IntroductionMesenchymal stromal cells (MSCs) have attracted particular attention in the field of regenerative medicine due to their promotion of tissue repair, their potent immunomodulatory properties, combined with their minimal ethical issues1,2,3. Their accessibility and ease of expansion have enabled widespread basic and clinical investigations across a broad range of diseases, from inflammatory disorders to acute organ failure. However, despite encouraging preclinical data, clinical efficacy remains limited due to rapid immune clearance, resulting in poor target tissue retention and short-lived therapeutic effect4,5,6.To address this, biomaterial-assisted delivery systems have emerged as a strategy to enhance MSC survival, persistence, and functionality. Among these, alginate hydrogels have emerged as clinically usable matrices owing to their biocompatibility, inertness, and adaptable or modifiable mechanical properties7,8,9,10. GMP-grade alginates have now been developed to align with regulatory requirements for translational applications. GMP-grade alginate was selected to ensure translational relevance and alignment with regulatory frameworks for advanced therapy medicinal products (ATMPs). Compared to research-grade materials, GMP-grade alginates offer (i) low endotoxin content and high purity, reducing the risk of unintended immune activation, (ii) improved batch-to-batch consistency, including certificate-backed documentation of RGD substitution, and (iii) traceability and quality documentation required for clinical product development. Alginate-based encapsulation offers physical immuno-protection by forming a semi-permeable barrier that enables the diffusion of nutrients and paracrine factors while preventing immune cell attack11. Although MSCs were once considered hypoimmunogenic, they are now recognised as immune-evasive but still susceptible to clearance by host NK and T cells12. By physically shielding transplanted MSCs from this immune pressure, alginate encapsulation can prolong their persistence and enable sustained paracrine activity essential for repair. MSC functions also rely on appropriate cell–matrix interactions that maintain their phenotype and therapeutic potency. Functionalisation of alginate with Arg-Gly-Asp (RGD) peptides enables integrin-mediated adhesion, better recapitulating the extracellular matrix environment and supporting MSC survival signalling13,14,15.At the same time, advances in MSC biology highlight the central role of metabolic programming in dictating therapeutic function. Native MSC niches, such as bone marrow or perivascular tissues, are typically hypoxic and mechanically dynamic, favouring glycolysis over oxidative phosphorylation (OXPHOS)16,17. In contrast, atmospheric 2D culture disrupts this balance, leading to a metabolic drift and functional modifications. The biophysical properties of the encapsulating matrix and cell–matrix interactions significantly impact MSC bioenergetics and therapeutic output. MSCs exhibit a high degree of metabolic plasticity, relying on either glycolysis or OXPHOS depending on their environmental conditions18,19. Under reduced oxygen tension, hypoxia-inducible factor 1-alpha (HIF1A) is stabilised, promoting a metabolic shift towards glycolysis to support cell survival20,21. In MSCs, such hypoxic adaptation has been shown to preserve their stemness and enhance their paracrine function21,22,23. Mitochondrial biogenesis, regulated by PGC1A, NRF1/2, and TFAM, plays a central role in maintaining mitochondrial integrity and energy production during metabolic adaptation24. However, its activation under hypoxia is context-dependent, mitochondrial biogenesis being suppressed or transiently induced depending on the intensity and duration of hypoxic exposure25.We and other groups have showed that MSC can support the function of a variety of cells when co-encapsulated26. However, we never focused on the specific response of MSCs to the encapsulation process. Because our group focuses on the development of new therapies, requiring adherence to GMP regulations, this study aimed at comparing the behaviour of MSCs - isolated, cultured and cryopreserved in a GMP-like fashion - in 2 GMP-compliant