Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes

Abstract

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.

Exosomes in serum‑free cultures of THP‑1 macrophages infected with Mycobacterium tuberculosis

“…NTA, flow cytometric MACSPlex exosome analysis of the THP-1 human monocytic cell line and western blot analysis were also used to characterize and confirm that the isolated vesicles were exosomes. In agreement with previous studies ( 5 , 19 , 21 ), the aforementioned methodologies allowed for the recovery of intact exosomes at high yields from THP-1 macrophages. Similar results have been reported for the human lung cancer cell line A549 ( 18 ), also with the same 10 ml sample volume input ( 13 ).…”

“…The size and concentration of the exosomes were then analyzed by NTA using a NanoSight LM10 apparatus. This instrument is able to detect and analyze small particles <30 nm in size by increasing the viscosity of the sample ( 19 ). This technology, however, is not able to differentiate exosomes from other synthetic nanoparticles or from large protein aggregates ( 20 ).…”

“…Furthermore, mycobacterial antigen 85 (Ag85) and the Mpt64 protein were selected to evaluate the presence of Mtb molecules within exosomes. The majority of the published studies on exosomes and protein surface markers to date have focused on the use of cells cultured in media supplemented with 10% fetal bovine serum (FBS) (17)(18)(19). Abramowicz et al (13) demonstrated that high abundance exosome-contaminating serum proteins are potential sources of contaminants that cause substantial drawbacks in terms of exosome harvest, isolation and processing.…”

The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids

“…Further purification of scUCF‐isolated EVs prior to proteomic analyses was performed by sucrose density gradient ultracentrifugation as previously described (Arteaga‐Blanco et al., 2020). Briefly, the scUCF‐purified EV pellet from HEK‐CCM was bottom loaded on a sucrose density gradient (90%‐10%, bottom to top) and subjected to ultracentrifugation at 200,000 × g for 16 h. Following centrifugation six fractions (F1‐F6) were collected from the top to the bottom of the gradient and transferred into new tubes.…”

Extracellular Vesicles in Innate Immune Cell Programming

“…Upon tissue recruitment peripheral blood monocytes can differentiate into macrophages. Monocyte-derived macrophages release small EV that are positive for EV markers ALIX, CD63 and CD81, which accumulate rapidly (< 3 h) in recipient macrophages [70] and induce differentiation of other macrophage via transfer of EV-miR-223 [71]. Macrophages internalise EV from a wide variety of sources including breast milk derived EV [72], tumour derived EV, which activate macrophages [73] and acute myeloid leukaemia (AML) derived EV, which induce a myeloid derived suppressor cell (MDSC) differentiation phenotype in recipient macrophages [74].…”