Isolation, Cultivation And Identification Of Bone Marrow Mesenchymal Stem Cells From Wistar Rats

Objective:To establish a method for isolation and cultivation of Wistar rats bone marrow mesenchymal stem cells(MSCs) in vitro. Flow cytometry was used to identify the purity of acquired bone marrow MSCs from Wistar rats with mouse-anti-rat CD34 and CD90 antibodies, which is in order to explore a method for the precise identification of bone marrow MSCs with as few antibodies as possible.Methods:Wistar rats of 4-6 weeks old were sacrificed by cutting off the head and soaked in 75% alcohol for 10min. Femurs and tibias were got under sterile conditions and muscle tissue was removed from the acquired long bones. Wash the long bones repeatedly with phosphate buffered saline(PBS) and then flush the bone marrow cavity with serum-free low glucose DMEM(LG-DMEM) medium. The acquired bone marrow cells were mixed sufficiently and made into single cell suspension. Discard the supernatant after being centrifugated at 1500rpm for 5min and resuspend the cells with PBS. Then the single cell suspension of bone marrow was slowly added to appropriate amount of 1.073g/ml Percoll separation and centrifuged at 2500rpm for 20min. After being centrifugated, mononuclear cells were collected in the white haze of boundary layer. The collected cells were subsequently diluted and washed twice with PBS by centrifugating it at 1500rpm for 5min every time. After that, the mononuclear cells were resuspended with LG-DMEM medium containing 10% fetal bovine serum(FBS) and were seeded in a 25cm2 flask at a density of 1×106/ml. Then the well-prepared cells were cultured in an incubator containing 5% CO2 at 37℃. The culture medium was renewed 72 hours later for the first time in order to discard the non-adherent cells. Then the adherent cells had medium changes every three to four days and were observed daily for cell proliferation and morphological characteristics under an inverted microscope. At 80%-90% confluence, the adherent cells were digested by 0.25% pancreatin and 0.02% EDTA(1:1) for about 5min at 37℃and were passaged in a ratio of one to two. The passaged cells were cultured like the primary cells until they passaged to the next generation for a total of three passages. The third generation of bone marrow MSCs were harvested with 0.25% pancreatin and 0.02% EDTA(1:1) and were made into single cell suspension. Subsequently, the cells were centrifugated at 1500rpm for 5min and the supernatant was discarded. After being washed repeatedly with PBS, the cells were made into single cell suspension again at a density of 1×106/ml and incubated with antiCD34, CD90 antibodies at room temperature in the dark for 30min. Then the cells were washed twice and the expression of surface molecule CD34, CD90 were identified by flow cytometry. Meanwhile, the percentage of CD90+ CD34- cells in the acquired cells was detected. The above experimental procedure was repeated three times so as to observe the accuracy of identification of MSCs with antiCD34, CD90 antibodies.Results:(1)MSCs with high purity can be acquired from Wistar rats bone marrow by the combination of density gradient centrifugation and adherence culture. The newly isolated bone marrow cells were round and variable in size. They mingled with the surrounding blood cells.24 hours later, there were some adherent cells at the bottom of the flask that presented to be round, oval and multi-angular. The culture medium was renewed 72 hours later for the first time and the non-adherent cells were discarded. The number and volume of the adherent cells increased obviously which had begun to divide and proliferate. Of the adherent cells, a few cells became short spindle. Cell colony which arranaged in a manner of radiation could be seen by about the sixth day. The cells were with large nucleus and stretched out some prominence variable in length and thickness. The cells reached 80%-90% confluence by the 14th day or so and grew in a manner of school of fish or whirlpool. The passaged cells adhered to the flask more quickly than the primary cells. All of them could be adherent and extense in 24 hours. The cells proliferated rapidly and were uniform in size and morphous.6-7d later, long spindle-shaped cells reached 80%-90% confluence. (2)In the three independent experiments, the positive expression rates of CD90 were all above 95.0%, and the positive expression rates of CD34 were all below 3.0%. The percentage of CD90+CD34- cells in the acquired cells were 93.7%,94.8% and 95.6% respectively, and the mean percentage was 94.7%.Conclusions:(1)MSCs with high purity can be acquired from Wistar rats bone marrow by the combination of density gradient centrifugation and adherence culture. (2)The accuracy of identification of MSCs with antiCD34, CD90 antibodies was high, and CD90 combined with CD34 could be a method for the precise identification of the isolated bone marrow MSCs.