A novel three-dimensional co-culture model for studying exosome-mediated cell interactions in glioblastoma

Abstract

Three-dimensional(3D) cell culture systems provide a larger space for cell proliferation, which is crucial for simulating cellular behavior and drug responses in the tumor microenvironment. In this study, we developed a novel 3D co-culture system for cell interactions, utilizing a commercialized bioreactor-microcarrier system. Mesenchymal stem cells (MSCs) were extracted via enzymatic digestion, and markers CD105 and CD31 were identified. Cell growth was observed using AO and immunofluorescence staining. No significant differences in Ki67 and GFAP expression were found between 2D and 3D cultures, though the 3D system offered more space for proliferation and reduced contact inhibition. Therefore, this 3D culture system may represent the tumor microenvironment more accurately than 2D cultures and will facilitate the investigation of the characteristics and functions of exosomes derived from this system. Exosomes are nanoscale vesicles that mediate intercellular communication by transferring molecules such as miRNAs between cells. Exosomes from 3D cultures were collected via ultra-high-speed centrifugation and characterized using nano-flow cytometry, transmission electron microscopy, and western blotting for markers CD9, Alix, and TSG101. PKH26 staining revealed peak exosome uptake by tumor cells at 24 h and complete metabolism by 72 h. Exosomes from 3D cultures inhibited GBM cell proliferation, migration, and invasion. Lastly, miRNA sequencing of exosomes was performed. This study emphasizes the importance of creating 3D co-culture systems to advance cancer research and offers a helpful tool for studying the complex cell interaction environment of GBM and other malignancies.