Adipose derived stem cell extracellular vesicles modulate primary human macrophages to an anti‐inflammatory phenotype in vitro

Abstract EVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue repair because of their purported immune‐regulatory properties. Extracellular vesicles (EVs) from ADSCs could be beneficial in improving graft retention rates for autologous fat grafting (AFG) post‐mastectomy as, currently, grafted tissue rates are variable. Enriching grafted tissue with ADSC‐EVs may improve retention rates by modulating macrophages resident within both the breast and lipoaspirate. We aimed to identify key macrophage phenotypes that are modulated by ADSC‐EVs in vitro. ADSCs were isolated from lipoaspirates of women undergoing AFG and characterised by flow cytometry and differentiation potential. ADSC‐EVs were isolated from culture media and characterised by tuneable resistive pulse sensing, transmission electron microscopy and Western blot. Primary monocyte‐derived macrophages were polarized to an M1‐like (GM‐CSF, IFNγ), M2‐like phenotype (M‐CSF, IL‐4) or maintained (M0‐like; M‐CSF) and ADSC‐EVs were co‐cultured with macrophages for 48 h. Flow cytometry and high‐dimensional analysis clustered macrophages post co‐culture. A manual gating strategy was generated to recapitulate these clusters and was applied to a repeat experimental run. Both runs were analysed to examine the prevalence of each cluster, representing a unique macrophage phenotype, with and without ADSC‐EVs. Following the addition of ADSC‐EVs, M0‐like macrophages demonstrated a reciprocal shift of cell distribution from a cluster with a ‘high inflammatory profile’ (CD36 +++ CD206 +++ CD86 +++ ; 16.5 ± 7.0%; p < 0.0001) to a cluster with a ‘lower inflammatory profile’ (CD36 +CD206+ CD86+; 35 ± 21.5%; p < 0.05). M1‐like macrophages shifted from a cluster with a ‘high inflammatory profile’ (CD206 ++ CD11b ++ CD36 ++ CD163 ++ ; 26.1 ± 9.4%; p = 0.0024) to a ‘lower inflammatory profile’ (CD206 + CD11b + CD36 +CD163+ ; 72.8 ± 8.7%; p = 0.0007). There was no shift in M2‐like clusters following ADSC‐EV treatment. ADSC‐EVs are complex regulators of macrophage phenotype that can shift macrophages away from a heightened pro‐inflammatory state.