Stem Cell Culture Research Articles

Activating the healing process: three-dimensional culture of stem cells in Matrigel for tissue repair

BackgroundTo establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration.MethodsThe biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A Sprague‒Dawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining.ResultsThree-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue.ConclusionThree-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.Graphical abstract

Rescue of impaired blood-brain barrier in tuberous sclerosis complex patient derived neurovascular unit

BackgroundTuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene and dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood–brain barrier (BBB).MethodsWe generated TSC disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies.ResultsUsing microphysiological systems, we demonstrate that a BBB generated from TSC2 heterozygous mutant cells shows increased permeability. This can be rescued by wild type astrocytes or by treatment with rapamycin, an mTOR kinase inhibitor.ConclusionOur results demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of cell lineages contributing to TSC pathogenesis and informs future therapeutics.

Electrospun fibers modified with polydopamine for enhancing human mesenchymal stem cell culture

In this study, we coated electrospun polycaprolactone (PCL) fibers with polydopamine (PDA) to modify their hydrophobicity and fabricated a matrix for culturing mesenchymal stem cells (MSCs). Additionally, we incorporated Arg-Gly-Asp (RGD) peptides into PDA to enhance MSCs culture performance on PCL fibers. PDA and RGD were successfully coated in one step by immersing the electrospun fibers in a coating solution, without requiring an additional surface activation process. The characteristics of functionalized PCL fibers were analyzed by scanning electron microscopy with energy-dispersive x-ray analysis, Fourier transform infrared spectroscopy, water contact angle measurement, and fluorescence measurements using a carboxylic-modified fluorescent microsphere. MSCs cultured on the modified PCL fibers demonstrated enhanced cell adhesion, proliferation, and osteogenic- and chondrogenic differentiation. This study provides insight into potential applications for scaffold fabrication in MSCs-based tissue engineering, wound dressing, implantation, and a deeper understanding of MSCs behavior in vitro.

Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth.

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which are present in both deciduous and permanent teeth, have emerged as a vital stem cell source due to their accessibility, adaptability, and innate differentiation capabilities. DPSCs offer a readily available and abundant reservoir of mesenchymal stem cells, showcasing impressive versatility and potential, particularly for regenerative purposes. Despite their promise, the main hurdle lies in effectively isolating and characterizing DPSCs, given their representation as a minute fraction within dental pulp cells. Equally crucial is the proper preservation of this invaluable cellular resource. The two predominant methods for DPSC isolation are enzymatic digestion (ED) and outgrowth from tissue explants (OG), often referred to as spontaneous growth. This protocol concentrates primarily on the enzymatic digestion approach for DPSC isolation, intricately detailing the steps encompassing extraction, in-lab processing, and cell preservation. Beyond extraction and preservation, the protocol delves into the differentiation prowess of DPSCs. Specifically, it outlines the procedures employed to induce these stem cells to differentiate into adipocytes, osteoblasts, and chondrocytes, showcasing their multipotent attributes. Subsequent utilization of colorimetric staining techniques facilitates accurate visualization and confirmation of successful differentiation, thereby validating the caliber and functionality of the isolated DPSCs. This comprehensive protocol functions as a blueprint encompassing the entire spectrum of dental pulp stem cell extraction, cultivation, preservation, and characterization. It underscores the substantial potential harbored by DPSCs, propelling forward stem cell exploration and holding promise for future regenerative and therapeutic breakthroughs.

Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation

Enteroendocrine cells (EECs) secrete serotonin (enterochromaffin [EC] cells) or specific peptide hormones (non-EC cells) that serve vital metabolic functions. The basis for terminal EEC diversity remains obscure. By forcing activity of the transcription factor (TF) NEUROG3 in 2D cultures of human intestinal stem cells, we replicated physiologic EEC differentiation and examined transcriptional and cis-regulatory dynamics that culminate in discrete cell types. Abundant EEC precursors expressed stage-specific genes and TFs. Before expressing pre-terminal NEUROD1, post-mitotic precursors oscillated between transcriptionally distinct ASCL1+ and HES6hi cell states. Loss of either factor accelerated EEC differentiation substantially and disrupted EEC individuality; ASCL1 or NEUROD1 deficiency had opposing consequences on EC and non-EC cell features. These TFs mainly bind cis-elements that are accessible in undifferentiated stem cells, and they tailor subsequent expression of TF combinations that underlie discrete EEC identities. Thus, early TF oscillations retard EEC maturation to enable accurate diversity within a medically important cell lineage.

