POTENTIAL OF HUMAN WHARTON'S JELLY MESENCHYMAL STEM CELLS-DERIVED EXOSOME CULTURED WITH NORMOXIC AND HYPOXIC CONDITIONS AS ANTI-INFLAMMATORY TOWARD L2 CELL LINE INDUCED BY LIPOPOLYSACCHARIDE

• Main Author: Adhani Sholihah, Ika
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/72062
• Institution: Institut Teknologi Bandung

Acute Lung Injury (ALI) is an acute inflammatory condition that causes disruption of lung epithelial and endothelial cells. The incidence of ALI is 25 cases per 100,000 population per year, and 9% of intensive care unit patients have the condition. Presently, in-vitro pharmacological research for ALI involves lipopolysaccharide (LPS) because LPS triggers the inflammatory response LPS binds to TLR4, activating the NF-B pathway and increasing production of pro-inflammatory cytokines like TNF-?, IL-6, and IL-1?. LPS can inhibit cell viability and promote apoptosis. Widespread usage of corticosteroids, such as dexamethasone, is accompanied by negative side effects. Subsequently, an alternative therapy that can suppress pro-inflammatory cytokines and has minimal side effects is required, one such therapy is the use of human Wharton's Jelly mesenchymal stem cells (hWJ-MSC). The purpose of this study was to investigate the potency of exosomes derived from hWJ-MSCs cells cultured under normoxic and hypoxic conditions which were examined on L2 cell lines induced by LPS. First, hWJ-MSCs are isolated and characterized using flow cytometry and tri-lineage differentiation, CD90, CD73, CD105, CD44, and negative lineage surface markers. The second stage was the characterization of hWJ-MSCs cell-derived exosomes in passage 4 using CD63 markers, Transmission Electron Microscopy (TEM), and Nanoparticle Tracking Analysis (NTA). The third phase was exosome internalization for 24 hours in L2 line cells. Next, induce L2 line cells for 18 hours with LPS at a concentration of 1 ?g/mL. Following induction using LPS, cells were treated with normoxic exosomes (21% oxygen level), hypoxic exosomes (5% oxygen level), and dexamethasone (dex) as positive controls. The MTT assay was used to determine the viability of the sample. Pro-inflammatory proteins such as TNF-?, 1L-1?, and IL-6 were measured using ELISA. TLR4 and NF-?? gene expression profiles were examined after isolating RNA from L2 cells. To assess the amount of nor-exo and hypo-exo proteins, liquid chromatography-tandem mass spectrometry (LC/MS-MS) was utilized. The results of this study reveal that the nor-exo and hypo-exo were successfully characterized, with the hypo-exo producing a larger yield of exosomes. In 24 hours, L2 cells successfully internalized nor-exo and hypo-exo at a concentration of 1x106 particles/mL. Variations in the concentrations of nor-exo and hypo-exo (3.125 x 104 particles/mL and 1x 106 particles/mL) can reduce the concentration of 1L-1? to 1981.72±50.55 pg/mg protein (p=0.032), TNF-? to 1865.19±42.92 pg/mg protein (p=0.050), and IL-6 to 139.29±12.11 pg/mg protein compared to positive control (dex) despite a decrease in concentration protein is not as good as the positive control. Furthermore, at a concentration of 1 x 106 particles per milliliter, nor-exo and hypo-exo particles can inhibit TLR4 and NF-?? gene expression in L2 line cells in a manner comparable to the positive control (dex). According to the results of proteomic study, the nor-exo contains 2,368 proteins and the hypo-exo contains 2,595 proteins. Compared to nor-exo, hypo-exo has a greater number of particles and a greater variety of proteins. This study concludes that exosomes derived from hWJ-MSCs have anti-inflammatory potential by lowering levels of pro-inflammatory proteins TNF-?, 1L-1?, and IL-6, as well as TLR4 and NF-?? gene expression in L2 line cells.