Chromatographyc scalable method to isolate engineered extracellular vesicles derived from human umbilical cord perivascular cells for the treatment of liver fibrosis.

DOMÍNGUEZ LM ; ALBORNOZ M; BAYO J; ONORATO A; CANTERO MJ; BUELONI B; ATORRASAGASTI C; GARCIA M; YANNARELLI G; FIORE E; MAZZOLINI G

Congreso; Reunión Conjunta SAIC SAI&FAIC SAFIS 2022; 2022

We previously demonstrated that extracellular vesicles (EV) mediate the therapeutic effect of human umbilical cord perivascular cells (HUCPVC) over-expressing hIGF1 on liver fibrosis in mice. Aim: To apply a scalable method by affinity chromatography to isolate engineered HUCPVC-derived EV loading therapeutic genes for liver fibrosis therapy. Methods: EVs were isolated by ion exchange chromatography from supernatants of HUCPVC infected with adenoviruses codifying for IGF1 (EV-IGF1) or green fluorescent protein (EV-GFP). EVs morphology was analyzed by electron microscopy,markers (CD9, CD63, CD81), and IGF1 cargo expression by flow cytometry (FC), and ELISA. Antifibrotic effect of EVs was determined in experimental mice model of liver fibrosis (thioacetamide for 8 weeks). The treatments were administered on week 6 (groups: saline, EV-IGF1 or EV-GFP, 3 doses, 15 mg/dose/mice every 5 days). Collagen deposition was measured by Sirius red staining; immunohistochemistry for PCNA proliferating cells, and aSMA for activated Hepatic Stellate Cells (HSC) was performed. In vitro effects of EVs on HSC pro-fibrogenic genes expression (CFSC-G2 cell line) were evaluated by qPCR. Results: EV-IGF1 isolated by chromatography show a typical and homogeneous morphology, and were CD9, CD63 and CD81 positive. Increased IGF1 levels on EV-IGF1 determined by ELISA and FC indicate its loading on EV. In vivo treatment with EV-IGF1 resulted in a further amelioration of collagen deposition (p