance readings, calculations, and experimental conditions for future reference.</li>
	</ol>
	</li>
	<li>ATP proliferation assay
	<ol>
		<li>In a sterile environment, mix 50 &#181;L of the recovered cells with 50 &#181;L of the ATP reagent in each well. 
		NOTE: Adjust the volume of cells and reagents based on the number of wells and experimental design. Maintain consistent ratios for accurate results.</li>
		<li>Place the plate on a shaker and shake vigorously in the dark for 5 min. This step ensures thorough mixing of the cells with the reagent. After shaking, incubate the plate for an additional 25 min at RT. This incubation period allows the reagent to lyse the cells and generate a luminescent signal proportional to the amount of ATP present.</li>
		<li>Set up the plate reader according to the manufacturer&#39;s instructions for luminescence detection. Measure luminescence from each well containing the cell lysate and ATP reagent. Ensure that the plate reader is set to measure luminescence with appropriate settings.</li>
		<li>Subtract background luminescence readings from both treatment groups and control wells. Obtain background readings from wells containing only the ATP reagent and PBS without cells.</li>
		<li>Analyze the corrected luminescence values for each sample to determine ATP levels, reflecting cell proliferation or viability.</li>
		<li>Ensure that each experimental group and control consisted of four repeats (step 3.2.2) for statistical accuracy. Calculate and import the average luminescence of each group and control into an appropriate statistical program.</li>
		<li>Compare the results between treatment groups and relevant controls to assess ATP proliferation accurately. Record all luminescence readings, calculations, and experimental conditions for future reference. 
		NOTE: For all biochemical analyses in kit form, follow the kit&#39;s manual or protocol provided by the manufacturer for specific details, such as recommended incubation times, concentrations, and any additional steps or considerations. Always adhere to laboratory safety guidelines when handling chemicals and biological materials.</li>
	</ol>
	</li>
</ol>

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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. P., 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=Synthetic+matrix+metalloproteinase-sensitive+hydrogels+for+the+conduction+of+tissue+regeneration:+Engineering+cell-invasion+characteristics.">Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics.</a> Proc Natl Acad Sci U S A. 100 (9), 5413-5418 (2003).</li><li>Robledo, F., 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=Spheroids+derived+from+the+stromal+vascular+fraction+of+adipose+tissue+self-organize+in+complex+adipose+organoids+and+secrete+leptin.">Spheroids derived from the stromal vascular fraction of adipose tissue self-organize in complex adipose organoids and secrete leptin.</a> Stem Cell Res Ther. 14 (1), 70 (2023).</li><li>Landry, J., Freyer, J. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+of+a+synthetic+extracellular+matrix+on+immortalized+vocal+fold+fibroblasts+in+3-d+culture.">Biocompatibility of a synthetic extracellular matrix on immortalized vocal fold fibroblasts in 3-d culture.</a> Acta Biomater. 6 (8), 2940-2948 (2010).</li><li>Crous, A., Van Rensburg, M. J., Abrahamse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+and+consecutive+application+of+near-infrared+and+green+irradiation+modulates+adipose+derived+stem+cell+proliferation+and+affect+differentiation+factors.">Single and consecutive application of near-infrared and green irradiation modulates adipose derived stem cell proliferation and affect differentiation factors.</a> Biochimie. 196, 225-233 (2022).</li></ol>

The authors declare no competing interests.