alginates: SLG20, an ultrapure sterile alginate, and an RGD-modified alginate (G-RGD). We specifically looked at the ability of the hydrogels to support umbilical cord-derived MSC (UC-MSC) viability, bioenergetics and cytokine secretion. While RGD‑modified alginate matrices have been widely explored in experimental MSC culture systems, most prior studies employ various types of research‑grade polymers, freshly harvested cells, or two‑dimensional and spheroid‑based formats. In contrast, the present study adopts a clinically constrained framework, integrating GMP‑grade alginates (SLG20 vs. G‑RGD), cryopreserved UC‑MSCs reflecting real‑world clinical workflows, and microbead formulations compatible with intraperitoneal delivery.Materials and methodsCulture and cryopreservation of UC-MSCsAll procedures involving human-derived biological materials were carried out in accordance with relevant institutional and national guidelines and regulations. The study was approved by the King’s College Hospital Research Ethics Committee (Reference: LREC 01–016). Human umbilical cord–derived mesenchymal stromal cells were obtained from the Anthony Nolan Trust (UK) with written informed consent from all donors or their legal guardians, in accordance with the Declaration of Helsinki. Human umbilical cord–derived mesenchymal stromal cells (UC-MSCs procured from the Anthony Nolan Trust, UK) were isolated from Wharton’s jelly as previously described26 and cryopreserved at temperatures lower than – 130 °C. UC-MSC vials were thawed and expanded for experimental use in α-Minimal Essential Medium (α-MEM; Thermo Fisher Scientific, UK cat# 32561094) supplemented with 5% Stemulate® pooled human platelet lysate (Cook Regentec, USA, Ref# G35220), 1% penicillin‐streptomycin (P/S; (cat# 15140122)), and 1 mM sodium pyruvate (Lonza, Switzerland, cat# 13‐115E). Cells were maintained at 37 °C in a humidified 5% CO₂ incubator. After reaching 70–80% confluence within a week, the cells were passaged and expanded, with an approximate density of 3000 cells per cm2. Passage 4 (P4) cells were used for all experiments to ensure optimal viability and functionality. For cryopreservation, cells were resuspended in α-MEM supplemented with 10% DMSO (Sigma‐Aldrich, UK, cat# D2650) and 5% human serum albumin (Zenalb 20, UK, PL 08801/0007) at a density of 1 × 10⁶ cells/mL and stored at − 140 °C. All reagents were obtained from Thermo Fisher Scientific, UK, unless otherwise specified.Encapsulation of cells in alginate hydrogelsCryopreserved UC-MSCs were thawed and encapsulated in alginate microbeads using a B-395 Pro encapsulator (Buchi Labortechnik AG, Flawil, Switzerland), while the IE-50R encapsulator (Inotech Encapsulation AG, Dottikon, Switzerland) was used in early optimisation steps, as previously described27. Click or tap here to enter text.Before encapsulation, ultrapure sodium alginate with low viscosity and high guluronate (PRONOVA™ SLG20; NovaMatrix, Sandvika, Norway) was dissolved in 0.9% (w/v) NaCl to reach a final concentration of 1.5% (w/v). For RGD-modified alginate, lyophilised NOVATACH™ MVG GRGDSP alginate (NovaMatrix, Sandvika, Norway) was used. This GMP-grade alginate is based on high-G sodium alginate covalently coupled to the GRGDSP peptide (Gly-Arg-Gly-Asp-Ser-Pro) via stable amide linkage, enabling integrin-specific binding that mimics natural extracellular matrix interactions. The degree of RGD peptide substitution is indicated in the manufacturer’s certificates of analysis for each batch. For batch BP-2109-22, the degree of substitution was 0.375%, and for batch BP-2311-20, the substitution degree was 0.382%. Both batches were used in this study. The alginate was reconstituted in 0.9% (w/v) NaCl to achieve a final concentration of 1.1%. This lower concentration was selected based on the manufacturer’s (NovaMatrix) recommendation to optimise viscosity and droplet formation for GRGDSP-functionalised alginate, as RGD coupling alters gelation properties. The adjustment ensured comparable bead size and structural integrity to 1.5% unmodified SLG20, in line with previous encapsulation reports26. The mixture was gently combined with the cells at a density of 0.8 × 10⁶ cells/ml of alginate, corresponding