Comparative investigation of exosome extraction from rat bone marrow mesenchymal stem cells using three different methodologies

Abstract Exosomes have been identified as crucial mediators in numerous physiological and pathological processes, emerging as a focal point of scientific inquiry. This study aims to compare three methods for isolating exosomes from rat bone marrow mesenchymal stem cells: ultracentrifugation (UC), ultrafast separation system (EXODUS), and commercial precipitation kit (EXO‐kit). First, the investigation compared exosomal morphology, particle size distribution, and expression of marker proteins. Subsequently, the RNA content, protein concentration, and purity of exosomes were evaluated. Finally, the impact of these exosomes on cellular metabolic viability and migration capacity was assessed. Results indicated that exosomes exhibited spherical or elliptical membrane structures, and most of the exosomes extracted by the three methods were in the range of 30 to 200 nm. UC‐extracted exosomes demonstrated the least impurities and clearest background, followed by EXODUS‐extracted exosomes, and lastly EXO‐kit‐extracted exosomes. The EXO‐kit‐extracted exosomes yielded the highest RNA and protein content, whereas those isolated through UC exhibited superior purity. Furthermore, exosomes extracted from EXODUS and EXO‐kit methods effectively enhanced the metabolic viability and migratory ability of osteoblast precursor cells compared to UC‐extracted exosomes. In conclusion, each of the three methodologies presents advantages and limitations. Therefore, the selection of an appropriate exosome extraction technique should be based on specific experimental objectives and requirements.