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Macrophage bioassay standardization to assess the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles

  • Ricardo Malvicini
    Correspondence
    Correspondence: Ricardo Malvicini and Natalia Pacienza Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería, Universidad Favaloro–Consejo Nacional de Investigaciones Científicas y Técnicas, Solís 453, Buenos Aires 1078, Argentina.
    Affiliations
    Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería, Universidad Favaloro–Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina

    Department of Women's and Children's Health, University of Padua, Padua, Italy

    Laboratory of Extracellular Vesicles as Therapeutic Tools, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy

    LIFELAB Program, Consorzio per la Ricerca Sanitaria, Padua, Italy
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  • Diego Santa-Cruz
    Affiliations
    Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería, Universidad Favaloro–Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
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  • Giada De Lazzari
    Affiliations
    Department of Women's and Children's Health, University of Padua, Padua, Italy

    Laboratory of Extracellular Vesicles as Therapeutic Tools, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy

    LIFELAB Program, Consorzio per la Ricerca Sanitaria, Padua, Italy
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  • Anna Maria Tolomeo
    Affiliations
    Department of Women's and Children's Health, University of Padua, Padua, Italy

    Laboratory of Extracellular Vesicles as Therapeutic Tools, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy

    LIFELAB Program, Consorzio per la Ricerca Sanitaria, Padua, Italy
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  • Cecilia Sanmartin
    Affiliations
    Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería, Universidad Favaloro–Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
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  • Maurizio Muraca
    Affiliations
    Department of Women's and Children's Health, University of Padua, Padua, Italy

    Laboratory of Extracellular Vesicles as Therapeutic Tools, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy

    LIFELAB Program, Consorzio per la Ricerca Sanitaria, Padua, Italy
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  • Gustavo Yannarelli
    Correspondence
    These authors contributed equally to this work.
    Affiliations
    Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería, Universidad Favaloro–Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
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  • Natalia Pacienza
    Correspondence
    Correspondence: Ricardo Malvicini and Natalia Pacienza Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería, Universidad Favaloro–Consejo Nacional de Investigaciones Científicas y Técnicas, Solís 453, Buenos Aires 1078, Argentina.These authors contributed equally to this work.
    Affiliations
    Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería, Universidad Favaloro–Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
    Search for articles by this author

      Abstract

      Background aims

      Owing to the lack of biological assays, determining the biological activity of extracellular vesicles has proven difficult. Here the authors standardized an in vitro assay to assess the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) based on their ability to prevent acquisition of the M1 phenotype in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Induction of tumor necrosis factor alpha, IL-1β, IL-6 and inducible nitric oxide synthase (iNOS) characterizes the M1 phenotype. Nitric oxide released by iNOS turns into nitrite, which can be easily quantitated in culture media by Griess reaction.

      Methods

      The authors first tested different assay conditions in 96-well plates, including two seeding densities (2 × 104 cells/well and 4 × 104 cells/well), four LPS doses (1 ng/mL, 10 ng/mL, 100 ng/mL and 1000 ng/mL) and two time points (16 h and 24 h), in order to determine the best set-up to accurately measure nitrite concentration as an index of M1 macrophage polarization.

      Results

      The authors found that seeding 2 × 104 cells/well and stimulating with 10 ng/mL LPS for 16 h allowed the inhibition of nitrite production by 60% with the use of dexamethasone. Using these established conditions, the authors were able to test different MSC-sEV preparations and generate dose–response curves. Moreover, the authors fully analytically validated assay performance and fulfilled cross-validation against other M1 markers.

      Conclusions

      The authors standardized a quick, cheap and reproducible in vitro macrophage assay that allows for the evaluation and estimation of the anti-inflammatory activity of MSC-sEVs.