to approximately 50–55 cells per 500 μm bead at day 0, based on bead volume (~ 65 nL). The suspension was carefully prepared to avoid bubble formation and subsequently filtered through a 100 μm mesh to ensure uniformity. Encapsulation was performed using a regularly maintained Buchi B-395 Pro encapsulator, which consistently produced highly uniform bead suspensions. Microbeads were generated using a 200 μm nozzle, cross-linked in a 100 mM CaCl₂ (1.2%w/v) solution for 10 min and washed twice with 0.9% (w/v) NaCl. Encapsulation efficiency was not directly quantified; however, the cells were premixed with alginate prior to extrusion, ensuring that the vast majority of cells were incorporated into microbeads during droplet formation, with free single cells rarely observed in the CaCl₂ crosslinking bath. Vibrational extrusion systems such as the Buchi B-395 Pro encapsulator have been reported to achieve encapsulation efficiencies exceeding 90–95% under optimised conditions28. For cell culture, the microbeads were maintained in Williams E medium (cat# A1217601) supplemented with 10% (v/v) heat‐inactivated foetal calf serum (Gibco, cat# 10270106), 2 mM L‐glutamine (cat# 25030081), 10 mM HEPES (cat# 15630056), 10 mg/L insulin (Sigma-Aldrich, UK, cat# I9278), 5.5 mg/L transferrin (Sigma-Aldrich, UK, cat# T8158), 670 µg/L sodium selenite (ITS-G supplement, cat# 41400045), 10⁻⁷ M dexamethasone (cat# D4902), 100 U/mL penicillin, and 100 µg/mL streptomycin (P/S, cat# 15140122). One millilitre of microbeads was cultured in 4 mL of complete medium per well of a 6‐well plate in a humidified incubator at 37 °C with 5% CO₂ for up to 5 days, with medium replacement every 2–3 days. Images of the microbeads were captured using an EVOS FL Auto 2 microscope (Thermo Fisher Scientific, UK), and an inverted microscope (DMi8, Leica Microsystems Ltd.) was used for higher-magnification imaging, including Z-stack acquisition for 3D assessment of encapsulated cells.Microbead size measurementMicrobead size was quantified using the Fiji (ImageJ 2.16.0/1.54 g) software, and the bead size distribution was compared between UC-MSCs encapsulated in SLG20 and G-RGD alginate formulations. Beads were imaged under consistent magnification, and diameter measurements were performed. All microbeads were generated using a GMP-compliant encapsulation process, and their diameters met predefined clinical release criteria, ensuring suitability for translational application.Cell viability assessmentA small volume of beads (100–200 µl) was placed in a 3 mL petri dish and gently washed with calcium- and magnesium-free DPBS (Gibco, cat# 14190144). FDA (10 µg/mL, Sigma #F7378, UK) staining solution was prepared from concentrated stocks and combined as specified. The beads were incubated with the staining solution for 15 min at 37 °C, washed three times with DPBS, and then imaged to assess cell viability.Phalloidin staining in alginate beadsAlginate beads (500 µl) were transferred into 35 mm petri dishes using a wide-bore pipette and fixed in 4% methanol-free formaldehyde (PFA) (Pierce™ 16% Formaldehyde (w/v), Methanol-free Cat# 28906) in DPBS for 20 min at RT with gentle shaking. The beads were then washed three times with DPBS before permeabilization of the cells in 0.1% Triton X-100 in DPBS for 10 min at RT. Following a further three washes with DPBS by shaking, the microbeads were incubated in the dark with phalloidin CF568 conjugate (Biotium, cat# 00044-T), prepared at a 1:40 dilution by mixing 5 µL of stock with 200 µL DPBS per well of a 24-well plate. Staining was performed for 60 min at room temperature (RT). Subsequently, the beads were rinsed in DPBS for 5 min. Finally, the beads were transferred to glass-bottom dishes containing a 50% glycerol solution to minimise movement during imaging using a fluorescence microscope.MTT assay for viability in alginate beadsA 5 mg/mL MTT solution was prepared in calcium- and magnesium-free DPBS, using MTT powder (Sigma-Aldrich, UK, cat# D8537). The solution was thoroughly vortexed, filter-sterilised through a 0.2 μm syringe filter, aliquoted, and stored at − 20 °C until use. Alginate beads containing UC-MSCs (100 µL packed volume including medium) were transferred