耳郭软骨细胞微载体的体外三维动态培养

Three-dimensional dynamic culture of auricular chondrocytes with microcarriers in vitro

背景:软骨细胞是软骨组织工程最常用的种子细胞,但是如何在体外扩增的同时又能维持其良好的软骨表型是研究的重点和难点之一。目的:通过微载体动态培养法,明确三维动态培养对耳郭软骨细胞增殖和分化的影响,为建立规模化扩增方法提供技术参考。方法:应用胰酶、胶原酶消化法分离、培养猪耳软骨细胞,并将获得的软骨细胞分为3组:培养皿单层培养、微载体三维静止培养和微载体三维动态培养。应用扫描电镜、荧光倒置显微镜、DIO荧光染色、冰冻切片DAPI染色观察细胞在微球上生长情况,DNA定量检测软骨细胞增殖能力,Real-time PCR检测软骨相关基因表达差异。结果与结论:①第1代软骨细胞可快速贴附于CultispherG多孔微载体表面,微载体动态培养组细胞数量多于微载体静止培养组,而且微载体动态培养组细胞分布更为均匀;②培养皿单层培养时,软骨细胞在4d时进入平台期,微载体动态培养组软骨细胞对数生长期延长为2-10 d,细胞数量在14 d达到高峰,微载体静止培养组细胞增殖速度缓慢;③在长期体外培养中,二维培养和微载体静止培养软骨表型丧失,而微载体三维动态培养在促进细胞增殖的同时,能够维持软骨表型;④结果表明,微载体联合旋转细胞培养系统三维动态培养可以作为耳郭软骨细胞规模化扩增的一种方法。

BACKGROUND:Chondrocytes are the most commonly used seed cells in cartilage tissue engineering.How to expand chondrocytes and maintain the cell phenotype in vitro has not been solved.OBJECTIVE:To clarify the effects of three-dimensional dynamic culture on the proliferation and differentiation of chondrocytes through microcarrier culture system,providing a technical reference for the establishment of large-scale amplification methods.METHODS:Auricular chondrocytes were isolated and purified from swine ear using trypsin and collagenase digestion methods.The chondrocytes were divided into three groups:monolayer culture in petri dish,three-dimensional static culture with microcarrier,and three-dimensional dynamic culture with microcarrier.Growth of chondrocytes on the microcarrier was observed by using inverted and scanning electron microscopes.Proliferation rate of chondrocytes was measured by DNA quantitative examination method.Expression of cartilage-related genes was measured by real-time PCR.RESULTS AND CONCLUSION:The passage 1 chondrocytes rapidly attached to the surface of Cultisphere G porous microcarriers.Chondrocytes proliferated faster and distributed more evenly under three-dimensional dynamic culture than static culture.Plateau-stage chondrocytes proliferating in the monolayer culture dish were observed at 4 days of culture.Monolayer-cultured chondrocytes proliferated slowly under three-dimensional static culture,while under three-dimensional dynamic culture the cells logarithmically grew at 2-10 days of culture with the growth peak reaching at 14 days of culture.In the long-term culture,chondrocytes lost phenotypes in monolayer culture and microcarrier static culture.Three-dimensional dynamic microcarrier culture could promote cell proliferation while maintaining the cartilage phenotype.Therefore,the three-dimensional culture with microcarriers and rotary cell culture system can be a feasible method to expand auricle chondrocytes in a large scale.