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

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.