into a 24-well plate (three wells per condition) and gently rinsed with calcium- and magnesium-free DPBS (Gibco, cat# 14190144) to remove residual culture medium prior to the MTT assay. Fifty microlitres of stock MTT solution was added to each well (final concentration 0.45 mg/ml in 500 µl serum-free Williams’ E medium), and the plate was incubated at 37 °C/5% CO₂ for 4 h, allowing purple formazan crystals to form within the beads. The medium was carefully removed, and 0.25 mL DMSO was added to each well to dissolve the formazan, with the plate then shaken for 30 min. The solubilised formazan was transferred to a 96-well plate, and absorbance at 550 nm was measured using the FLUOstar® Omega microplate reader (BMG LABTECH, Germany).Oxidative phosphorylation analysisEncapsulated MSCs (0.5 × 106 cells) were assessed for oxidative phosphorylation (OXPHOS) using an Oxygraph-2k high-resolution respirometer (Oroboros Instruments, Innsbruck, Austria). The two chambers (2 mL each) were filled with Williams E culture medium at 37 °C under gentle agitation (750 rpm). Oxygen consumption was measured at baseline, after adding 2.5 µM oligomycin (leak state), and following successive titrations of 1 µM CCCP (uncoupled state). Non-mitochondrial respiration was assessed after the addition of 2.5 µM antimycin A. Basal respiration, maximal respiratory capacity, reserve capacity, ATP-linked respiration, and the respiratory control ratio were determined. MSCs were either in cell suspension from 2D culture or encapsulated, with freshly thawed MSCs serving as controls.Fluorescence microscopy-based hypoxia measurements in microbeadsMicrobeads were cultured in 6-well plates for up to 5 days at 37 °C under standard CO₂ incubation. The microbeads were stained with 5 µM Image-iT™ Green Hypoxia Reagent (Invitrogen™), with sodium dithionite at 1.5 mM concentration (Sigma-Aldrich, cat# 7775-14-6) serving as a positive control for mimicking hypoxic conditions. After 4 h of incubation, the beads were imaged by fluorescent microscopy.Depolymerisation of alginate capsulesThe alginate beads were settled to remove the medium and resuspended in a solution containing a calcium chelator (0.2 M sodium citrate, 0.1 M EDTA, 10 mM HEPES, 0.1% glucose) on ice for 10 min to break down the alginate scaffold. The digestion solution was filtered through a 70-µm cell strainer to remove any remaining cross-linked alginate, followed by centrifugation at 300 g for 5 min to collect the released cells.RNA isolation and one-step RT- qPCRAt the desired time point, the culture medium was removed, and microbeads were washed twice with DPBS. The encapsulated cells were then retrieved by depolymerisation. Total RNA was then isolated using the Qiagen RNeasy Mini Kit (cat# 74104) following the manufacturer’s guidelines. The concentration and purity of the extracted RNA were determined using a Nanodrop spectrophotometer, and samples were stored at − 80 °C until further use. The mRNA levels of the target genes were quantified using the SuperScript™ III Platinum™ One-Step qRT-PCR Kit (Thermo Fisher Scientific, Life Technologies) according to the manufacturer’s instructions and run on an ABI Quant Studio 5384. Reactions utilised a 4 ng RNA template, with gene expression levels normalised to the housekeeping genes β-actin, GAPDH, and B2M at the specified time points. Reference gene stability was assessed across all experimental conditions, and β-actin, GAPDH, and B2M showed minimal Ct variability (coefficient of variation ≤ ~ 3%); therefore, the mean expression of these three genes was used for normalisation. The mRNA expression fold change relative to controls was calculated using the ΔΔCt method. The TaqMan® probes (Thermo Fisher Scientific, USA) employed for quantitative PCR are listed as follows: HIF1A (Hs00153153_m1), PGC1A (Hs00173304_m1), NRF1 (Hs00602161_m1), NRF2 (Hs00975961_g1), TFAM (Hs00273372_s1), TOMM40 (Hs01587378_m1), COX4I1 (Hs00971639_m1); CD90 (THY1) (Hs06633377_s1) GAPDH (Hs02786624_g1), B2M (Hs00187842_m1), ACTB (β-actin; Hs01060665_g1).Mitochondrial membrane potential assessment via JC-1 staining (2D and 3D encapsulated cultures)The cell mitochondrial membrane potential (ΔΨm) was assessed using the