      Graphical Abstract

      Key Words

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      References

        • Bianco P.
        “Mesenchymal” stem cells.
        Annu Rev Cell Dev Biol. 2014; 30: 677-704
        • Heldring N
        • Mäger I
        • Wood MJA
        • Le Blanc K
        • Andaloussi SEL
        Therapeutic Potential of Multipotent Mesenchymal Stromal Cells and Their Extracellular Vesicles.
        Hum Gene Ther. 2015; 26: 506-517
        • Davies JE
        • Walker JT
        • Keating A.
        Concise Review: Wharton's Jelly: The Rich, but Enigmatic, Source of Mesenchymal Stromal Cells.
        Stem Cells Transl Med. 2017; 6: 1620-1630
        • Trounson A
        • McDonald C.
        Stem Cell Therapies in Clinical Trials: Progress and Challenges.
        Cell Stem Cell. 2015; 17: 11-22
        • Wang LT
        • Ting CH
        • Yen ML
        • Liu KJ
        • Sytwu HK
        • Wu KK
        • et al.
        Human mesenchymal stem cells (MSCs) for treatment towards immune- and inflammation-mediated diseases: review of current clinical trials.
        J Biomed Sci. 2016; 23: 1-13
        • Mastrolia I
        • Foppiani EM
        • Murgia A
        • Candini O
        • Samarelli AV
        • Grisendi G
        • et al.
        Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review.
        Stem Cells Transl Med. 2019; 8: 1135
        • Shammaa R
        • El-Kadiry AEH
        • Abusarah J
        • Rafei M.
        Mesenchymal Stem Cells Beyond Regenerative Medicine.
        Front Cell Dev Biol. 2020; 872
        • Dayan V
        • Yannarelli G
        • Billia F
        • Filomeno P
        • Wang XH
        • Davies JE
        • et al.
        Mesenchymal stromal cells mediate a switch to alternatively activated monocytes/macrophages after acute myocardial infarction.
        Basic Res Cardiol. 2011; 106: 1299-1310
        • DJ P.
        Concise review: two negative feedback loops place mesenchymal stem/stromal cells at the center of early regulators of inflammation.
        Stem Cells. 2013; 31: 2042-2046
        • Phinney DG
        • Pittenger MF.
        Concise Review: MSC-Derived Exosomes for Cell-Free Therapy.
        Stem Cells. 2017; 35: 851-858
        • Gomzikova MO
        • James V
        • Rizvanov AA.
        Therapeutic Application of Mesenchymal Stem Cells Derived Extracellular Vesicles for Immunomodulation.
        Front Immunol. 2019; 10: 1-9
        • Witwer KW
        • Van Balkom BWM
        • Bruno S
        • Choo A
        • Dominici M
        • Gimona M
        • et al.
        Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications.
        J Extracell Vesicles. 2019; 81609206
        • Pegtel DM
        • Gould SJ
        Exosomes.
        Annu Rev Biochem. 2019; 88: 487-514
        • Liang Y
        • Eng WS
        • Colquhoun DR
        • Dinglasan RR
        • Graham DR
        • Mahal LK.
        Complex N-linked glycans serve as a determinant for exosome/microvesicle cargo recruitment.
        J Biol Chem. 2014; 289: 32526-32537
        • Tomasoni S
        • Longaretti L
        • Rota C
        • Morigi M
        • Conti S
        • Gotti E
        • et al.
        Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem cells.
        Stem Cells Dev. 2013; 22: 772-780
        • Wang X
        • Gu H
        • Qin D
        • Yang L
        • Huang W
        • Essandoh K
        • et al.
        Exosomal miR-223 Contributes to Mesenchymal Stem Cell-Elicited Cardioprotection in Polymicrobial Sepsis.
        Sci Rep. 2015; 513721
        • Tkach M
        • Kowal J
        • Théry C.
        Why the need and how to approach the functional diversity of extracellular vesicles.
        Philos Trans R Soc Lond B Biol Sci. 2018; 37320160479
        • Kordelas L
        • Schwich E
        • Dittrich R
