The Primary Investigation Of The Culturation Of Mesenchymal Stem Cells (MSCs) Under The Modified Gas Microenviroment

During the research of tissue engineering heart valve(TEHV), how to abtain a large number of good functional MSCs,how to maintain the primary stage of MSCs ,and how to control the differentiation are three hot sopts,nowadays. There are many factors correlated with MSCs in growth, cell function status, and differentiation, including oxygen concentration, hydrogenion concentration(PH), temperature, growth factor, nutrient medium, shearing force, and cell density, etc. Oxygen(O2) and PH are two very important factors: O2 has many effects on MSCs, for example, morderate concentration of oxygen can benefit MSCs on proliferation, differentiation and maintenance of primary status; while unsuitable O2 concentration will lead to bad effects on cells growth and functions. MSCs are very sensitive to the change of medium PH, which can significantly affects the proliferation, differentiation and biological functions of MSCs. Nowadays, tranditional cells culturation adopts a vitro circumstancecomprised of 21% O2 and 5%CO2, and aopts medium with PH between 7.2 and 7.4. Whereas, in our bodies, O2 in normal tissue and tissue space is about 3% ~ 9%, and about 1% ~ 7% in cavum ossis; CO2 is about 5% in artery, about 6% in vein, and about 7% in cavum ossis; PH in blood and in cavum ossis stays between 7.2 and 7.5,constantly. At present, some researches indicated that low oxygen concentration can significantly affects MSCs in proliferation and in secretion of growth factors. However, no study has been found concerned about the effects of low oxygen concentration on MSCs adhesion function. Meanwhile, in view of the problem that tranditional mediums such as DMEM, DF-12 and M199 will step up in PH obviously along with the time; we first time try to use 7% CO2 to enhance CO2/HCO3- buffer capacity in medium, keeping medium PH, and to learn about the effects of this new circumstance on MSCs in proliferation and primary status maintenance, in order to find a suitabile condition for cell culture. Part I: Isolation, purification, amplification and identification of mouse MSCs: MSCs were isolated and purified from the mouse bone marrow by Percoll density gradient centrifugation and by adhering to the culture plastic. The morphology was observed under phase contrast microscope, and the some special antigens were examined by immunohistochemistry stain and flow cytometry. Results: we chose 6 group of MSCs for immunohistochemistry stain, and found thatɑ-SMA and Vimentin were positive expression in MSCs. Moreover, flow cytometry indicated that the MSCs had high positive epression in CD29(92.7%), CD44(99.6%), CD90(99.5%) and CD105(99.4%),and low positive expression in CD34(4.6%) and CD45(3.2%). The results show that the cells obtained by our methods were identified to be BMSCs which had rich quantity and well activity.Part II: Comparison of cells proliferation, adhesion and ECM secretion between 6%O2 group and 21%O2 group: By choosing 6 groups of passage 3 MSCs in log phase to go on with proliferation tests, adhesion tests, and Western-blotting of FN and VN, we primary estimated MSCs functions in proliferation, secretion and adhesion under different O2 concentration. Results: mouse MSCs cultured under 6%O2 are much more enhanced in proliferation(p<0.05), adhesion (p<0.05)and secretion(p<0.05), compare to cells under 21% O2.Part III: Comparison of proliferation and protein OCT-4 expression of mouse MSCs between 7%CO2 group and 5%CO2 group: By choosing 6 groups of passage 3 MSCs in log phase to go on with proliferation tests, and Western-blotting of OCT-4, we firstly learned about the effects of different CO2 concentrations on MSCs proliferation and maintenance of primary status. Results: Mouse MSCs cultured under 7%CO2 are much more enhanced in proliferation (p<0.05), than cells under 5%CO2; Meanwhile, under 7%CO2 MSCs had a higher expression of OCT-4 (p<0.05), which indicated that 7%CO2 is more suitable to maintain MSCs primary status. Part IV: Comparison of cells proliferation, adhesion and protein OCT-4 expression of mouse MSCs between 7%CO2 + 6%O2 group and 5%CO2 + 6%O2 group: By choosing 6 groups of passage 3 MSCs in log phase to go on with proliferation tests, adhesion tests and Western-blotting of OCT-4, we investigated the abilities of MSCs in proliferation, adhesion and primary status maintenance, under two different gas environment. Result: compared to 5%CO2 + 6%O2, the condition of 7%CO2 +6%O2 is more benifitial to MSCs in proliferation (p<0.05)and adhesion(p<0.05), as well as a higher expression of OCT-4(p<0.05). Conclusion: O2 and PH are two important key factors to the growth of cells, which have great influences on MSCs proliferation and differentiation. According to our modified gas environment, we could rapidly obtain ideal MSCs in quantity and in quality in vitro cell culture. And this will play a positive role in tissue engineering research.