Rresed Pontificia Universidad Cat ica de Chile; University Health-related 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 Medical 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 All-natural Sources and Life Sciences, PI3Kγ manufacturer Vienna (BOKU), Vienna, AustriaIntroduction: Human cells are of ever escalating value as in vitro test system to represent the in vivo situation. Moreover, very differentiated cells are also important production systems for complicated biopharmaceuticals. On the other hand, the usage of such cell systems are restricted because of the reality that the cells enter replicative life span and for that reason can only be propagated to get a restricted number of population doublings in vitro, which limited standardization of experiments also as production processes. Additionally, reports have shown that the number of secreted vesicles significantly decreased with escalating age of typical cells.Introduction: Background: Transition from isolated steatosis (IS) to non-alcoholic steatohepatitis (NASH) is actually a essential problem in non-alcoholic fatty liver disease (NAFLD). Current observations in individuals with obstructive sleep apnea syndrome (OSAS), suggest that hypoxia may perhaps contribute to disease progression primarily by way of activation of hypoxia inducible element 1 (HIF-1)-related pathways. Release of extracellular vesicles (EV) by injured hepatocytes could be involved in NAFLD progression. Aim: To discover whether hypoxia modulates the release of EV from free fatty acid (FFA)-exposed hepatocytes and assess cellular crosstalk amongst hepatocytes and LX-2 cells (human hepatic stellate cell line). Procedures: HepG2 cells were treated with FFAs (250 M palmitic acid + 500 M oleic acid) and chemical hypoxia (CH) was induced with Cobalt (II) Chloride, that 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 had been treated with 15 g/ml of EV from hepatocytes obtained from different groups and markers of pro-fibrogenic NK3 Species signalling had been determined by quantitative PCR (qPCR), WB and immunofluorescence (IF). Benefits: FFA and CH-treatment of HepG2 cells elevated gene expression of IL-1 and TGF-1 inJOURNAL OF EXTRACELLULAR VESICLESHepG2 cells and improved the release of EV in comparison to non-treated HepG2 cells. Remedy of LX-2 cells with EV from FFA-treated hypoxic HepG2 cells elevated gene expression of TGF-1, CTGF, -SMA and Collagen1A1 when compared 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. In addition, EV from FFA-treated hypoxic HepG2 cells enhanced Collagen1A1 and -SMA protein levels.Summary/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 beneath hypoxia are necessary to further.