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The MSC Secretome: Conditioned Medium, Growth Factors and EVs

Por Benn Bluestein-Veyra, M.Sc. en Química Orgánica · Actualizado el 20 de octubre de 2026

The MSC secretome is the full set of molecules that mesenchymal stromal cells release into their surroundings, and MSC conditioned medium is the culture medium that collects them.^[1] It contains soluble growth factors, cytokines and chemokines together with extracellular vesicles that enclose proteins and RNA.^[1][2]

This reference describes what the secretome contains, how conditioned medium is produced, how culture conditions change it, how vesicle fractions are separated from it and how each fraction is measured. Witwer and colleagues, writing for four scientific societies, framed these measurements as quantifiable metrics that let one laboratory compare its preparations with another.^[3]

Understanding the paracrine model of MSC biology

In 2006 Caplan and Dennis described marrow-derived mesenchymal stem cells as trophic mediators. Beyond their capacity to differentiate into osteoblasts, chondrocytes, adipocytes and other mesenchymal phenotypes, the cells secrete a variety of cytokines and growth factors with paracrine and autocrine activity.^[4]

Caplan and Dennis grouped the reported activities of these secreted factors, including modulation of local immune responses, support of angiogenesis and stimulation of mitosis and differentiation in neighboring cells, under the term trophic effects. They described these effects as distinct from the direct differentiation of the cells themselves.^[4]

Experimental support for soluble mediators came from co-culture work. Di Nicola and colleagues separated bone marrow stromal cells from T cells with a Transwell membrane and still observed suppressed T cell proliferation, and neutralizing antibodies identified TGF-beta1 and hepatocyte growth factor (HGF) as the mediators.^[5]

Konala and colleagues describe the secretome as the combined repertoire of soluble factors and factors released in extracellular vesicles such as exosomes and microvesicles.^[1] Research on MSC conditioned medium therefore studies both classes of material, together and apart.^[6]

The MSC secretome combines soluble proteins, matrix components, metabolites and vesicles

MSC conditioned medium holds soluble growth factors, cytokines, chemokines, matrix proteins and metabolites alongside a population of extracellular vesicles (Fig. 1).^[1][7] The vesicles enclose their own proteins, RNA and metabolites within a lipid bilayer.^[8]

Sze and colleagues produced one of the first systematic catalogs. They conditioned a chemically defined, serum-free medium with MSCs derived from human embryonic stem cells, analyzed it by multidimensional protein identification technology and cytokine antibody arrays, and identified 201 unique gene products, 86 to 88 percent of which had detectable transcripts in the cells.^[9]

Computational analysis of that protein set predicted activation of signaling pathways including Jak-STAT, MAPK, Toll-like receptor, TGF-beta and mTOR.^[9] Later proteomic studies extended this catalog to MSCs from bone marrow, adipose tissue, placenta and Wharton’s jelly.^[10]

Growth factors: VEGF-A, HGF, IGF-1 and TGF-beta1

Hsiao and colleagues compared paracrine factor expression in MSCs from bone marrow, adipose and dermal tissue. VEGF-A, angiogenin, basic fibroblast growth factor and nerve growth factor were expressed at comparable levels across the populations, while IGF-1, VEGF-D and IL-8 were higher in adipose-derived cells.^[11]

Protein measurements in their conditioned media confirmed comparable VEGF-A and angiogenin secretion across all the populations. In endothelial tube formation assays, neutralizing antibodies identified VEGF-A and VEGF-D as two of the main secreted factors supporting tubulogenesis by adipose-derived cell media.^[11]

VEGF-A belongs to the PDGF and VEGF growth factor family and is a heparin-binding protein that exists as a disulfide-linked homodimer (Fig. 2a). It induces proliferation and migration of vascular endothelial cells.^[12]

HGF binds its receptor to regulate cell growth, cell motility and morphogenesis in many cell and tissue types, and it is made as a preproprotein that is proteolytically processed.^[13] In the co-culture experiments of Di Nicola and colleagues, HGF acted together with TGF-beta1 as a soluble stromal cell signal.^[5]

TGF-beta1 is secreted as a preproprotein that is processed into a latency-associated peptide and a mature peptide. The mature homodimer binds TGF-beta receptors to recruit and activate SMAD transcription factors (Fig. 2b).^[14]

Cytokines and chemokines

Park and colleagues profiled the media of human bone marrow MSCs on an antibody array recognizing 120 cytokines and chemokines. IL-6 gave the predominant signal, with moderately elevated signals for IL-8, TIMP-2, MCP-1, VEGF and osteoprotegerin.^[15]

The same profile appeared in cells from three different donors and was similar, with some differences, to that of umbilical cord blood-derived cells. The authors suggested that the profile might depend slightly on cell origin, with little effect of the individual donor.^[15]

Matrix proteins and metabolites

The secretome also carries matrix and matrix-regulating proteins. Park and colleagues detected the metalloproteinase inhibitor TIMP-2 among the abundant secreted factors, and Lai and colleagues found fibronectin containing the EDA domain on one class of MSC vesicles.^[8][15]

