左旋聚乳酸多孔微球体外动态培养耳郭软骨细胞的研究

Dynamic Culture of Auricular Chondrocytes with L-Polylactic Acid Porous Microspheres in Vitro

目的明确PLLA(左旋聚乳酸)、Cultispher G两种多孔微球作为耳郭软骨细胞体外培养载体的可行性,为软骨细胞三维动态培养寻找合适的微球支架。方法分离猪耳郭软骨、培养软骨细胞,将获得的第1代软骨细胞分别接种于PLLA和Cultispher G多孔微球上,并于RCCS三维培养系统中进行培养。应用电镜和荧光倒置显微镜观察细胞在微球上的生长情况,CCK-8法检测软骨细胞增殖情况,Real-time PCR检测相关基因表达。结果软骨细胞可贴附于两种微球表面,PLLA和Cultispher G两种微球接种率分别为(37.67%±2.33%)和(73.67%±3.53%)。培养21 d后,PLLA多孔微球细胞总数高于Cultispher G多孔微球。Real-time PCR检测发现PLLA多孔微球维持软骨细胞表型能力优于Cutispher G多孔微球。结论与Cultispher G多孔微球相比,PLLA多孔微球更适合软骨细胞体外三维培养。

Objective To clarify the feasibility of PLLA and Cultispher G porous microspheres as an in vitro culture material for auricular chondrocytes,and to find a suitable microsphere scaffold for the three-dimensional dynamic culture of chondrocytes.Methods Aricular chondrocytes were isolated and purified from swine ear.P1 chondrocytes were inoculated on PLLA microspheres and Cultispher G microspheres,then cultured in a rotary cell culture system(RCCS).Growth of chondrocytes was observed on the microcarriers by using inverted and scanning electron microscopes.Proliferation rate of chondrocyte was measured by CCK-8 examination method.Relevant genes were measured by real-time PCR.Results Inverted and scanning electron microscopes showed chondrocytes could grow on PLLA microspheres and Cultispher G microspheres.Inoculation rates of chondrocytes were(37.67%±2.33%)and(73.67%±3.53%)respectively.Increasing fold of chondrocytes on PLLA microspheres was higher than that on Cultispher G microspheres after cultured for 21 days.Real-time PCR showed that PLLA microspheres maintained chondrocyte phenotype better than Cultispher G microspheres.Conclusion Compared with Cultispher G microsphere,PLLA porous microspheres are more suitable for three-dimensional culture of chondrocytes in vitro.