左旋聚乳酸多孔微球体外动态培养耳郭软骨细胞的研究
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摘要
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.
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.
引文
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[2]Bonnier F,Keating ME,Wrobel TP,et al.Cell viability assessment using the Alamar blue assay:a comparison of 2D and 3D cell culture models[J].Toxicol In Vitro,2015,29(1):124-131.
[3]Xie L,Mao M,Zhou L,et al.Signal factors secreted by 2D and spheroid mesenchymal stem cells and by cocultures of mesenchymal stem cells derived microvesicles and retinal photoreceptor neurons[J].Stem Cells Int,2017,2017:2730472.
[4]Kudva AK,Luyten FP,Patterson J.Initiating human articular chondrocyte re-differentiation in a 3D system after 2D expansion[J].J Mater Sci Mater Med,2017,28(10):156.
[5]Mujeeb A,Miller AF,Saiani A,et al.Self-assembled octapeptide scaffolds for in vitro chondrocyte culture[J].Acta Biomater,2013,9(1):4609-4617.
[6]Sheehy EJ,Buckley CT,Kelly DJ.Chondrocytes and bone marrowderived mesenchymal stem cells undergoing chondrogenesis in agarose hydrogels of solid and channelled architectures respond differentially to dynamic culture conditions[J].J Tissue Eng Regen Med,2011,5(9):747-758.
[7]Costa AR,Withers J,Rodrigues ME,et al.The impact of microcarrier culture optimization on the glycosylation profile of a monoclonal antibody[J].Springerplus,2013,2(1):25.
[8]Marlovits S,Tichy B,Truppe M,et al.Collagen expression in tissue engineered cartilage of aged human articular chondrocytes in a rotating bioreactor[J].Int J Artif Organs,2003,26(4):319-330.
[9]常彬,肖统光.Cultispher微载体构建组织工程软骨修复关节软骨缺损[J].中国组织工程研究,2017,21(30):4793-4798.
[10]Chung HJ,Kim IK,Kim TG,et al.Highly open porous biodegradable microcarriers:in vitro cultivation of chondrocytes for injectable delivery[J].Tissue Eng Part A,2008,14(5):607-615.
[11]Kim TK,Yoon JJ,Lee DS,et al.Gas foamed open porous biodegradable polymeric microspheres[J].Biomaterials,2006,27(2):152-159.
[12]Kang SW,La WG,Kim BS.Open macroporous poly(lactic-coglycolic Acid)microspheres as an injectable scaffold for cartilage tissue engineering[J].J Biomater Sci Polym Ed,2009,20(3):399-409.