DOI: 10.5281/zenodo.17242428
title: Supporting data for the article "METABOLIC REPROGRAMMING OF MULTIPOTENT MESENCHYMAL STROMAL CELLS IN THREE-DIMENSIONAL SPHEROIDS"

description: The data set includes the following files:

Figure 1. Formation of spheroids from AT MSCs and size characterization over time. (A) Representative images of spheroid formation in BiofloatTM plates from the seeding point to day 7. Scale bar = 100 μm. (B) Quantitative evaluation of spheroid diameter. Data are presented as mean±SD, n=10 spheroids per time point. Note: **** - p<0.0001 compared to day 0 (seeding point). (C) Comparison of cell size following isolation from spheroids versus cells cultured in monolayer conditions and subsequently trypsinized. The values are expressed as mean±SD, n=32 per group. Note: **** - p<0.0001 compared to monolayer-cultured cells.

Figure 2. Spheroid culture-driven changes in secretory protein expression and release (N=4 in duplicates). (A) Expression of TSG6, HGF, and VEGFA in monolayer-cultured and spheroid-cultured cells. Box plots show median, interquartile range, 5th and 95th percentiles. Note: * - p<0.05 compared to monolayer, # - p<0.05 compared to day 3 of spheroid culture. (B) Alteration in the release of cytokines and growth factors during spheroid culture compared to monolayer conditions. Box plots show median, interquartile range, 5th and 95th percentiles. Note: * - p<0.05 compared to monolayer, # - p<0.05 compared to day 3 of spheroid culture. (C) Representative immunofluorescence images of spheroids at day 3 and day 7 of culture, stained with anti-HIF-1 antibody. Scale bar = 200 μm.

Figure 3. Metabolic activity of AT MSCs in monolayer and spheroids (N=4 per condition). (A) Resazurin reduction by cells cultured in spheroids and monolayers. Box plots show median, interquartile range, 5th and 95th percentiles. Note: * - p<0.05, ** - p<0.01, *** - p<0.001, **** - p<0.0001 compared to day 1 of culture in corresponding conditions. (B) The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of monolayer-cultured AT MSCs, spheroid-derived cells, and cells in spheroids. AT MSCs in each group were assessed using the Seahorse assay, and results were normalized to equal cell number. Note: X and Y axes scales and measurement conditions for spheroids differ from other panels because of protocol adjustments for 3D structures. (C) AMP and ADP levels in monolayer and spheroid cultures. Note: * - p<0.05, ** - p<0.01, *** - p<0.001.

Figure 4. Metabolomic reprogramming of cells in spheroids cultured for 3 and 7 days (N=4 per condition). (A) Principal component analysis (PCA) of metabolomic profiles from monolayer-cultured cells (white circles) and spheroid-cultured cells (blue – 3 days; violet – 7 days of 3D culture). (B) Volcano plot showing changes of key metabolite classes and subclasses, including amino acids, purine/pyrimidine metabolites, phospholipids, glycerides, fatty acids, and lysophospholipids. (C) KEGG pathway enrichment analysis of significantly altered polar metabolites performed in MetaboAnalyst. Each dot represents a metabolic pathway, with colour indicating p-value, and the dot size reflecting the pathway’s impact values.

Figure 5. Lipid profile of cells cultured in monolayer and spheroids for 3 and 7 days. A doughnut chart illustrates the distribution of 664 identified lipid species grouped into 12 most abundant lipid classes. Lipid classes representing less than 3% of the total lipid profile were combined in the category “Others”. The proportion of upregulated (white segments) and downregulated (grey segments) lipid species within each class at days 3 and 7 of spheroid culture, relative to monolayer conditions, is presented as horizontal bar charts (N=4 per condition).

Figure 6. Changes in phospholipid and fatty acid selected classes and expression of corresponding metabolic enzymes between day 3 and day 7 of spheroid culture (N=4 per condition). (A) Total TIC-normalized abundances of main phospholipid subclasses in spheroids and monolayer cultures. Box plots show median, interquartile range, 5th and 95th percentiles. Note: * - p<0.05, ** - p<0.01, *** - p<0.001, **** - p<0.0001. (B) Heatmap illustrating the changes in enzymes related to phospholipid and triacylglycerol synthesis, fatty acid synthesis, and desaturation occurring during spheroid culture. (C) Comparison of the fatty acid saturation profiles between monolayer-cultured and spheroid-cultured cells.

Figure 7. Spheroid culture is associated with the release of extracellular vesicles (EVs). (A) Scanning electron microscopy image of a smaller-sized (5,000 cells) spheroid, showing the entire structure, which allows for clear visualization of EVs on the spheroid surface. Scale bar = 100 μm. (B) A higher-density 50,000 cells spheroid, which is larger in size, allows only the surface to be imaged, revealing EVs present on the outer layer. Scale bar = 20 μm. (C) Confocal microscopy and (D) Spinning disk super-resolution microscopy images of EVs in the secretome released by spheroids formed from PKH26-labelled AT-MSCs (cyan LUT pseudocolour is applied for enhanced contrast and overall presentation quality).  (E) Size distribution of EVs produced by spheroids.    

Figure 8. Comparison of lipid composition between spheroids and their secreted extracellular vesicles (EVs). (A) Overlap of lipid species identified in spheroids (day 3 of culture) and their secreted EVs. (B) Pearson correlation between absolute abundances of individual lipid species in spheroids and EVs. (C) Pearson correlation between absolute abundances of key lipid classes in spheroids and EVs. (D) Pearson correlation between fold changes in individual lipid species in spheroids relative to monolayer cultures and their corresponding absolute levels in EVs. (E) Pearson correlation between fold changes in lipid classes in spheroids relative to monolayer cultures and their corresponding absolute levels in EVs. 

Supplemental materials - file includes: Figure 1S. Total TIC-normalized abundances of glycerols in spheroids and monolayer cultures. Box plots display the median, interquartile range, and the 5th and 95th percentiles. Note: * - p<0.05, ** - p<0.01, *** - p<0.001, **** - p<0.0001. Figure 2S. Total TIC-normalized abundances of main lysophospholipid subclasses in spheroids and monolayer cultures. Box plots display the median, interquartile range, and the 5th and 95th percentiles. Note: * - p<0.05, ** - p<0.01, *** - p<0.001, **** - p<0.0001. Table 1. Lipid class composition and detected lipid species count in spheroids and spheroid-derived extracellular vesicles (EVs).

Additional data:



Metabolomic analysis of MSCs cultured in spheroids and in monolayer.

Metabolomic analysis of EVs derived from MSCs cultured in spheroids.

Analysis of cell size following isolation from spheroids versus cells cultured in monolayer conditions and subsequently trypsinized.

Analysis of changes of AT MSC-based spheroid size over time.

Confocal microscopy and spinning disk super-resolution microscopy images of EVs in the secretome released by spheroids 

Representative immunofluorescence images of spheroids at day 3 and day 7 of culture, stained with anti-HIF-1 antibody