Focal adhesion and actin orientation regulated by cellular geometry determine stem cell differentiation via mechanotransduction

Tuning cell adhesion geometry can affect cytoskeleton organization and the distribution of cytoskeleton forces, which play critical roles in controlling cell functions. To elucidate the geometrical relationship with cytoskeleton force distribution, it is necessary to control cell morphology. In this study, a series of dextral vortex micropatterns were prepared to precisely control cell morphology for investigating the influence of the curvature degree of adhesion curves on intracellular force distribution and stem cell differentiation at a sub-cellular level. Peripherial actin filaments of micropatterned cells were assembled along the adhesion curves and showed different orientations, filament thicknesses and densities. Focal adhesion and cytoskeleton force distribution were dependent on the curvature degree. Intracellular force distribution was also regulated by adhesion curves. The cytoskeleton and force distribution affected the osteogenic differentiation of mesenchymal stem cells through a YAP/TAZ-mediated mechanotransduction process. Thus, regulation of cell adhesion curvature, especially at cytoskeletal filament level, is critical for cell function manipulation. Statement of significanceIn this study, a series of dextral micro-vortexes were prepared and used for the culture of human mesenchymal stem cells (hMSCs) to precisely control adhesive curvatures (0°, 30°, 60°, and 90°). The single MSCs on the micropatterns had the same size and shape but showed distinct focal adhesion (FA) and cytoskeleton orientations. Cellular nanomechanics were observed to be correlated with the curvature degrees, subsequently influencing nuclear morphological features. As a consequence, the localization of the mechanotransduction sensor and activator-YAP/TAZ was affected, influencing osteogenic differentiation. The results revealed the pivotal role of adhesive curvatures in the manipulation of stem cell differentiation via the machanotransduction process, which has rarely been investigated.

Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture

BackgroundHyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications.MainHere we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former.ConclusionCultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.

Comparison of the efficacy of two-dimensional and three-dimensional cultures of human rotator cuff-derived mesenchymal stem cells for rotator cuff regeneration

Comparison of the efficacy of two-dimensional and three-dimensional cultures of human rotator cuff-derived mesenchymal stem cells for rotator cuff regeneration

Laminin as a Key Extracellular Matrix for Proliferation, Differentiation, and Maturation of Porcine Muscle Stem Cell Cultivation.

Extracellular matrix (ECM) proteins play a crucial role in culturing muscle stem cells (MuSCs). However, there is a lack of extensive research on how each of these proteins influences proliferation and differentiation of MuSCs from livestock animals. Therefore, we investigated the effects of various ECM coatings-collagen, fibronectin, gelatin, and laminin-on the proliferation, differentiation, and maturation of porcine MuSCs. Porcine MuSCs, isolated from 14-day-old Berkshire piglets, were cultured on ECM-coated plates, undergoing three days of proliferation followed by three days of differentiation. MuSCs on laminin showed higher proliferation rate than others (p<0.05). There was no significant difference in the mRNA expression levels of PAX7, MYF5, and MYOD among MuSCs on laminin, collagen, and fibronectin (p>0.05). During the differentiation period, MuSCs cultured on laminin exhibited a significantly higher differentiation rate, resulting in thicker myotubes compared to those on other ECMs (p<0.05). Also, MuSCs on laminin showed higher expression of mRNA related with maturated muscle fiber such as MYH1 and MYH4 corresponding to muscle fiber type IIx and muscle fiber type IIb, respectively, compared with MuSCs on other ECM coatings (p<0.05). In summary, our comparison of ECMs revealed that laminin significantly enhances MuSC proliferation and differentiation, outperforming other ECMs. Specifically, muscle fibers cultured on laminin exhibited a more mature phenotype. These findings underscore laminin's potential to advance in vitro muscle research and cultured meat production, highlighting its role in supporting rapid cell proliferation, higher differentiation rates, and the development of mature muscle fibers.