potentiometric dye JC-1 following the manufacturer’s instructions. (Thermo Fisher Scientific, Cat#T3168). Briefly, encapsulated MSCs and 2D-cultured MSCs were incubated with 5 µM JC-1 dye (Thermo Fisher Scientific) in serum-free WE medium at 37 °C with 5% CO₂ for 30 min. Following incubation, microbeads were washed twice with DPBS to remove excess dye prior to fluorescence analysis. Z-stack confocal images were acquired and subsequently subjected to maximum intensity projection. A threshold was applied to remove background fluorescence, and the mean fluorescence intensity within individually defined bead regions (ROIs) was quantified using the FIJI software (https://fiji.sc/).Enzyme-linked immunosorbent assays (ELISA)The conditioned medium was collected and centrifuged at 300 g for 5 min, then immediately frozen in liquid nitrogen and stored at − 80 °C for subsequent analysis. The concentrations of Human IL-6, VEGF, IL-10, and TNF-α were measured using DuoSet ELISA kits (R&D Systems, USA): IL-6 (cat# DY206-05), VEGF (cat# DY293B), IL-10 (cat# DY217B), and TNF-α (cat# DY210-05), following the manufacturer’s instructions.Measurement of lactate and pyruvate in cell cultureFollowing microbead cell culture, conditioned media was collected and stored at − 80 °C until further analysis. Lactate was quantified using an NADH/NAD+-independent enzymatic assay based on lactate dehydrogenase (LDH) activity, as described by Bergmeyer (1963). A reaction solution (pH 9) containing 0.75 mM NAD+, 0.4 M hydrazine hydrate, and 0.4 mM glycine was prepared. For each sample, 20 µL was added to a Greiner F-bottom 96-well plate, followed by 180 µL of reaction buffer with LDH (calculated to deliver 5 IU per 2 mL reaction volume from a 550 IU/mL stock). Absorbance was measured at 572 nm, and lactate concentrations were determined via a standard curve generated with sodium DL-Lactate (Cat# L9700-100mL). Pyruvate was measured similarly, using NADH (0.2 mM in PBS, pH 7.4) and 1.5 IU LDH per mL; 20 µL samples were incubated with 180 µL reaction buffer (0.9 IU LDH/well), and absorbance was recorded at 340 nm after 15 min. Lactate and pyruvate concentrations were calculated from standard curves generated using known concentrations of sodium L-lactate and sodium pyruvate standards. A standard curve was prepared using lactate and pyruvate standards (1.6–0.025 mM) in a 96-well plate. Optical density at 570 nm was measured, and sample concentrations were determined by interpolation from the standard curve.Quantification and statistical analysisData are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 10. Normality of data distribution was assessed using the Shapiro–Wilk test. For non-normally distributed data (e.g., microbead size), the Mann–Whitney U test was applied. For parametric data, one-way or two-way ANOVA was used as appropriate, followed by Tukey’s multiple comparisons test. Significance was defined as: ns (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.00001. Sample size selection was guided by a power calculation for paired analyses, assuming a standard deviation not exceeding 20% of the mean, indicating that n = 3 would be sufficient to detect differences of approximately 30% between group means.ResultsCharacterisation, viability, and actin network morphology of MSCs encapsulated in clinical-grade alginateTo investigate the impact of alginate formulations on MSCs, cells were encapsulated in either unmodified SLG20 or RGD-modified alginate (G-RGD) and cultured for up to five days. We used low-viscosity ultrapure sodium alginate with a guluronate (G) content above 60%, specifically the PRONOVA™ SLG20 or NOVATACH™ MVG GRGDSP formulations from NovaMatrix. As shown in Fig. 1A, both formulations produced morphologically clear, uniform, and well-defined spherical microbeads.The diameters of SLG20 and G-RGD microcapsules ranged between 431 and 673 μm. The mean diameter of SLG20 microcapsules was 504.34 ± 1.45 μm, while G-RGD microcapsules exhibited a mean diameter of 500.71 ± 2.02 μm (mean ± SEM), n = 66 beads per group from 3 independent experiments. No statistically significant difference was observed between the groups. Figure 1B shows violin plots illustrating the size distribution and