CAJ | 학술논문

目的探讨灌注培养对骨髓基质干细胞(BMSCs)在大段材料上增殖与分布的影响.方法在大段多孔磷酸三钙材料上种植转染了增强型绿色荧光蛋白基因的大鼠BMSCs(eGFP-BMSCs),分别采用灌注式生物反应器(实验组)和静止培养法(对照组)进行培养.培养7、28 d行扫描电镜观察;培养7、14 d行荧光显微镜观察;动态临测葡萄糖日耗量;培养7、14、28 d测定支架上各层细胞的数量,观察eGFP-BMSCs在支架上的增殖与分布情况.结果扫描电镜和荧光显微镜下观察发现各时间点实验组eGFP-BMSCs均可在支架边缘和内部分布与增殖,而对照组细胞仅能在支架边缘分布与增殖.两组葡萄糖日耗量均随着培养时间的延长而增加,培养28 d时实验组葡萄糖日耗量[(36.33±3.14)mg/d]是对照组[(9.82±1.33)mg/d]的3.7倍,两组分别于20、15 d进入平台期.实验组培养7、28 d时支架各层细胞数量差异无统计学意义(P>0.05),14 d时各层细胞数量的若异有统计学意义(P<0.05);而对照组各时间点支架各层细胞的数量差异均有统计学意义(P<0.05).结论灌注培养法能促进BMSCs在大段多孔材料上增殖,并能使其在大段材料上均匀分布.
목적 탐토관주배양대골수기질간세포(BMSCs)재대단재료상증식여분포적영향.방법 재대단다공린산삼개재료상충식전염료증강형록색형광단백기인적대서BMSCs(eGFP-BMSCs),분별채용관주식생물반응기(실험조)화정지배양법(대조조)진행배양.배양7、28 d행소묘전경관찰;배양7、14 d행형광현미경관찰;동태림측포도당일모량;배양7、14、28 d측정지가상각층세포적수량,관찰eGFP-BMSCs재지가상적증식여분포정황.결과소묘전경화형광현미경하관찰발현각시간점실험조eGFP-BMSCs균가재지가변연화내부분포여증식,이대조조세포부능재지가변연분포여증식.량조포도당일모량균수착배양시간적연장이증가,배양28 d시실험조포도당일모량[(36.33±3.14)mg/d]시대조조[(9.82±1.33)mg/d]적3.7배,량조분별우20、15 d진입평태기.실험조배양7、28 d시지가각층세포수량차이무통계학의의(P>0.05),14 d시각층세포수량적약이유통계학의의(P<0.05);이대조조각시간점지가각층세포적수량차이균유통계학의의(P<0.05).결론관주배양법능촉진BMSCs재대단다공재료상증식,병능사기재대단재료상균균분포.
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.