An In Vitro Potency Assay for Monitoring the Immunomodulatory Potential of Stromal Cell-Derived Extracellular Vesicles

Abstract

Abstract:The regenerative and immunomodulatory activity of mesenchymal stromal cells (MSCs) is partially mediated by secreted vesicular factors. Extracellular vesicles (EVs) exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies. We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reports the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. Our results support the application of a cell-based in vitro potency assay for reproducibly determining the immunomodulatory potential of EVs. Validation of this assay can help establish reliable release criteria for EVs for future clinical studies.

A functional corona around extracellular vesicles enhances angiogenesis during skin regeneration and signals in immune cells

“…We therefore plan a more detailed analysis, particularly of the immune-related proteomic profile of EVs vs. donor cells vs. soluble factors, and validation of results in additional experimental models towards rational potency-based selection of cell-and EVbased therapeutics. This will include comprehensive genomic analysis focusing on small RNA species as initiated previously in a study for monitoring the immunomodulatory potential of neonatal umbilical cord stromal cell-derived EVs [18]. Our quantitative EV purification approach will also allow for combining lipidomics, glycomics and metabolomics for a more broad view on PLX-EV biology using upcoming technology [35]- [37].…”

“…We next devised a standardized high content process for EV production (Fig.1) based on previous results [18]. Large volumes of fibrinogen-depleted -MEM* (n x 500 mL) were first depleted for platelet-rich plasma-and serum-derived particles (including EVs) before use in large-scale PLX culture ( Fig.1A).…”

“…For ECFC culture EGM-2 was supplemented with hydrocortisone, hFGF, VEGF, IGF, EGF and ascorbic acid from the supplied bullet Kit (all Lonza) and FBS was replaced by an equivalent volume of pHPL [26], [41]. For particle depletion media were prepared by clotting supplemented -MEM as described without heparin to avoid possible inhibition of EV function [15], [18], [19], [27], [42]. The collapsed fibrin clot was removed by centrifugation for 10 min, 3000 x g at room temperature.…”

Mesenchymal Stem Cell-Derived Exosomes Reduce A1 Astrocytes via Downregulation of Phosphorylated NFκB P65 Subunit in Spinal Cord Injury

“…Exosomes carry complex cargo including proteins, lipids and nucleic acids, and transfer them to recipient cells to exert function. MSC-derived exosomes (MSC-exo) are reportedly capable of mimicking most biological functions of MSCs including anti-inflammatory and anti-apoptotic effects and can increase levels of anti-inflammatory cytokines, decrease levels of proinflammatory cytokines [18,19], and inhibit activation of astrocytes [17] and macrophages [20]. Similar to MSCs, MSC-exo infusion can inhibit NFκB activation [21,22].…”

Extracellular Vesicles isolated from Mesenchymal Stromal Cells Modulate CD4+ T Lymphocytes Toward a Regulatory Profile

“…The literature is controversial regarding the effects of MSC-EVs on lymphocyte proliferation. Some studies have shown inefficiency of MSC-EVs in inhibiting T cell proliferation [52][53][54], others have demonstrated lower efficiency of EVs when compared to MSCs [55][56][57][58] while others have reported ability to inhibit lymphocyte proliferation [59][60][61]. The difference between these studies may be due to variations in the dose and types of EVs and T cell sources used.…”