mean diameter of microbeads for each alginate formulation, highlighting the tight size distribution and consistency across different compositions.To assess the metabolic activity and cell viability of encapsulated MSCs, fluorescein diacetate (FDA) staining and an MTT assay were performed at the indicated time points. Cell viability was evaluated using FDA staining on days 1 and 5. As illustrated in Fig. 1C, G-RGD alginate enhanced cell viability and significantly increased oxidative function over time compared to SLG20 within 24 h of production (Fig. 1D).The RGD peptide present in the G-RGD alginates serves as an anchoring motif for the encapsulated cells, which are absent in the unmodified SLG20 alginate. To gain further insight into the cell-matrix interactions and the morphology changes of encapsulated cells, a phalloidin staining was carried out to visualise the cellular actin network morphology (Fig. 1E). Within SLG20 beads, cells predominantly exhibited a rounded morphology with minimal cytoskeletal development, suggesting limited interaction with the surrounding alginate matrix. In contrast, some MSCs encapsulated in G-RGD alginate exhibited cytoplasmic spreading, with evidence of actin network morphology. This was distinct from the more diffuse and uniform cortical F-actin staining observed in SLG20 encapsulated cells. While the current image resolution does not visualise distinct stress fibres or filopodia-like protrusions, the observed cytoskeletal pattern and cell elongation suggest enhanced integrin-mediated adhesion and interaction with the RGD-modified matrix. Although some cells maintained a rounded morphology, the overall cellular architecture suggested integrin-mediated adhesion and enhanced interaction with the RGD-modified matrix. The morphology of the cells, however, was entirely different from the classical morphology of MSCs when they were cultured on 2D conventional tissue culture plastic vessels (not shown). There were also no visible direct contact cell-to-cell interactions within the microbeads.Fig. 1The alternative text for this image may have been generated using AI.Full size imageViability, metabolic activity, and morphology of post-thawed MSCs encapsulated in clinical-grade alginates. (A) Following microbead fabrication, bright-field images were taken on day 0 using the EVOS™ FL Auto Imaging System (Thermo Fisher Scientific). Both unmodified SLG20 and modified G-RGD alginate produced morphologically uniform, well-defined spherical beads. Scale bar: 2000 μm. (B) Quantification of microbead diameters using FIJI software (https://fiji.sc/). The violin plot shows bead size distribution for SLG20 and G-RGD groups (n = 66 beads per group, n = 3). Mean bead size is indicated. (C) FDA staining of encapsulated MSCs in SLG20 and G-RGD alginate beads on days 1 and 5. Cell viability was assessed by fluorescence imaging. Bright-field and fluorescence images were captured using the EVOS™ FL Auto Imaging System. Scale bar: 2000 μm. (D) MTT assay. MSCs encapsulated in SLG20 and G-RGD alginate beads were tested at specific time points over a 5-day culture period. Data are presented as mean ± SEM (n = 3). (E) Fluorescence imaging of the actin network morphology in MSCs encapsulated in SLG20 and G-RGD alginate beads using phalloidin staining. Images were acquired using a Leica live-cell fluorescence microscope with a 20x objective lens. Brightness and contrast were adjusted using Fiji. (n = 3). The white rectangular box indicates the region shown in the inset, presenting a magnified view of cell morphology within the microbeads.Encapsulation reduces OXPHOS and induces HIF1A upregulationTo investigate how encapsulation and alginate composition influence MSC bioenergetics, mitochondrial function was assessed by high-resolution respirometry using an Oroboros instrument (Fig. 2A). Basal respiration, assessed in William’s E medium supplemented with 10% FBS, was significantly higher in 2D-cultured MSCs (40.0 ± 4.5 pmol O₂·s⁻¹·10⁻⁶ cells) than in thawed MSCs (23.4 ± 3.0 pmol O₂·s⁻¹·10⁻⁶ cells). The encapsulation resulted in a 58% decrease oxygen consumption rate as cultured MSCs encapsulated in G-RGD showed a respiration of 17 ± 2.0pmol O₂·s⁻¹·10⁻⁶ cells (p