Abstract:

BACKGROUND: During culture of bone marrow mesenchymal stem cells (BMSCs), a certain serum is commonly added in the basic medium, such as calf serum and fetal bovine serum, but there are potential biological safety risks.
OBJECTIVE: To study the effects of different serum microenvironments on in vitro culture of rat BMSCs.
METHODS: BMSCs were harvested from adult rat bone marrow, and cultured in vitro by whole bone marrow adherence method. The cells were cultured under the following serum microenvironment. The primary cells of autoserum group were cultured with autoserum, changing the medium with fetal bovine serum after passage. The primary cells of homogeneity foreign serum group were cultured with homogeneity foreign serum, changing the medium with fetal bovine serum after passage. The primary cells of fetal bovine serum group were cultured with fetal bovine serum, and cultured with fetal bovine serum after passage. The primary cells of Dulbecco's modified Eagle's medium (DMEM) group were cultured with serum-free DMEM, changing the medium with fetal bovine serum after passage. The morphologic changes in BMSCs were detected under an inverted phase contrast microscope. Attachment rate and growth curve were measured. Surface marker CD11b, CD45 and CD90 expression was analyzed by flow cytometry.
RESULTS AND CONCLUSION: In autoserum and homogeneity foreign serum groups, the homogenicity and degrees of fusion of cell morphology were improved in comparison with other two groups and the day of first passage was less than other groups. The attachment rate was greater in the autoserum, homogeneity foreign serum and fetal bovine serum groups than the DMEM group at 24, 48, 72 hours (P < 0.01). Doubling rate was fastest in the growth curve of autoserum group, followed by homogeneity foreign serum group and fetal bovine serum group. However, no doubling phenomenon was found in the DMEM group. Flow cytometry results demonstrated that the rates of CD11b-positive and CD45-positive cells at passage 3 were above 98% under medium containing serum, and CD90-positive rate was less than 2%. We could obtain BMSCs of higher purity. However, CD11b-positive rate was 95.83%, CD90-positive rate was 2.07%, but CD45 positive rate was only 64.79% under serum-free microenvironment. BMSC purity was significantly lower under serum-free microenvironment than under serum microenvironment. Results indicated that the microenvironment of rat autoserum can improve the attachment rate, growth and purification of BMSCs.