灌注培养对骨髓基质干细胞在大段材料上增殖与分布的影响

Objective To study the role of perfusion bioreactor in proliferation and distribution of rat bone marrow stromal cells (BMSCs) in a large-scale scaffold. Methods SD rat BMSCs transfected with enhanced green fluorescent protein (eGFP) (eGFP-BMSCs) were planted in large-scale porous β-tricalcium phosphate (β-TCP) scaffolds. In the dynamic perfusion culture group, the scaffold with eGFP-BMSCs was continuously cultured in our self-designed three-dimensional perfusion bioreactor for 7, 14 and 28 days. In the static culture group, the scaffold was put into a medium reservoir without perfusion for 7, 14 and 28 days.Proliferation and distribution of the cells in the scaffold were examined by scanning electronic microscopy (SEM), fluorescence microscopy (FM), measuring daily glucose consumption, and counting eGFP-BMSCs in each layer. Results SEM and FM showed that eGFP-BMSCs distributed and proliferated throughout the scaffold in dynamic perfusion culture, but distributed and proliferated only in the peripheral pores of the scaffold in static culture. The daily glucose consumption in both groups increased with time. Cell proliferation reached the plateau phase after culture for 14 days in the static culture group, but after culture for 21 days in the perfusion culture group. The rate and margin of increase were much more evident in the perfusion culture group. On day 28, glucose consumption in the perfusion culture group was 36. 33 ± 3. 14 mg/d, 3. 7 times as large as that in the static culture group (9. 82 ± 1. 33 mg/d). The eGFP-BMSCs counting revealed there were no significant differences in cell number between layers of the scaffold on days 7 and 28 d ( P > 0. 05), but there were significant differences on day 14 in the perfusion culture group ( P < 0. 05); while in the static culture group, there were significant differences in cell number between layers of the scaffold ( P < 0. 05),with most of the cells assembled at the bottom of the scaffold.Conclusion Our self-designed three-dimensional perfusion bioreactor may help eGFP-BMSCs proliferate and distribute uniformly in a large-scale porous scaffold.

The role of perfusion bioreactor in proliferation and distribution of bone marrow stromal cells in a large-scale scaffold