What if the culture medium used to grow therapeutic cells quietly changes the quality of the vesicles they release? This study tests whether a GMP-compliant, serum-free medium can make production more standardized without sacrificing anti-fibrotic activity.

The therapeutic potential of extracellular vesicles derived from human mesenchymal stromal cells is limited by the lack of standardized, Good Manufacturing Practice-compliant production protocols. The study compares MSC-Brew, a commercially available Good Manufacturing Practice-grade medium, with conventional cultures using Dulbecco’s Modified Eagle Medium supplemented with ten percent fetal bovine serum.

The central question is whether Brew-grown cells retain their characteristic surface markers and produce vesicles that meet extracellular-vesicle criteria, including particle size, concentration, marker expression, and minimal inflammatory cytokine content.

The key findings are a higher particle-to-protein ratio, largely conserved protein composition, only four differentially expressed microRNAs, and reduced collagen secretion in transforming growth factor beta one-activated LX-2 cells. Together, these findings support MSC-Brew as a standardized, serum-free platform for producing high-quality extracellular vesicles for therapeutic applications.

Standardized manufacturing and Good Manufacturing Practice are being adopted to improve reproducibility, scalability, and regulatory compliance for mesenchymal stromal cell extracellular vesicles. A crucial choice is the culture medium, because it must support mesenchymal stromal cell isolation and expansion while maintaining extracellular-vesicle quality and therapeutic efficacy.

MSC-Brew GMP Medium is among the few Good Manufacturing Practice-compliant options reported to support mesenchymal stromal cell isolation and expansion from multiple tissue sources. However, earlier studies mainly examined mesenchymal stromal cell expansion, leaving the effects of Brew medium on mesenchymal stromal cell-derived extracellular vesicles unexplored.

This study evaluates both the characteristics and biological activity of extracellular vesicles derived from adult-dermis mesenchymal stromal cells, addressing limited data on clinically relevant serum-free production. Mesenchymal stromal cells were isolated and expanded either in MSC-Brew GMP Medium or in standard Dulbecco’s Modified Eagle Medium supplemented with fetal bovine serum.

Extracellular vesicles were isolated from the serum-free secretome by differential ultracentrifugation, and assessment focused on purity, identity, and biological activity. The workflow followed a published Good Manufacturing Practice-compliant process for mesenchymal stromal cell extracellular-vesicle production.

Characterization combined Nanoparticle Tracking Analysis and Atomic Force Microscopy with protein quantification, Western blotting, and Luminex cytokine profiling. The study also included informative tests: extracellular-vesicle proteomics and a disease-relevant evaluation of anti-fibrotic microRNA content.

Biological activity was tested in an in vitro model using immortalized hepatic stellate cells called LX-2, activated with pro-fibrogenic transforming growth factor beta one. After activation, LX-2 cells become myofibroblast-like cells characterized by increased collagen production and alpha smooth muscle actin expression.

L-ascorbic acid was added alongside transforming growth factor beta one because it is a cofactor of prolyl four-hydroxylases involved in collagen biosynthesis. The addition of L-ascorbic acid was intended to simulate pathological collagen deposition, while dimethyloxalylglycine was included as an anti-fibrotic treatment control.

Human skin biopsies measuring fifteen by fifteen square centimeters came from healthy adult donors aged twenty to forty who were undergoing plastic surgery, with review-board approval and signed informed consent. Mesenchymal stromal cells were isolated from the dermis using a non-enzymatic cell outgrowth method.

The biopsy fragments were placed dermis-side down on treated tissue-culture dishes and incubated at room temperature for twenty minutes before medium was added. The cultures were maintained at thirty-seven degrees Celsius with five percent carbon dioxide, the medium was refreshed every four days, and outgrown cells were harvested after fifteen days for expansion.

When adult-dermis mesenchymal stromal cell cultures reached eighty percent confluence, the growth medium was removed and the cells were washed three times with phosphate-buffered saline. Serum-free alpha Minimum Essential Medium was added, and the secretome was collected after forty-eight hours.

Initial debris removal used centrifugation at two thousand times gravity for ten minutes. Extracellular vesicles from six preparations were isolated from the secretome by differential ultracentrifugation. The protocol used sequential spins at twenty thousand times gravity for forty minutes and one hundred sixty thousand times gravity for two hours at four degrees Celsius.

Figure one verifies the immunophenotype of adipose-derived mesenchymal stromal cells at passage two in both Brew medium and DMEM with ten percent FBS. The flow-cytometry workflow first selects cells by forward- and side-scatter, removes doublets, and excludes non-viable cells using seven-AAD.

The histograms then assess the positive markers CD105, CD73, and CD90, alongside negative markers CD45, CD34, and HLA-DR, with red showing isotype controls and blue showing antibody staining. Flow cytometry found consistent surface-antigen expression in adult-dermis mesenchymal stromal cells cultured in either MSC-Brew GMP medium or Dulbecco’s Modified Eagle Medium with ten percent fetal bovine serum.

