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…
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