Maryam Adelipour (충남대학교 화학과)

Maryam Adelipour (충남대학교 화학과)

Maryam Adelipour (충남대학교 화학과)

김정권 (충남대학교 화학과)

Mesenchymal stem cells (MSCs) are promising in regenerative medicine due to their immunomodulatory, anti-inflammatory properties, and low immunogenicity. This study focuses on extracting and analyzing exosomes derived from MSCs, which share these beneficial properties without tumorigenic concerns. MSCs were cultured to 70% confluency, then switched to serum-free media. After 48 hours, the media were collected, centrifuged, and filtered to remove large particles. Exosomes were extracted using 15% polyethylene glycol (PEG) 3300 Da, followed by overnight incubation at 4˚C and centrifugation at 1500×g. The pellet was re-suspended in 10% PEG/PBS and centrifuged, repeating this process twice more. Exosome characterization involved western blotting to identify positive markers (CD63) and confirm the absence of negative markers (calnexin, calreticulin). Nanoparticle tracking analysis (NTA) measured exosome size (~150 nm), and scanning electron microscopy (SEM) confirmed their spherical shape (<200 nm). Proteomic analysis was conducted by lysing exosomes with 5% SDS, digesting proteins into peptides using the S-Trap method with trypsin, and analyzing them with nanoLC-MS/MS on a Q Exactive Plus Orbitrap mass spectrometer. The gradient for the buffer was 2-20-32% buffer B over 90 minutes at 0.3 mL/min. Results showed 170 protein groups in the exosome fraction, including immune-related proteins. This study successfully extracted exosomes from MSCs using PEG and identified their protein content through mass spectrometry-based proteomics, revealing their potential for therapeutic applications. In conclusion, MSC-derived exosomes, characterized by their specific protein profiles and beneficial properties, hold promise for clinical use in treating degenerative diseases.

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