Cultivation of Bovine Mesenchymal Stem Cells on Plant-Based Scaffolds in a Macrofluidic Single-Use Bioreactor for Cultured Meat.

Reducing production costs, known as scaling, is a significant obstacle in the advancement of cultivated meat. The cultivation process hinges on several key components, e.g., cells, media, scaffolds, and bioreactors. This study demonstrates an innovative approach, departing from traditional stainless steel or glass bioreactors, by integrating food-grade plant-based scaffolds and thermoplastic film bioreactors. While thermoplastic films are commonly used for constructing fluidic systems, conventional welding methods are cost-prohibitive and lack rapid prototyping capabilities, thus inflating research and development expenses. The developed laser welding technique facilitates contamination-free and leakproof sealing of polyethylene films, enabling the efficient fabrication of macrofluidic systems with various designs and dimensions. By incorporating food-grade plant-based scaffolds, such as rice seeded with bovine mesenchymal stem cells, into these bioreactors, this study demonstrates sterile cell proliferation on scaffolds within macrofluidic systems. This approach not only reduces bioreactor prototyping and construction costs but also addresses the need for scalable solutions in both research and industrial settings. Integrating single-use bioreactors with minimal shear forces and incorporating macro carriers such as puffed rice may further enhance biomass production in a scaled-out model. The use of food-grade plant-based scaffolds aligns with sustainable practices in tissue engineering and cultured-meat production, emphasizing its suitability for diverse applications.

In Vitro Evaluation of Biocompatibility of Two Dental Alloys on Stem Cells from Apical Papilla – a Comparative Study

In the present study we performed a comparative in vitro evaluation of biocompatibility of two nickel-containing dental alloys produced by two different methods – conventional and powder metallurgy. Stem cells from apical papilla (SCAPs) were isolated, cultured and incubated in 6% solution of alloys powders in DMSO for 48 hours and further examined for apoptosis, proliferation and cellular metabolism. Our findings suggest that the alloys produced by the traditional method (Crystalloy N) release more electrically charged products of electrochemical corrosion, resulting in increased ROS production in SCAPs, increased percentage of apoptoic cells, decreased proliferative potential and tend to increase antioxidant enzymatic defence. No allergic pro-inflammatory effect of the metal alloys on stem cell cultures was confirmed.

Energy Metabolism in Human Pluripotent Stem and Differentiated Cells Compared Using a Seahorse XF96 Extracellular Flux Analyzer.

Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous, a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult, resulting in inaccuracies. In this study, we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts, human induced PSCs, and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis. We evaluated normalization methods using glucose and selected the most suitable for the metabolic analysis of heterogeneous PSCs during the reprogramming stage. The response of fibroblasts to glucose increased with starvation time, with oxygen consumption rate and extracellular acidification rate responding most effectively to glucose 4 hours after starvation and declining after 5 hours of starvation. Fibroblasts and PSCs achieved appropriate responses to glucose without damaging their metabolism 2∼4 and 2∼3 hours after starvation, respectively. We developed a novel method for comparing basal metabolic rates of fibroblasts and PSCs, focusing on quantitative analysis of glycolysis and oxidative phosphorylation using glucose without enzyme inhibitors. This protocol enables efficient comparison of energy metabolism among cell types, including undifferentiated PSCs, differentiated cells, and cells undergoing cellular reprogramming, and addresses critical issues, such as differences in basal metabolic levels and sensitivity to normalization, providing valuable insights into cellular energetics.