Showalter and colleagues reported that MSCs exposed to culture conditions designed to mimic the in vivo microenvironmental niche underwent glycolytic reprogramming. Their exosomes were packaged with metabolites previously associated with immunomodulation, including M2 macrophage polarization and regulatory T cell induction.^[7]

Extracellular vesicles and their RNA cargo

Chen and colleagues studied conditioned medium from MSCs derived from human embryonic stem cells. It held small RNAs enclosed in cholesterol-rich phospholipid vesicles, which protected them from RNase until the membranes were disrupted. MicroRNAs such as let-7b and let-7g were present with a high ratio of precursor to mature forms.^[2]

Baglio and colleagues sequenced the small RNAs of exosomes from bone marrow and adipose MSCs. MicroRNAs made up only 2 to 5 percent of the exosomal small RNA, a defined subset was enriched relative to the cells, and tRNA species, in particular tRNA halves, were preferentially released.^[16]

Vesicles in the secretome are themselves heterogeneous. Lai and colleagues separated at least three MSC vesicle types by their affinity for lipid-binding ligands, and each carried a different combination of membrane lipid, protein and RNA.^[8]

Fig. 2 | VEGF-A receptor-binding domain homodimer and TGF-beta1 bound to its type II and type I receptors.

MSC conditioned medium is produced through a defined culture workflow

Conditioned medium production starts with expanded cells, continues with a conditioning period in defined medium, and ends with harvest and clarification of the supernatant (Fig. 3).^[17] Witwer and colleagues list the diversity of MSC preparations and of vesicle production and separation methods among the main sources of variation between studies.^[3]

Expansion media and serum-free conditioning

Serum and platelet lysate supplements carry vesicles of their own. Pachler and colleagues depleted vesicles from pooled human platelet lysate medium by ultracentrifugation, and bone marrow MSCs grown in it retained their surface markers, morphology, viability and osteogenic and adipogenic differentiation in vitro.^[18]

Vesicles from cells in that depleted medium showed a more clearly defined pattern of vesicle-associated proteins. Reducing the platelet lysate from 10 to 2 percent, or removing it, strongly altered MSC characteristics and the RNA content of released vesicles.^[18]

For the conditioning step itself, Sze and colleagues used a chemically defined serum-free medium.^[9] Mitchell and colleagues incubated adipose-derived MSCs in a saline solution to obtain a secretome free of exogenous molecules, and Kehl and colleagues collected secretomes under xeno-free conditions.^[6][19]

Harvest, clarification and concentration

Culture supernatants contain several types of lipid membranes, and Théry and colleagues describe sequential purification steps and purity checks for isolating exosomes from them.^[17] Clarified medium can then be concentrated and kept whole, or fractionated into vesicle and soluble components.^[6][20]

Bari and colleagues purified secretome from culture supernatants by ultrafiltration in a validated good manufacturing practice cell factory, added a cryoprotectant and freeze-dried it. The powder contained a non-aggregated vesicle population by nanoparticle tracking analysis, and infrared spectra showed protein and lipid together.^[20]

Passage number

Serra and colleagues collected conditioned media from adipose-derived cells at passages 3, 6, 9 and 12. Proteomic analysis found that secretome composition did not vary significantly across that range.^[21]

Culture conditions and source tissue reshape secretome composition

Oxygen tension, inflammatory signals, culture format and source tissue each change what MSCs secrete. Paquet and colleagues compared conditioned media from cells at 21 percent, 5 percent and near-anoxic 0.1 percent oxygen.^[22]

Near anoxia, the cells increased secretion of VEGF-A, VEGF-C, IL-8, RANTES and MCP-1 and decreased several inflammatory and immunomodulatory mediators. Media from those cells differed in chemotactic and angiogenic activity in the authors’ assays.^[22]

Inflammatory priming affects soluble and vesicle fractions differently

Gorgun and colleagues stimulated MSCs with hypoxia and pro-inflammatory cytokines together and tested each secretome fraction separately. Inflammatory priming inhibited the pro-angiogenic capacity of the soluble fraction at every oxygen level, while the vesicle fraction kept its activity.^[23]

Primed cells released proteins involved in interaction with innate immune cells and in tissue remodeling. The microRNA cargo of the vesicle fractions was less sensitive to the external stimuli than the soluble proteins.^[23]

Three-dimensional culture raises vesicle yield

Haraszti and colleagues found that umbilical cord-derived MSCs gave the highest exosome yield of the sources they tested. Microcarrier-based three-dimensional culture produced 20-fold more exosomes than two-dimensional culture, and tangential flow filtration added a further 7-fold gain over ultracentrifugation.^[24]

Source tissue shapes the secreted proteome

Comparative proteomic studies report distinct secretome profiles for each tissue source, with shared functional themes. The table summarizes three such comparisons by liquid chromatography and mass spectrometry.^[10][19][25]

Shin and colleagues found that, despite compositional differences, secretomes from all four sources shared functional annotations such as cell migration.^[10] Pires and colleagues likewise reported that all three cell populations secreted neuroregulatory molecules.^[25]

