Isolation,Extraction And Identification Of Bone Marrow Mesenchymal Stem Cell-Derived Exosomes From SD Rats

Objective:Bone marrow mesenchymal stem cells(BMSCs)from SD rats were successfully cultured in vitro,and extracellular vesicles were isolated and collected by differential velocity and ultra-high velocity centrifugation.It was further identified as SD rat BMSCsderived exosomes.Methods:1.Bone extraction: SD rats were sacrificed after cervical vertebrae removal at 4 weeks,and the metaphysis of the femur,tibia,humerus and radius were removed from both sides to expose the medullary cavity.2.Irrigation: appropriate amount of complete culture solution(DMEM-F12 containing10% fetal bovine serum and 1% penicillin-streptomycin)was taken into a sterile beaker,and the complete culture solution was extracted from a 5ml syringe and rinsed repeatedly into the medulary cavity until the shaft turned white.3.Filter centrifugation: Complete culture medium containing bone marrow is filtered and centrifuged.4.Inoculate: Counted cells were inoculated in a flask and cultured in a CO2 incubator(37,5%CO2).5.Liquid change: first semi-quantitative liquid change 48 h after inoculation to remove impurities.6.Passing: Passing to the third generation,reserve when the cell fusion reaches more than 95%.7.Identification:(1)Growth characteristics were observed by inverted microscope;(2)MSCs were detected by flow cytometry.8.Cell cryopreservation: cells at exponential growth stage were taken and cryopreservation was made in liquid nitrogen of-196℃.9.When the proliferation and fusion of the third generation BMSCs reached 95% or more,exosome-free serum medium was replaced for overnight culture(14h).10.The next day,about 100 ml of conditional culture medium(DMEM containing exosomes)was collected as samples for reserve.11.The cell survival rate of BMSCs after overnight culture of exosomal free serum was determined.12.Exosomes were extracted by differential centrifuge.13.Identification of exosomes.Conclusion:Transmission electron microscopy(TEM)NTA and WB identification of exosomes obtained by differential centrifuge showed that exosomes were successfully separated.