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 harvested, are well characterized, and are homogenous populations ensuring consistent results31. The aim of this study was to determine the augmentative potential of PBM on the proliferation and cytotoxicity of ADSCs, using 3D cell culture. The manufacturer&#39;s protocol for a 30 &#181;L hydrogel has been adapted to a 10 &#181;L to save cost32. However, this reduced volume made the preparation and handling of the gel more difficult.
ADSCs have been shown to self-aggregate within a 3D environment33. Similarly in this study, the cells formed cell aggregates with a grape-like appearance. However, single-laying cells were also identified. Cell shedding is a known property of spheroids in which cells become detached from the main spheroid or aggregate body and are released into the culture environment. Cell shedding can result from cellular proliferation, in which viable cells are shed during mitosis and allow for migration34. Alternatively, shedding can result due to cell death and loss of spheroid or aggregate shape35, especially in very large spheroids due to a growing necrotic core. The cells adopted a rounded morphology in 3D culture, compared to a spindle shape in 2D cultures. In keeping with the findings of similar studies, these results are due to a lack of attachment sites or extracellular matrix for the cells to adhere to6,9. Furthermore, the cells showed no significant migration in the gel, further implying the need for attachment sites36. It is recommended to add an adhesion peptide or another extracellular matrix protein to facilitate attachment, adhesion, and migration. A limitation of the study was the use of inverse light microscopy to observe the 3D morphology of the ADSCs instead of Z-stacking. PBM exposure and 3D culture had no significant effect on the cytotoxicity of the ADSCs both at 24 h and 10 days post exposure, indicating that hydrogel culture and PBM are safe and have no negative impact on the cells6,26. In 3D cell culture, ADSCs have been associated with slower proliferation rates compared to 2D cell culture. This is supported by the low proliferation rates of the normal controls in the current study6. PBM (525 nm and 825 nm) significantly increased the proliferation rates of ADSCs in 3D culture. Initially, the green light (525 nm) at both 5 J/cm2 and 10 J/cm2 fluencies elicited the highest proliferation rates compared to the near-infrared light (825 nm). However, at the 10-day mark, the near-infrared light (825 nm) has the highest proliferation rates at both 5 J/cm2 and 10 J/cm2 fluencies. Similarly, PBM (525 nm and 825 nm) has been demonstrated to increase proliferation rates in 2D cultures37. It is recommended to also include a wavelength combination group, as other studies have reported a synergistic effect of green and near-infrared light.
In conclusion, the hydrogel used is a synthetic hydrogel with tremendous customization capacity to suit the needs of various applications. The current study has demonstrated that the gel has no cytotoxic effect on cells in culture over a 10-day period. Furthermore, PBM demonstrated a positive augmentative potential by significantly upregulating the proliferation rates of ADSCs, despite being in a 3D culture environment. Thus, this hydrogel can serve as a possible biomaterial carrier for transplantation into wounds. Future investigations should be aimed at testing the potential ability of the gel to assist in wound healing using animal studies. If the animal trial outcomes are favorable, it is envisioned that patient-derived ADSCs may be harvested, encapsulated, and augmented with PBM prior to transplantation into wounds to assist in wound healing in clinical applications.

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><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&amp;rsquo;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 CO&lt;sub&gt;2&lt;/sub&gt; 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 &amp;deg;C</td></tr><tr><td>Olympus CellSens Entry</td><td>Olympus</td><td>Version 3.2 (23706)&amp;nbsp;</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 &amp;ndash; True3</td><td>Sigma-Aldrich</td><td>TRUE3-1KT</td><td>10 &amp;micro;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.