        • Horn P
        • Beelen D
        • Börger V
        • et al.
        Individual Immune-Modulatory Capabilities of MSC-Derived Extracellular Vesicle (EV) Preparations and Recipient-Dependent Responsiveness.
        Int J Mol Sci. 2019; 20: 1642
        • Mantovani A
        • Sica A
        • Sozzani S
        • Allavena P
        • Vecchi A
        • Locati M.
        The chemokine system in diverse forms of macrophage activation and polarization.
        Trends Immunol. 2004; 25: 677-686
        • Yannarelli G
        • Dayan V
        • Pacienza N
        • Lee CJ
        • Medin J
        • Keating A.
        Human umbilical cord perivascular cells exhibit enhanced cardiomyocyte reprogramming and cardiac function after experimental acute myocardial infarction.
        Cell Transplant. 2013; 22: 1651-1666
        • Dominici M
        • Le Blanc K
        • Mueller I
        • Slaper-Cortenbach I
        • Marini FC
        • Krause DS
        • et al.
        Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.
        Cytotherapy. 2006; 8: 315-317
        • Pacienza N
        • Lee RH
        • Bae EH
        • ki Kim D
        • Liu Q
        • Prockop DJ
        • et al.
        In Vitro Macrophage Assay Predicts the In Vivo Anti-inflammatory Potential of Exosomes from Human Mesenchymal Stromal Cells.
        Mol Ther - Methods Clin Dev. 2019; 13: 67-76
        • Visnovitz T
        • Osteikoetxea X
        • Sódar BW
        • Mihály J
        • Lőrincz P
        • Vukman KV.
        • et al.
        An improved 96 well plate format lipid quantification assay for standardisation of experiments with extracellular vesicles.
        J Extracell Vesicles. 2019; 81565263
        • Kim DK
        • Nishida H
        • SY An
        • Shetty AK
        • Bartosh TJ
        • Prockop DJ.
        Chromatographically isolated CD63+CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI.
        Proc Natl Acad Sci U S A. 2016; 113: 170-175
        • Théry C
        • Clayton A
        • Amigorena S
        • Raposo G.
        Isolation and Characterization of Exosomes from Cell Culture Supernatants.
        Curr Protoc Cell Biol. 2006; Chapter 3: Unit 3.22
        • Livak KJ
        • Schmittgen TD.
        Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
        Methods. 2001; 25: 402-408
        • Green LC
        • Wagner DA
        • Glogowski J
        • Skipper PL
        • Wishnok JS
        • Tannenbaum SR.
        Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.
        Anal Biochem. 1982; 126: 131-138
        • Patel MI
        • Tuckerman R
        • Dong Q.
        A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay due toevaporation in wells on the edge of a 96 well plate.
        Biotechnol Lett. 2005; 27: 805-808
        • McWhorter FY
        • Wang T
        • Nguyen P
        • Chung T
        • Liu WF
        Modulation of macrophage phenotype by cell shape.
        Proc Natl Acad Sci U S A. 2013; 110: 17253-17258
        • Padgett EL
        • Pruett SB.
        Evaluation of nitrite production by human monocyte-derived macrophages.
        Biochem Biophys Res Commun. 1992; 186: 775-781
        • Stavely R
        • Nurgali K.
        The emerging antioxidant paradigm of mesenchymal stem cell therapy.
        Stem Cells Transl Med. 2020; 9: 985-1006
        • François M
        • Romieu-Mourez R
        • Li M
        • Galipeau J.
        Human MSC suppression correlates with cytokine induction of indoleamine 2,3-dioxygenase and bystander M2 macrophage differentiation.
        Mol Ther. 2012; 20: 187-195
        • Reis M
        • Mavin E
        • Nicholson L
        • Green K
        • Dickinson AM
        • Wang X.
        Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function.
        Front Immunol. 2018; 9: 2538
        • Zhang B
        • Wu X
