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<p>Mesenchymal stromal cells (MSCs) have been widely studied for their regenerative and immunomodulatory properties. However, clinical translation is hindered by a lack of, or limited, in vivo retention due to immune-mediated clearance. Biomaterial-based encapsulation, particularly using alginate hydrogels, offers a promising strategy to enhance MSC persistence and functionality. This study aimed to evaluate the impact of unmodified and RGD-functionalized GMP-grade alginate matrices on umbilical cord-derived MSCs (UC-MSCs) viability, mitochondrial function, and cytokine secretion profile. Cryopreserved UC-MSCs were encapsulated in GMP-compatible ultrapure alginates, an unmodified alginate (SLG20) and (G-RGD) modified with Arg-Gly-Asp (RGD) peptides, and compared to 2D cultures over five days for viability, metabolic activity, cytokine secretion, and mitochondrial function. MSCs encapsulation in G-RGD alginate significantly enhanced viability and modified cytoskeletal organisation compared to SLG20. While encapsulation resulted in 58% reduction in OXPHOS, relative to 2D culture at Day 0 (<i>p</i> &lt; 0.01), with no significant difference between SLG20 and G-RGD. G-RGD-encapsulated cells maintained significantly higher basal, ATP-linked, and maximal respiration (<i>p</i> &lt; 0.01) than SLG20-encapsulated cells for up to five days. Notably, encapsulation triggered a 60-fold upregulation of <i>PGC1A</i> and a twofold increase in <i>HIF1A</i> expression by Day 3, indicating metabolic adaptation and mitochondrial biogenesis signalling. Early cytokine profiling showed that encapsulation increased VEGF secretion by approximately 11-fold compared to 2D MSCs. IL-6 secretion was 34.5% higher in G-RGD than in SLG20 at Day 1 and was markedly higher in encapsulated MSCs than in 2D cultures. Although TNF-α secretion remained low overall, levels were 49% higher in G-RGD than in SLG20 at Day 5. IL-10 levels were similar between matrices. Encapsulation reduced glycolytic output by 67% compared to 2D cultures and lowered <i>THY1 (CD90)</i> expression over time. This work was translation-focused, testing whether established RGD-related benefits are preserved under GMP-grade materials and can be clinically deployable with cryopreserved cells. Our findings reveal that a simple encapsulation in alginate microbeads, within 500&#xa0;μm beads, creates a hypoxic environment for MSCs, similar to their natural niche, which strongly alters their functions as compared to the classical 2D plastic culture. G-RGD alginate provided modest but consistent advantages over unmodified SLG20 in maintaining mitochondrial function and modulating cytokine secretion. Matrix composition remains a critical factor in shaping MSC behaviour, and G-RGD ultrapure alginate represents a promising material for optimising cell-based therapies under clinically relevant conditions.</p>

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RGD-modified alginate enhances viability, metabolic reprogramming, and cytokine secretion profiles in encapsulated mesenchymal stromal cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月13日

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  • Kadriye Güven,
  • Anil Dhawan,
  • Celine Filippi

摘要

Mesenchymal stromal cells (MSCs) have been widely studied for their regenerative and immunomodulatory properties. However, clinical translation is hindered by a lack of, or limited, in vivo retention due to immune-mediated clearance. Biomaterial-based encapsulation, particularly using alginate hydrogels, offers a promising strategy to enhance MSC persistence and functionality. This study aimed to evaluate the impact of unmodified and RGD-functionalized GMP-grade alginate matrices on umbilical cord-derived MSCs (UC-MSCs) viability, mitochondrial function, and cytokine secretion profile. Cryopreserved UC-MSCs were encapsulated in GMP-compatible ultrapure alginates, an unmodified alginate (SLG20) and (G-RGD) modified with Arg-Gly-Asp (RGD) peptides, and compared to 2D cultures over five days for viability, metabolic activity, cytokine secretion, and mitochondrial function. MSCs encapsulation in G-RGD alginate significantly enhanced viability and modified cytoskeletal organisation compared to SLG20. While encapsulation resulted in 58% reduction in OXPHOS, relative to 2D culture at Day 0 (p < 0.01), with no significant difference between SLG20 and G-RGD. G-RGD-encapsulated cells maintained significantly higher basal, ATP-linked, and maximal respiration (p < 0.01) than SLG20-encapsulated cells for up to five days. Notably, encapsulation triggered a 60-fold upregulation of PGC1A and a twofold increase in HIF1A expression by Day 3, indicating metabolic adaptation and mitochondrial biogenesis signalling. Early cytokine profiling showed that encapsulation increased VEGF secretion by approximately 11-fold compared to 2D MSCs. IL-6 secretion was 34.5% higher in G-RGD than in SLG20 at Day 1 and was markedly higher in encapsulated MSCs than in 2D cultures. Although TNF-α secretion remained low overall, levels were 49% higher in G-RGD than in SLG20 at Day 5. IL-10 levels were similar between matrices. Encapsulation reduced glycolytic output by 67% compared to 2D cultures and lowered THY1 (CD90) expression over time. This work was translation-focused, testing whether established RGD-related benefits are preserved under GMP-grade materials and can be clinically deployable with cryopreserved cells. Our findings reveal that a simple encapsulation in alginate microbeads, within 500 μm beads, creates a hypoxic environment for MSCs, similar to their natural niche, which strongly alters their functions as compared to the classical 2D plastic culture. G-RGD alginate provided modest but consistent advantages over unmodified SLG20 in maintaining mitochondrial function and modulating cytokine secretion. Matrix composition remains a critical factor in shaping MSC behaviour, and G-RGD ultrapure alginate represents a promising material for optimising cell-based therapies under clinically relevant conditions.