All cells were positive for the classical mesenchymal stromal cell markers CD90, CD105, and CD73, and negative for the hematopoietic markers CD34, CD45, and HLA-DR. These results were obtained from cells at passage two. Figure two validates the identity and physical characteristics of AD-MSC extracellular vesicles produced in either Brew medium or DMEM with ten percent FBS.

NTA shows mostly small, relatively homogeneous particles, while AFM displays vesicle-like structures and the PBS control lacks this pattern. Western blots detect the EV markers Alix and CD81, but not calnexin or beta-actin, and both EV preparations contain fibronectin and collagen-related bands, including cleavage fragments.

Particle-size histograms showed homogeneous populations with minimal contamination from larger particles. Brew extracellular vesicles averaged one hundred twenty-five plus or minus eighty nanometers, while Dulbecco’s Modified Eagle Medium extracellular vesicles averaged one hundred fifteen plus or minus seventy nanometers; both fit the MISEV definition of small extracellular vesicles below two hundred nanometers.

From twenty-eight milliliters of secretome, concentrations were three point four plus or minus one point zero times ten to the tenth particles per milliliter for Brew vesicles and three point one six plus or minus zero point seven five times ten to the tenth for Dulbecco’s Modified Eagle Medium vesicles.

Atomic Force Microscopy confirmed isolated spherical or dome-shaped particles, with particle heights ranging from sixty to one hundred nanometers. No significant differences in morphology, size distribution, or surface cleanliness appeared between the two preparations, and no particles were detected in the phosphate-buffered saline control.

Both Brew and Dulbecco’s Modified Eagle Medium extracellular vesicles were positive for Alix and CD81 and showed no expression of calnexin or beta-actin, unlike their parent cells. Both extracellular-vesicle types were enriched in extracellular-matrix proteins, including fibronectin one and collagen.

Collagen showed prominent bands near fifty and thirty-seven kilodaltons, likely corresponding to collagen cleavage products, while procollagen bands at two hundred and one hundred eighty kilodaltons were weakly detected. Collagen enrichment was significantly higher in adult-dermis extracellular vesicles than in extracellular vesicles from non-dermal sources such as umbilical-cord mesenchymal stromal cells.

Figure three profiles twenty inflammatory markers in EVs produced with Brew medium or DMEM plus ten percent FBS, using a Luminex assay. Most pro-inflammatory cytokines appear near the baseline, while adhesion molecules and selectins are clearly detected; the chart also marks a significant difference for ICAM-1 with three asterisks.

This matters because it shows that culture conditions can shape the inflammatory and adhesion-related cargo of AD-MSC-EVs. Luminex analysis found a near absence of pro-inflammatory cytokines in extracellular vesicles from both culture conditions. Adhesion molecules and selectins were present in both extracellular-vesicle types, with higher concentrations in the Dulbecco’s Modified Eagle Medium group.

Brew vesicles had lower E-selectin, P-selectin, and ICAM-one levels than Dulbecco’s Modified Eagle Medium vesicles: four hundred fifty versus nine hundred ninety-seven, three hundred thirteen versus one thousand eight hundred sixty-one, and one thousand seven hundred sixty-eight versus five thousand three hundred seventy-one picograms per milliliter, respectively.

Only ICAM-one showed a statistically significant difference, with a p-value below zero point zero zero one. Figure four compares the proteomes of EVs and secretomes from AD-MSCs cultured in Brew medium or DMEM with ten percent FBS. The volcano plots show twenty-eight proteins enriched in Brew-EVs versus eight in DMEM-EVs, while the secretome comparison identifies one hundred thirty-nine and two hundred twenty-eight, respectively.

Venn diagrams and STRING enrichment indicate that shared Brew proteins relate to extracellular organization and blood vessel morphogenesis, whereas EV-exclusive proteins associate with skin and epidermis development, highlighting distinct cargo patterns between EVs and secretome.

Mass spectrometry identified one thousand two hundred fifty-eight proteins in adult-dermis mesenchymal stromal cell extracellular vesicles, with twenty-eight significantly more abundant in Brew vesicles and eight enriched in Dulbecco’s Modified Eagle Medium vesicles.

The secretome contained one thousand nine hundred ten proteins, including one hundred thirty-nine enriched in the Brew condition and two hundred twenty-eight enriched in the Dulbecco’s Modified Eagle Medium condition. The secretome therefore displayed a broader protein repertoire than the extracellular-vesicle fractions.

Both conditions shared general biological-process enrichment, while specific patterns emerged in the Brew condition, including enrichment related to wound healing among proteins shared between Brew vesicles and the Brew secretome. Figure five compares miRNA expression in Brew-EVs and DMEM-EVs using a volcano plot: each point combines log two fold change with statistical significance, shown as minus log ten of the p-value.

