A Study On The Purification Of Bone Marrow Mesenchymal Stem Cells From Rat And Their Identification By CD45 And CD90

AimsTo explore the method of purifying bone marrow mesenchymal stem cells from rats and identify the cells by combination of mouse-anti-rat CD45 and CD90 by flow cytometry.MethodsFemur and tibia were got from male rats, and then bone marrow was flushed out by DMEM/F12. And then the bone marrow was mixed by proper amount of 1.073g/ml Percoll and centrifuged at 4℃2000rpm for 30 min. After centrifugation, mononuclear cells were gathered in the interface. The cells were collected and then washed by PBS twice. The well prepared cells were cultured in 5%CO2 at 37℃by DMEM/F12(1:1) which contained 20% FBS on the concentration of 5 x 104/cm2 in the 75ml flask. The fluid and the unattached cells were discarded 24 h later and the fresh culture fluid was added every 3 days. Cells were observed everyday by inverted microscope. The cells were detached by the mixture of 0.1% tripsin and 0.01%EDTA when they were 80% confluent. Then the cells were washed by PBS with 2%FBS and suspended to the concentration of 107/ml. Cells were stained with mouse-anti-rat PE conjugated CD45, FITC conjugated CD90, or both antibodies. Cells were protected from light and cultured at 4℃for 30 min and then analyzed by flow cytometry.Results1 Mesenchymal stem cells can be collected from rat bone marrow by the combination of density gradient centrifugation and adherence culture. The newly separated cells were round, and not equal in size. While 24 h later, the cells began to be adherent to the bottom of the flask and became oval or even multiangluar. When the cells were cultured for 48 h, they became bigger and the border of nuclus and cytoplasm was clear. Most of the cells were mononuclear and the nuclus resided in the center. The cells were oval and some spindle-like cells showed up.10-12 dayslater, most of the cells were spindle-like while a small part were multiangular. The cells reached 80% confluence by about the 12th day.2 Stained with mouse-anti-rat CD45 and CD90, mesenchymal stem cells could be identified by flow cytometry. And in this study, the percentage were 40.02%,43.80%,42.63% in 3 independent experiments.Conclusions1 Mesenchymal stem cells can be collected from rat bone marrow by the combination of density gradient centrifugation and adherence culture.2 Stained with mouse-anti-rat CD45 and CD90, mesenchymal stem cells could be identified by flow cytometry and the mean percentage was about 42.15%.