Purified EV fractions separate vesicles from the soluble secretome

Whole conditioned medium holds soluble proteins and vesicles together, and separation methods divide it into a vesicle fraction and a soluble fraction. Mitchell and colleagues reported that bioactive factors were distributed across both, and that each fraction affected different aspects of the processes they studied.^[6]

Four separation principles

• Differential ultracentrifugation pellets vesicles by size and density after low-speed steps remove cells and debris.^[17]
• Size-exclusion chromatography elutes vesicles ahead of smaller proteins and lipoproteins. Böing and colleagues recovered particles larger than 70 nm in early fractions with less than 5 percent of HDL and less than 1 percent of protein.^[26]
• Tangential flow filtration retains vesicles while medium passes across a membrane, which scales to large culture volumes.^[24]
• Ion exchange chromatography captures negatively charged vesicles on an anion exchange resin and elutes them with salt.^[27]

Malvicini and colleagues applied anion exchange chromatography to MSC conditioned media and recovered most vesicles in a single elution fraction. The vesicles measured under 150 nm on average and carried CD9, CD63, CD81 and TSG101 (Fig. 4).^[27]

MISEV2023 terminology describes what a fraction contains

The International Society for Extracellular Vesicles updated its minimal information guidance as MISEV2023, covering production, separation and characterization of vesicles from cell culture, body fluids and tissues. The document addresses nomenclature and the separation of vesicles from non-vesicular extracellular particles.^[28]

Applied to the secretome, this vocabulary names conditioned medium as the starting material and the separated product as an extracellular vesicle preparation with a stated separation method. The MISEV2023 compliance and exosome references on this site explain the terms in detail.^[3][28]

Proteomics, immunoassays and particle counting characterize the secretome

Each fraction of the MSC secretome is measured with methods suited to its contents. Proteomics catalogs the proteins, immunoassays quantify named factors, and particle analysis counts and sizes the vesicles.^[3]

Mass spectrometry proteomics

Kehl and colleagues analyzed secretomes by liquid chromatography tandem mass spectrometry and found biological processes related to angiogenesis enriched in the proteomic profiles.^[19] Bari and colleagues used proteomic characterization to identify proteins involved in immune response, stress response, cytoskeleton and metabolism in their freeze-dried secretome.^[20]

Multiplex immunoassays and ELISA

Antibody arrays measure many named factors in parallel, as in the IL-6, IL-8, MCP-1 and VEGF profile of Park and colleagues.^[15] Targeted immunoassays quantify a single analyte, and Malvicini and colleagues used a CD63 ELISA to follow vesicles through their chromatography fractions.^[27]

Particle counting and sizing

Nanoparticle tracking analysis follows the Brownian motion of individual particles in a light-scattering video and calculates their size and concentration. Dragovic and colleagues measured vesicles as small as about 50 nm, below the roughly 300 nm limit of conventional flow cytometry, and labeled them with antibody-conjugated quantum dots to determine phenotype.^[29]

A characterization panel for a conditioned medium or vesicle preparation typically reports:

• Protein identity and relative abundance, from mass spectrometry proteomics.^[9][19]
• Concentrations of named growth factors and cytokines, from antibody arrays or ELISA.^[11][15]
• Particle concentration and size distribution, from nanoparticle tracking or resistive pulse sensing.^[27][29]
• Vesicle morphology by electron microscopy and vesicle markers such as CD63, CD81, CD9 and TSG101.^[27][28]
• Small RNA content by sequencing, where vesicle cargo is studied.^[16]

Shared reporting standards make MSC-EV data comparable

Witwer and colleagues, on behalf of SOCRATES, ISEV, ISCT and ISBT, proposed harmonization criteria for small vesicles from MSCs. Preparations should be defined by quantifiable metrics for the cellular origin of the vesicles, the presence of lipid membrane vesicles and their physical and biochemical integrity.^[3]

Gimona and colleagues describe MSC small vesicles as 50 to 200 nm in diameter and note that the cells producing them respond to changes in their microenvironment. They argue that identity and potency metrics must be specified during development, with potency assays built around the vesicle attribute most relevant to the intended biological activity.^[30]

Together with MISEV2023, these papers give secretome studies a common vocabulary for source cells, culture conditions, separation methods and measurements.^[3][28][30]

Where to go from here

The ISCT minimal criteria reference explains how the cells behind a secretome are defined, and the source tissues reference compares bone marrow, adipose and perinatal MSCs. The immunobiology reference follows HLA, IDO, PGE2 and TSG-6 into immune cell signaling, and the stem versus stromal cell reference explains the terminology.

The perinatal tissue donor eligibility reference covers how placental and umbilical cord tissue is screened under federal rules. The exosome and MISEV2023 compliance references describe vesicle biology and reporting, and the surface receptor chemistry reference covers the tetraspanins used as vesicle markers.

Verified practices can request lot documentation, and our team is glad to discuss the methods described on this page.