        • Zhang X
        • Sun Y
        • Yan Y
        • Shi H
        • et al.
        Human Umbilical Cord Mesenchymal Stem Cell Exosomes Enhance Angiogenesis Through the Wnt4/β-Catenin Pathway.
        Stem Cells Transl Med. 2015; 4: 513-522
        • Del Fattore A
        • R Luciano
        • Pascucci L
        • Goffredo BM
        • Giorda E
        • Scapaticci M
        • et al.
        Immunoregulatory effects of mesenchymal stem cell-derived extracellular vesicles on T lymphocytes.
        Cell Transplant. 2015; 24: 2615-2627
        • Chen W
        • Huang Y
        • Han J
        • Yu L
        • Li Y
        • Lu Z
        • et al.
        Immunomodulatory effects of mesenchymal stromal cells-derived exosome.
        Immunol Res. 2016; 64: 831-840
        • Zhang B
        • Yin Y
        • Lai RC
        • Tan SS
        • Choo ABH
        • Lim SK.
        Mesenchymal stem cells secrete immunologically active exosomes.
        Stem Cells Dev. 2014; 23: 1233-1244
        • Khare D
        • Or R
        • Resnick I
        • Barkatz C
        • Almogi-Hazan O
        • Avni B.
        Mesenchymal Stromal Cell-Derived Exosomes Affect mRNA Expression and Function of B-Lymphocytes.
        Front Immunol. 2018; 9: 3053
        • Tolomeo AM
        • Castagliuolo I
        • Piccoli M
        • Grassi M
        • Magarotto F
        • De Lazzari G
        • et al.
        Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis.
        Front Immunol. 2021; 12627605
        • Kordelas L
        • Rebmann V
        • Ludwig AK
        • Radtke S
        • Ruesing J
        • Doeppner TR
        • et al.
        MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease.
        Leukemia. 2014; 28: 970-973
        • Nassar W
        • El-Ansary M
        • Sabry D
        • Mostafa MA
        • Fayad T
        • Kotb E
        • et al.
        Umbilical cord mesenchymal stem cells derived extracellular vesicles can safely ameliorate the progression of chronic kidney diseases.
        Biomater Res. 2016; 20: 1-11
        • García-Bernal D
        • García-Arranz M
        • Yáñez RM
        • Hervás-Salcedo R
        • Cortés A
        • Fernández-García M
        • et al.
        The Current Status of Mesenchymal Stromal Cells: Controversies, Unresolved Issues and Some Promising Solutions to Improve Their Therapeutic Efficacy.
        Front Cell Dev Biol. 2021; 9: 609
        • Del Fattore A
        • Luciano L
        • Saracino R
        • Battafarano G
        • Rizzo C
        • Pascucci L
        • et al.
        Differential effects of extracellular vesicles secreted by mesenchymal stem cells from different sources on glioblastoma cells.
        Expert Opin Biol Ther. 2015; 15: 495-504
        • Lopez-Verrilli MA
        • Caviedes A
        • Cabrera A
        • Sandoval S
        • Wyneken U
        • Khoury M.
        Mesenchymal stem cell-derived exosomes from different sources selectively promote neuritic outgrowth.
        Neuroscience. 2016; 320: 129-139
        • Gámez-Valero A
        • Monguió-Tortajada M
        • Carreras-Planella L
        • Franquesa M
        • Beyer K
        • Borràs FE.
        Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles’ characteristics compared to precipitating agents.
        Sci Rep. 2016; 6: 1-9
        • Paolini L
        • Zendrini A
        • Di Noto G
        • Busatto S
        • Lottini E
        • Radeghieri A
        • et al.
        Residual matrix from different separation techniques impacts exosome biological activity.
        Sci Rep. 2016; 6: 1-11
        • Sahoo S
        • Klychko E
        • Thorne T
        • Misener S
        • Schultz KM
        • Millay M
        • et al.
        Exosomes from human CD34(+) stem cells mediate their proangiogenic paracrine activity.
        Circ Res. 2011; 109: 724-728