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.
To assess the cytotoxic effects of PBM exposure and 3D culture using hydrogel, LDH leakage was measured (Figure 3). There were no significant differences between the experimental groups and controls at 24 h or 10 days, indicating that PBM exposure and hydrogel culture had no detrimental effect on the cells.
Cellular proliferation rates were measured by measuring the ATP content of ADSCs (Figure 4). PBM exposure resulted in increased proliferation rates in all experimental groups compared to the controls. At 24 h post-PBM exposure, the 525 nm wavelength stimulated the highest proliferation rates. However, at the 10 day mark, the 825 nm wavelength demonstrated superior proliferation rates compared to the 525 nm wavelength. At the end of the 10-day culture period, the 825 nm, 10 J/cm2 group showed the highest proliferation rate.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66616/66616fig01.jpg" /> 
Figure 1: Overview of the protocol. Summarized protocol highlighting the preparation of the hydrogel in 96 well plates, PBM exposure, and the measurement of morphology and biochemical assays at 24 h and 10 days post-PBM exposure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66616/66616fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66616/66616fig02.jpg" /> 
Figure 2: ADSCs self-aggregated after seeding. The cells retained a round-to-oval morphology and were distributed in the hydrogel as either single cells or aggregates with a grape-like cluster appearance at (A) 24 h and (B) 10 days after seeding and PBM exposure. No definitive change was observed after 10 days of incubation. Using controls (1 and 4), 525 nm at 5 J/cm2 (2) and 10 J/cm2 (5) or 825 nm at 5 J/cm2 (3) and 10 J/cm2 (6), respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66616/66616fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66616/66616fig03.jpg" /> 
Figure 3: Cellular LDH leakage as a measurement of cytotoxicity 24 h and 10 days post-PBM exposure, using a 525 nm and 825 nm diode (5 J/cm2 and 10 J/cm2) in the hydrogel. No significant increase in cytotoxicity was observed compared to the experimental control. The positive control was representative of complete cell death. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66616/66616fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66616/66616fig04.jpg" /> 
Figure 4: Cellular ATP as a measurement of proliferation 24 h and 10 days post-PBM exposure, using a 525 nm and 825 nm diode (5 J/cm2 and 10 J/cm2) in the hydrogel. PBM exposure stimulated increased proliferation rates in all experimental groups at both time points and both fluencies. Significant differences (P &#60; 0.001) are indicated by ***. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66616/66616fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume (&#181;L) per well</td>
		</tr>
		<tr>
			<td>SLO-Dextran polymer (30 mmol/L)</td>
			<td>0.7</td>
		</tr>
		<tr>
			<td>Cell suspension</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td>0.8</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>5.5</td>
		</tr>
		<tr>
			<td>Crosslinker</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>10</td>
		</tr>
	</tbody>
</table>
Table 1: Hydrogel (10 &#181;L) reagent volumes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Laser Parameters</td>
			<td>Green (G)</td>
			<td>Near infra-red (NIR)</td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>Diode Laser</td>
			<td>Diode Laser</td>
		</tr>
		<tr>
			<td>Wavelength (nm)</td>
			<td>525</td>
			<td>825</td>
		</tr>
		<tr>
			<td>Power Output (mW)</td>
			<td>553</td>
			<td>515</td>
		</tr>
		<tr>
			<td>Power Density (mW/cm2)</td>
			<td>57.48</td>
			<td>53.53</td>
		</tr>
	</tbody>
</table>
Table 2: Laser parameters.

Watch this Scientific Journal Video about Advancements in Stem Cell Regenerative Therapy Through Photobiomodulation at JoVE.com

Author Spotlight: Advancements in Stem Cell Regenerative Therapy Through Photobiomodulation

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.

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 ...

three-dimensional-cell-culture-adipose-derived-stem-cells-hydrogel

Nanyang Technological University

Research

JoVE Journal

Biology

822 Views.  University of Johannesburg. Het Laser Research Centre leidt de inspanningen om fototherapie of laserstraling van biologisch weefsel te onderzoeken, met de nadruk op fotobiomodulatie van stamcellen voor regeneratieve therapie. In deze studie onderzochten we de fotobiomodulatie van stamcellen in van vet afgeleide stamcellen met behulp van driedimensionale celkweek. Recent onderzoek heeft zich gericht op het combineren van fotobiomodulatie en van vet afgeleide stamcellen voor regeneratieve stamceltherapie.Studies he...

Video: Auteur in de schijnwerpers: vooruitgang in stamcelregeneratieve therapie door middel van fotobiomodulatie

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Developmental Biology

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Bioengineering

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Medicine

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

Behavior

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

Mechanical Stromal Vascular Fraction in Fibrin Hydrogel for Regenerative Medicine

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

Author Spotlight: Exploring the Potential of Fat-Derived Stromal Vascular Fraction for Wound Healing

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

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

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