Improving the mechanical and biological functions of cell sheet constructs: The interplay of human-derived periodontal ligament stem cells, endothelial cells and plasma rich in growth factors

ObjectiveThe aim of this study was to produce and characterize triple-layered cell sheet constructs with varying cell compositions combined or not with the fibrin membrane scaffold obtained by the technology of Plasma Rich in Growth Factors (mPRGF). Materials and methodsHuman primary cultures of periodontal ligament stem cells (hPDLSCs) were isolated, and their stemness nature was evaluated. Three types of triple-layered composite constructs were generated, composed solely of hPDLSCs or combined with human umbilical vein endothelial cells (HUVECs), either as a sandwiched endothelial layer or as coculture sheets of both cell phenotypes. These three triple-layered constructs were also manufactured using mPRGF as cell sheets’ support. Necrosis, glucose consumption, secretion of extracellular matrix proteins and synthesis of proangiogenic factors were determined. Histological evaluations and proteomic analyses were also performed. ResultsThe inclusion of HUVECs did not clearly improve the properties of the multilayered constructs and yet hindered their optimal conformation. The presence of mPRGF prevented the shrinkage of cell sheets, stimulated the metabolic activity and increased the matrix synthesis. At the proteome level, mPRGF conferred a dramatic advantage to the hPDLSC constructs in their ability to provide a suitable environment for tissue regeneration by inducing the expression of proteins necessary for bone morphogenesis and cellular proliferation. ConclusionshPDLSCs’ triple-layer construct onto mPRGF emerges as the optimal structure for its use in regenerative therapeutics. Clinical relevanceThese results suggest the suitability of mPRGF as a promising tool to support cell sheet formation by improving their handling and biological functions.

Reprogramming fibroblast into human iBlastoids.

The study of early human embryogenesis has relied on the use of blastocysts donated to research or simple stem cell culture systems such as pluripotent and trophoblast stem cells, which have been seminal in shedding light on many key developmental processes. However, simple culture systems lack the necessary complexity to adequately model the spatiotemporal, cellular and molecular dynamics occurring during the early phases of embryonic development. As such, an in vitro model of the human blastocyst is advantageous in many aspects to decipher human embryogenesis. Here we describe a step-by-step protocol for the generation of induced blastoids (iBlastoids), an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This protocol details the workflow for reprogramming of human dermal fibroblasts and subsequent generation of iBlastoids using the reprogramming intermediates, which together takes ~27 days (21 days for reprogramming and 6 days for iBlastoid generation). We also discuss several characterization/functional assays that can be used on the iBlastoids. We believe that a person trained in cell culture with ~1 year of experience with human somatic cell and reprogramming/cell differentiation assays would be able to perform this protocol. In short, the iBlastoids present an alternative tool as a model to the blastocyst to facilitate the scientific community in the exploration of early human development.

Osteopontin drives neuroinflammation and cell loss in MAPT-N279K frontotemporal dementia patient neurons

Frontotemporal dementia (FTD) is an incurable group of early-onset dementias that can be caused by the deposition of hyperphosphorylated tau in patient brains. However, the mechanisms leading to neurodegeneration remain largely unknown. Here, we combined single-cell analyses of FTD patient brains with a stem cell culture and transplantation model of FTD. We identified disease phenotypes in FTD neurons carrying the MAPT-N279K mutation, which were related to oxidative stress, oxidative phosphorylation, and neuroinflammation with an upregulation of the inflammation-associated protein osteopontin (OPN). Human FTD neurons survived less and elicited an increased microglial response after transplantation into the mouse forebrain, which we further characterized by single nucleus RNA sequencing of microdissected grafts. Notably, downregulation of OPN in engrafted FTD neurons resulted in improved engraftment and reduced microglial infiltration, indicating an immune-modulatory role of OPN in patient neurons, which may represent a potential therapeutic target in FTD.

Anti-apoptotic protein Bcl-2 contributes to the determination of reserve cells during myogenic differentiation of C2C12 cells.

