WP3, Validation of cell communication in 3D Systems: the 3D secretome

Organoids obtained from cultures at different time points, will be centrifuged at 2000g for 15min and supernatant will be stored at -80°C until use.  We will isolate EVs from CM by ultracentrifugation at 100.000g for 1h. The EVs present in the pellet will be characterized by electron microscopy analyses (SEM, crioTEM), NTA, western blotting and FACS analyses according to MISEV guidelines.[18] The supernatant recovered by ultracentrifugation at 100.000g, called processed CM (pCM), will be recovered and used for the analysis of protein profiles. The isolated EVs will be biochemically characterized by profiling their protein and lipid cargo.

The presence of IDO1, Arginase 1, IL4i, GR, GC, GILZ and RAGE in secretome/EVs will be determined through mass spectrometry and ELISA. HPLC and mass spectrometry will be used to quantify the main immunoregulatory metabolites deriving from L-tryptophan, arginine and L-phenylalanine.

Peripheral venous blood will be obtained from three healthy donors. PBMCs will be isolated on a Ficoll-Hypaque gradient and cultured for 24 hours in vitro in complete medium (RPMI with 10% FCS, 10 mM HEPES, and 50 μM 2-ME), as activated or not (with anti-CD3 and anti-CD28) and cultured in the presence or absence of the organoids-derived whole secretome or isolated EVs. After 24 hours the immunological profile (regulatory or immunostimulatory), the differentiation of Th1, Th2, Th17, and Treg cells of the PBMCs will be evaluated by Flow cytometry, multiplex and real time qPCR analysis. Human myotubes will be cultured with whole secretome or EVs for different time periods and analyzed for myotube diameters by immunofluorescence for MyHC; RAGE and MyHC expression, pathways for protein synthesis/degradation by western blotting; markers of proteolytic system activation by real-time PCR; the presence of pro-inflammatory cytokines and tumor-derived factors known to induce muscle catabolism in culture medium by ELISA.