Rresed Pontificia Universidad Cat ica de Chile; University Healthcare Center of Groningen, Groningen, Netherlands; bUMCG, Groningen, Netherlands; Pontificia Universidad Cat ica de Chile/Universidad Bernardo O iggins, SANTIAGO, Chile; dPontificia Universidad Cat ica de Chile, Santiago, Chile; eUniversity Health-related Center Groningen, Groningen, Netherlandsc aPS01.Human telomerized cells for production of extracellular vesicles Regina Grillaria, Susanne Neubertb, Matthias Wiesera and Johannes GrillaribaEvercyte GmbH, Vienna, Austria; bChristian Doppler Laboratory on Biotechnology of Skin Aging, University of Organic Resources and Life Sciences, Vienna (BOKU), Vienna, AustriaIntroduction: Human cells are of ever growing significance as in vitro test technique to represent the in vivo circumstance. Additionally, extremely differentiated cells are also essential production systems for complex biopharmaceuticals. Even so, the usage of such cell systems are limited due to the truth that the cells enter replicative life span and hence can only be propagated for any limited quantity of population doublings in vitro, which restricted standardization of experiments at the same time as production processes. Furthermore, reports have shown that the amount of secreted vesicles substantially lowered with rising age of normal cells.Introduction: Background: Transition from isolated steatosis (IS) to non-alcoholic steatohepatitis (NASH) is usually a important challenge in non-alcoholic fatty liver illness (NAFLD). Recent observations in patients with obstructive sleep apnea syndrome (OSAS), recommend that hypoxia might contribute to disease progression mostly through activation of hypoxia inducible aspect 1 (HIF-1)-related pathways. Release of extracellular vesicles (EV) by injured hepatocytes may be involved in NAFLD progression. Aim: To explore no matter if hypoxia modulates the release of EV from cost-free fatty acid (FFA)-exposed hepatocytes and assess cellular crosstalk between hepatocytes and LX-2 cells (human hepatic stellate cell line). Strategies: HepG2 cells have been treated with FFAs (250 M palmitic acid + 500 M oleic acid) and chemical hypoxia (CH) was induced with Cobalt (II) Chloride, which is an inducer of HIF-1. Induction of CH was confirmed by Western blot (WB) of HIF-1. EV isolation and quantification was performed by ultracentrifugation and nanoparticle tracking evaluation respectively. EV characterization was performed by electron microscopy and WB of CD-81 marker. LX-2 cells have been treated with 15 g/ml of EV from hepatocytes obtained from unique groups and markers of pro-fibrogenic signalling have been determined by quantitative PCR (qPCR), WB and immunofluorescence (IF). Outcomes: FFA and CH-treatment of HepG2 cells increased gene CD66a Proteins Storage & Stability expression of IL-1 and TGF-1 inJOURNAL OF EXTRACELLULAR VESICLESHepG2 cells and increased the release of EV when compared with non-treated HepG2 cells. Treatment of LX-2 cells with EV from FFA-treated hypoxic HepG2 cells increased gene expression of TGF-1, CTGF, -SMA and Collagen1A1 in comparison with LX-2 cells treated with EV from non-treated hepatocytes or LX-2 cells exposed to EV-free supernatant from FFA-treated hypoxic HepG2 cells. Moreover, EV from FFA-treated hypoxic HepG2 cells improved Collagen1A1 and -SMA protein levels.Summary/Glycophorin-A/CD235a Proteins Biological Activity conclusion: CH promotes EV release from HepG2 cells. EV from hypoxic FFA-treated HepG2 cells evoke pro-fibrotic responses in LX-2 cells. Additional genomic and proteomic characterization of EV released by steatotic cells below hypoxia are essential to additional.
