A Protocol for Large-Scale Isolation of Rat Primary Osteoblasts Suitable for Exosome Extraction

Abstract

Objective: This study aims to introduce an optimized method for the large-scale isolation of rat primary osteoblasts, suitable for exosome extraction. Methods: An advancement on the traditional secondary enzyme digestion method, manual bone grinding to clean connective tissue and ophthalmic cutting into fragments have been replaced with oscillating vortex rinsing and a 14 cm straight head shear in a centrifuge tube to cut bone fragments, significantly reducing the experimental time. The experiment was conducted on forty suckling rats. The experimental time of both the optimized and traditional methods were compared. The osteoblasts obtained through the optimized method were observed morphologically, identified by Alkaline phosphatase and alizarin red staining, and their proliferation activity was detected using the EdU method. Sufficient cell supernatant was collected to extract osteoblast-derived exosomes, identified by employing transmission electron microscopy, nanoparticle tracking analysis, and Western blot. Result: The total experimental time of the improved method (5.5±0.17 hours) was significantly shorter than the traditional method (7.25±0.23 hours) with a notable statistical difference (P <0.001); the osteoblasts obtained by the improved method displayed good growth status. On the seventh day, the cells matured and exhibited positive ALP staining. By the 21st day, mineralized nodules were formed, and alizarin red staining was positive. The EdU results showed that osteoblasts demonstrated healthy growth at both 24 and 48 hours, with a proliferation rate of (96.2%±1.3%) at 48 hours, slightly higher than that of (94.6%±2.8%) at 24 hours, with no significant statistical difference (P >0.05); Western blot showed positive results for CD9, CD63, and TSG101, and negative results for Calnexin. Conclusion: The optimized primary osteoblast isolation method can significantly improve experimental efficiency and is suitable for extracting osteoblast-derived exosomes.