oroboros high resolution respirometry  (Oroboros Instruments)

Journal: Scientific Reports

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

doi: 10.1038/s41598-026-42864-7

Figure Lengend Snippet: OXPHOS activity, oxygen availability, and HIF1A gene expression in MSCs under various encapsulation conditions. ( A ) OXPHOS Parameters, including basal respiration, ATP-linked respiration, maximal respiratory capacity, and reserve capacity, were assessed using Oroboros high-resolution respirometry. Analyses were conducted on passage 4 MSCs maintained in 2D culture ( n = 6), cryopreserved-thawed MSCs ( n = 9), and MSCs that were either thawed and encapsulated in SLG20 or G-RGD alginate ( n = 3 per group), or cultured and subsequently encapsulated in G-RGD alginate ( n = 4), all assessed immediately after encapsulation (Day 0). Cells or alginate microbeads were sequentially exposed to defined substrates and inhibitors to interrogate distinct components of the mitochondrial respiratory chain. OXPHOS were recorded in real-time and normalised to cell number, expressed as pmol O₂·s⁻¹·10⁻⁶ cells (specific flux). ( B ) Microbeads were stained with 5 µM Image-iT™ Green Hypoxia Reagent (Invitrogen™) and incubated for 4 h. Sodium dithionite-treated beads (top left) showed strong fluorescence, confirming probe sensitivity under hypoxic conditions. Minimal fluorescence signal was observed in MSCs encapsulated in G-RGD alginate at all time points (up to Day 5); representative images from Day 3 microbeads are shown here. Images were acquired using an inverted fluorescence microscope. Scale bars = 2000 μm. ( C ) HIF1A mRNA was assessed via qRT-PCR in encapsulated MSCs over time ( n = 3). Fold changes in MSCs encapsulated in SLG20 versus G-RGD, normalised to SLG20 Day 0. Comparison including 2D-cultured MSCs, highlighting time-dependent upregulation of HIF1A expression in 3D-encapsulated conditions. Statistical analysis by one-way ANOVA with Tukey’s post hoc test; data are presented as mean ± SEM from at least three independent experiments ( n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns, not significant (GraphPad Prism10).

Article Snippet: Fig. 3 Time-Dependent OXPHOS Dynamics and Glycolytic output in Encapsulated MSCs ( A ) OXPHOS—including basal, ATP-linked, maximal, and reserve capacity—was assessed in thawed MSCs encapsulated in G-RGD or SLG20 alginate at day 0 and subsequent time points using Oroboros high-resolution respirometry.

Techniques: Activity Assay, Gene Expression, Encapsulation, Cell Culture, Staining, Incubation, Fluorescence, Microscopy, Quantitative RT-PCR, Comparison, Expressing

Journal: Scientific Reports

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

doi: 10.1038/s41598-026-42864-7

Figure Lengend Snippet: Time-Dependent OXPHOS Dynamics and Glycolytic output in Encapsulated MSCs ( A ) OXPHOS—including basal, ATP-linked, maximal, and reserve capacity—was assessed in thawed MSCs encapsulated in G-RGD or SLG20 alginate at day 0 and subsequent time points using Oroboros high-resolution respirometry. Microbeads were exposed to defined mitochondrial substrates and inhibitors, and oxygen consumption was measured in real-time, normalised to cell number and expressed as pmol O₂·s⁻¹·10⁻⁶ cells (specific flux). ( B ) Total extracellular lactate + pyruvate levels and lactate-to-pyruvate ratio (L/P) were measured on days 1, 3, and 5 in MSCs encapsulated in SLG-20, G-RGD, and cultured in 2D. ( A , B ) Data are shown as mean ± SEM ( n = 3). Statistical analysis used two-way ANOVA with Tukey’s post hoc test. Significance levels are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = not significant.

Article Snippet: Fig. 3 Time-Dependent OXPHOS Dynamics and Glycolytic output in Encapsulated MSCs ( A ) OXPHOS—including basal, ATP-linked, maximal, and reserve capacity—was assessed in thawed MSCs encapsulated in G-RGD or SLG20 alginate at day 0 and subsequent time points using Oroboros high-resolution respirometry.

Techniques: Cell Culture