OPTIMIZATION OF CONDITIONED MEDIUM PRODUCTION FROM HUMAN UMBILICAL CORD MESENCHYMAL STEM CELL FOR EXOSOME ISOLATION AND CHARACTERIZATION

• Main Author: Aurelia Pranata, Gabrielle
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/57487
• Institution: Institut Teknologi Bandung

Mesenchymal stem cells (MSC) are used in cell therapy for treatment of degenerative diseases through cell replacement and paracrine effect mechanisms on the damaged tissue. However, the paracrine effect especially from MSC-derived exosomes have advantages in safety, production, and storage therefore having a higher potential to be studied as a cell-free therapy agent. There are some issues in exosome research for therapy such as limited yield and isolation methods. The aim of this research is to optimize an exosome production method from human umbilical cord mesenchymal stem cell (hUC-MSC), isolate exosomes, and characterize the isolated exosomes. hUC-MSC is cultured and characterized using fluorescence-activated cell sorting (FACS) and a multipotency assay. Exosome production from hUC-MSC is induced by starving using basal medium in hypoxia condition (5% O2, 90% N2, 5% CO2), and culture parameters such as medium volume, starving duration, and collection method are optimized on a small scale. Isolation of exosomes is done by sequential filtration consisting of syringe filtration and Tangential Flow Filtration (TFF). Protein concentration of the samples from optimization and isolation is measured by BCA protein assay. Exosome is characterized with flow cytometry, Transmission Electron Microscope (TEM), Scanning Electron Microscope (SEM), and Dynamic Light Scattering (DLS). Results of BCA protein assay shows that medium volume does not significantly affect exosome production however starving for 72 hours with collection and total medium replacement every 24 hours significantly increases exosome production. Exosome is successfully isolated from MSC conditioned medium by sequential filtration. Flow cytometry shows the presence of exosome in isolated samples which is confirmed by the expression of CD63 marker on the isolated sample. TEM and SEM visualization shows the spherical morphology of exosome. DLS reading shows the average diameter of exosomes on 24, 48, and 72 hours treatment groups as 158.53±2.51 nm, 187.1±8.31nm, and 193.33±5.98 nm respectively. In conclusion, this research shows that the optimum method for exosome production is starving for 72 hours with minimum medium volume and total medium replacement every 24 hours. Exosome is successfully isolated by sequential filtration and shows a spherical morphology with diameter ranging from 156.02 to 199.31 nm, which is larger than the size range of exosomes.