Skeletal muscle's regenerative ability is vital for maintaining muscle function, but chronic diseases like Duchenne muscular dystrophy can deplete this capacity. Muscle satellite cells, quiescent in normal situations, are activated during muscle injury, expressing myogenic regulatory factors, and producing myogenic progenitor cells. It was reported that muscle stem cells in primary culture and reserve cells in C2C12 cells express anti-apoptotic protein Bcl-2. Although the role of Bcl-2 expressed in myogenic cells has been thought to be to enhance cell viability, we hypothesized that Bcl-2 may promote the formation of reserve cells. The expression pattern analysis showed the expression of Bcl-2 in undifferentiated mononucleated cells, emphasizing its usefulness as a reserve cell marker and reminding us that cells expressing Bcl-2 have low proliferative potential. Silencing of Bcl-2 by transfection with siRNA decreased cell viability and the number of reserve cells, while overexpression of Bcl-2 not only increases cell viability but also inhibits muscle differentiation and proliferation. These results emphasize dual roles of Bcl-2 in protecting cells from apoptosis and contributing to reserve cell formation by regulating myoblast proliferation and/or differentiation. Overall, the study sheds light on the multifaceted role of Bcl-2 in the maintenance of skeletal muscle regeneration.

Modeling HIV-1 infection and NeuroHIV in hiPSCs-derived cerebral organoid cultures

The human immunodeficiency virus (HIV) epidemic is an ongoing global health problem affecting 38 million people worldwide with nearly 1.6 million new infections every year. Despite the advent of combined antiretroviral therapy (cART), a large percentage of people with HIV (PWH) still develop neurological deficits, grouped into the term of HIV-associated neurocognitive disorders (HAND). Investigating the neuropathology of HIV is important for understanding mechanisms associated with cognitive impairment seen in PWH. The major obstacle for studying neuroHIV is the lack of suitable in vitro human culture models that could shed light into the HIV-CNS interactions. Recent advances in induced pluripotent stem cell (iPSC) culture and 3D brain organoid systems have allowed the generation of 2D and 3D culture methods that possess a potential to serve as a model of neurotropic viral diseases, including HIV. In this study, we first generated and characterized several hiPSC lines from healthy human donor skin fibroblast cells. hiPSCs were then used for the generation of microglia-containing human cerebral organoids (hCOs). Once fully characterized, hCOs were infected with HIV-1 in the presence and absence of cART regimens and viral infection was studied by cellular, molecular/biochemical, and virological assays. Our results revealed that hCOs were productively infected with HIV-1 as evident by viral p24-ELISA in culture media, RT-qPCR and RNAscope analysis of viral RNA, as well as ddPCR analysis of proviral HIV-1 in genomic DNA samples. More interestingly, replication and gene expression of HIV-1 were also greatly suppressed by cART in hCOs as early as 7 days post-infections. Our results suggest that hCOs derived from hiPSCs support HIV-1 replication and gene expression and may serve as a unique platform to better understand neuropathology of HIV infection in the brain.

Image‐based hybrid model incorporating initial spatial distribution for mesenchymal stem cell cultivation process design

AbstractThis work presents an image‐based hybrid model incorporating the initial spatial distribution for mesenchymal stem cell (MSC) cultivation process design. First, three levels of seeding bias in static MSC cultivation were experimentally investigated, and the resulting initial distribution was quantified using phase contrast microscopy image analysis. Second, the observed spatial heterogeneity was defined as a new parameter by calculating the standard deviation of the seeded cell number fraction among numerical 8 × 8 tiles on two‐dimensional plates. Third, the parameter was incorporated into kinetic models to consider spatial growth limitation. The model was then applied to simulate MSC cultivation processes with experimental data. Using dynamic and stochastic simulation outputs, feasible ranges of cell‐harvesting time could be determined to satisfy a given requirement for the minimum cell number and acceptable confluency level. The developed hybrid model could serve as a basis for quantitative decision‐making in the design of MSC cultivation processes.

CD32 captures committed haemogenic endothelial cells during human embryonic development

During embryonic development, blood cells emerge from specialized endothelial cells, named haemogenic endothelial cells (HECs). As HECs are rare and only transiently found in early developing embryos, it remains difficult to distinguish them from endothelial cells. Here we performed transcriptomic analysis of 28- to 32-day human embryos and observed that the expression of Fc receptor CD32 (FCGR2B) is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and human pluripotent stem cell-derived endothelial cells revealed that robust multilineage haematopoietic potential is harboured within CD32+ endothelial cells and showed that 90% of CD32+ endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a haematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and human pluripotent stem cell cultures, thus allowing the efficient generation of haematopoietic cells in vitro.

Three-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 µL 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.