Only four of roughly seven hundred fifty miRNAs were differentially expressed, including Brew-EV-associated miR-199a-5p and three miRNAs associated with DMEM-EVs: miR-99b-3p, miR-591, and miR-572. This matters because the largely similar profiles support comparable anti-fibrotic miRNA content across the two culture conditions, while miR-199a-5p may relate to extracellular-matrix signaling.

The microRNA profiles were largely similar: only four microRNAs differed out of approximately seven hundred fifty analyzed. MicroRNA one hundred ninety-nine-a-five-prime was upregulated in Brew vesicles, while microRNA ninety-nine-b-three-prime, microRNA five hundred ninety-one, and microRNA five hundred seventy-two were downregulated.

Both Brew and Dulbecco’s Modified Eagle Medium extracellular vesicles showed comparable anti-fibrotic microRNA profiles, with similar expression patterns and intensities. Figure six tests AD-MSC-EVs in TGF-beta-one-activated LX-two liver stellate cells, using Western blots and densitometry for secreted and intracellular COL1A1, plus alpha-SMA.

The authors report that two sequential EV doses reduced secreted collagen, while the blot showed no detectable effect on intracellular collagen; alpha-SMA levels were also quantified after Brew-EV and DMEM-EV treatment. This matters because it provides an in-vitro readout of anti-fibrotic activity in a liver fibrosis-relevant model.

The LX-2 assay compared vehicle control, recombinant human transforming growth factor beta one, and transforming growth factor beta one plus zero point one seven millimolar L-ascorbic acid. Adult-dermis mesenchymal stromal cell extracellular vesicles were tested at fifty thousand vesicles per cell, while one millimolar dimethyloxalylglycine served as a positive anti-fibrotic control.

Extracellular vesicles and dimethyloxalylglycine were administered together with transforming growth factor beta one or transforming growth factor beta one plus L-ascorbic acid. The extracellular vesicles were given in two doses on days one and two, whereas dimethyloxalylglycine was given once, and protein samples were collected forty-eight hours after the first treatment dose.

Because collagen was detected in the extracellular vesicles themselves, the study also evaluated whether collagen contamination could contribute to the observed effect. Adult-dermis mesenchymal stromal cell extracellular vesicles significantly reduced secreted collagen in transforming growth factor beta one-activated LX-2 cells, but they did not produce a detectable reduction in intracellular collagen.

One dose of fifty thousand vesicles per cell did not reduce collagen secretion, while two sequential doses at the same concentration were required for effective inhibition. A single dose of dimethyloxalylglycine was sufficient to suppress collagen secretion.

Extracellular-vesicle treatment also reduced alpha-smooth-muscle-actin expression, with a stronger effect in cells co-activated with transforming growth factor beta one and L-ascorbic acid than with transforming growth factor beta one alone. Brew and Dulbecco’s Modified Eagle Medium extracellular vesicles showed comparable anti-fibrotic activity.

Figure seven shows LX-2 cell morphology across four conditions. Untreated cells appear in panel A, while TGF-beta one with L-ascorbic acid produces the network-like morphology shown on day three in panel B. Panels C and D show cells co-treated with the same activators and two doses of either Brew-derived or DMEM-derived extracellular vesicles, with visibly altered patterns relative to activated cells.

This matters because the figure provides a direct morphological readout of stellate-cell activation and EV treatment, using a one-hundred-micrometre scale bar. The results suggest that culture conditions did not significantly influence the anti-fibrotic potential of adult-dermis mesenchymal stromal cell extracellular vesicles.

The microRNA profiles were largely comparable, with only four microRNAs showing differential expression, including microRNA one hundred ninety-nine-a-five-prime, which was upregulated in Brew vesicles. MicroRNA one hundred ninety-nine-a-five-prime is predicted to target genes in the extracellular-matrix-receptor-interaction pathway, whose genes are involved in fibrosis.

Its role remains uncertain because studies have described both protective and detrimental effects in liver disease, so its upregulation may contribute to, but does not establish, anti-fibrotic activity. MSC-Brew GMP Medium provides a robust and reproducible platform for deriving adult-dermis mesenchymal stromal cells and producing high-quality extracellular vesicles for therapeutic use.

The serum- and xeno-free medium preserves mesenchymal stromal cell properties and enhances extracellular-vesicle purity, making it promising for fibrosis-related disorders. The study addresses a critical gap by comprehensively evaluating how MSC-Brew affects the functional characteristics of mesenchymal stromal cell-derived extracellular vesicles.

Further studies should evaluate therapeutic efficacy and long-term stability across various disease models before the clinical potential is fully assessed. The work is presented as a step toward standardizing mesenchymal stromal cell extracellular-vesicle production for clinical use.

MSC-Brew preserved the identity and anti-fibrotic activity of dermal MSC-derived extracellular vesicles while producing a higher particle-to-protein ratio than serum-containing culture. That makes it a promising manufacturing platform, although long-term and disease-model testing is still needed.