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Abstract BackgroundMesenchymal stem cells (MSCs) secrete a diversity of factors with broad therapeutic potential, yet current culture methods limit potency outcomes. In this study, we used topographical cues on polystyrene films to investigate their impact on the secretory profile and potency of bone marrow-derived MSCs (hBM-MSCs). hBM-MSCs from four donors were cultured on topographic substrates depicting defined roughness, curvature, grooves and various levels of wettability. MethodsThe topographical PS-based array was developed using razor printing, polishing and plasma treatment methods. hBM-MSCs from four donors were purchased from RoosterBio and used in co-culture with peripheral blood mononuclear cells (PBMCs) from Cell Applications Inc. in an immunopotency assay to measure immunosuppressive capacity. Cells were cultured on low serum (2%) for 24–48 h prior to analysis. Image-based analysis was used for cell quantification and morphology assessment. Metabolic activity of BM-hMSCs was measured as the mitochondrial oxygen consumption rate using an extracellular flux analyzer. Conditioned media samples of BM-hMSCs were used to quantify secreted factors, and the data were analyzed using R statistics. Enriched bioprocesses were identify using the Gene Ontology toolenrichGOfrom theclusterprofiler.One-way and two-way ANOVAs were carried out to identify significant changes between the conditions. Results were deemed statistically significant for combinedP < 0.05 for at least three independent experiments. ResultsCell viability was not significantly affected in the topographical substrates, and cell elongation was enhanced at least twofold in microgrooves and surfaces with a low contact angle. Increased cell elongation correlated with a metabolic shift from oxidative phosphorylation to a glycolytic state which is indicative of a high-energy state. Differential protein expression and gene ontology analyses identified bioprocesses enriched across donors associated with immune modulation and tissue regeneration. The growth of peripheral blood mononuclear cells (PBMCs) was suppressed in hBM-MSCs co-cultures, confirming enhanced immunosuppressive potency. YAP/TAZ levels were found to be reduced on these topographies confirming a mechanosensing effect on cells and suggesting a potential role in the immunomodulatory function of hMSCs. ConclusionsThis work demonstrates the potential of topographical cues as a culture strategy to improve the secretory capacity and enrich for an immunomodulatory phenotype in hBM-MSCs.
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Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells
BackgroundMesenchymal stem cells (MSCs) hold great promise for treating a variety of human diseases; however, their clinical translation is hindered by challenges in large‐scale expansion while preserving therapeutic potency and maintaining small cell size. Conventional 2D culture on rigid substrates induces MSC senescence and enlargement, compromising their function and biodistribution. MethodsWe present an alternating 2D/3D culture strategy that combines adherent monolayer expansion with transient spheroid formation to mitigate these limitations. Placenta‐derived MSCs were cultured under optimized spheroid conditions, with extracellular matrix supplementation and chemically defined media to enhance viability. To address scalability, we developed RGD-functionalized alginate hydrogel tubes (AlgTubes) that enable dynamic transitions between adherent and spheroid states for continuous culture. ResultsSpheroid culture significantly reduced cell size and enhanced immunomodulatory function. The alternating 2D/3D protocol slowed MSC enlargement and senescence over multiple passages while preserving anti-inflammatory activity. Extracellular matrix supplementation and chemically defined media further improved cell viability. AlgTubes successfully supported the alternating culture strategy in a continuous and scalable format. ConclusionsThe alternating 2D/3D culture system effectively overcomes limitations of conventional MSC expansion by mitigating enlargement, delaying senescence, and preserving both proliferative capacity and immunoregulatory potency. Combined with AlgTube technology, this work demonstrates a promising strategy for MSC manufacturing
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- Award ID(s):
- 2143151
- PAR ID:
- 10662966
- Publisher / Repository:
- Frontier
- Date Published:
- Journal Name:
- Frontiers in Bioengineering and Biotechnology
- Volume:
- 13
- ISSN:
- 2296-4185
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.3389/fbioe.2025.1632810
