Rresed Pontificia Universidad Cat ica de Chile; University Healthcare Center of Groningen, Groningen, Netherlands; bUMCG,

February 14, 2023

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 Healthcare 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 Natural Resources and Life Sciences, Vienna (BOKU), Vienna, AustriaIntroduction: Human cells are of ever increasing importance as in vitro test method to represent the in vivo circumstance. On top of that, hugely differentiated cells are also vital production systems for complicated biopharmaceuticals. On the other hand, the use of such cell systems are limited due to the fact that the cells enter replicative life span and for that reason can only be propagated for any restricted number of population doublings in vitro, which limited standardization of experiments as well as production processes. Furthermore, reports have shown that the number of secreted vesicles significantly decreased with growing age of standard cells.Introduction: Background: Transition from isolated steatosis (IS) to non-alcoholic steatohepatitis (NASH) is really a crucial situation in non-alcoholic fatty liver disease (NAFLD). Recent RGS4 custom synthesis observations in individuals with obstructive sleep apnea syndrome (OSAS), suggest that hypoxia may possibly contribute to disease progression primarily by way of activation of hypoxia inducible aspect 1 (HIF-1)-related pathways. Release of extracellular vesicles (EV) by injured hepatocytes may perhaps be involved in NAFLD progression. Aim: To explore whether hypoxia modulates the release of EV from free of charge fatty acid (FFA)-exposed hepatocytes and assess cellular crosstalk between hepatocytes and LX-2 cells (human hepatic stellate cell line). Approaches: 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 unique groups and markers of pro-fibrogenic signalling were determined by quantitative PCR (qPCR), WB and immunofluorescence (IF). Outcomes: FFA and CH-treatment of HepG2 cells RelB Source elevated gene expression of IL-1 and TGF-1 inJOURNAL OF EXTRACELLULAR VESICLESHepG2 cells and improved the release of EV in comparison 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. In addition, EV from FFA-treated hypoxic HepG2 cells elevated 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. Further genomic and proteomic characterization of EV released by steatotic cells beneath hypoxia are essential to additional.