中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (21): 3336-3341.doi: 10.3969/j.issn.2095-4344.1755

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

耳郭软骨细胞微载体的体外三维动态培养

孙 恒，马世泽，蒋海越，刘 霞，滕 利   

  1. 中国医学科学院整形外科医院，北京市 100043
  • 修回日期:2019-03-11 出版日期:2019-07-28 发布日期:2019-07-28
  • 通讯作者: 刘霞，博士，副研究员，中国医学科学院整形外科医院，北京市 100043; 并列通讯作者：滕利，博士，主任医师，中国医学科学院整形外科医院，北京市 100043
  • 作者简介:孙恒，男，1988年生，河南省安阳县人，汉族，在读博士，主要从事软骨组织工程研究。
  • 基金资助:

    中国医学科学院医学与健康科技创新工程项目(2017-I2M-1-007)，项目负责人：蒋海越；国家自然科学基金(81471804)，项目负责人：刘霞

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

Sun Heng, Ma Shize, Jiang Haiyue, Liu Xia, Teng Li   

  1. Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Beijing 100043, China
  • Revised:2019-03-11 Online:2019-07-28 Published:2019-07-28
  • Contact: Liu Xia, MD, Associate researcher, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Beijing 100043, China; Teng Li, MD, Chief physician, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Beijing 100043, China
  • About author:Sun Heng, Doctorate candidate, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Beijing 100043, China
  • Supported by:

    the Medical and Health Science and Technology Innovation Project of Chinese Academy of Medical Sciences, No. 2017-I2M-1-007 (to JHY); the National Natural Science Foundation of China, No. 81471804 (to LX)

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摘要：

文章快速阅读：

文题释义：
微载体：是指能适用于贴壁细胞生长的微珠，直径为60-250 μm。一般是由天然葡聚糖或者各种合成的聚合物组成，可以提供较高的表面积体积比，可用于贴壁细胞大规模培养。
旋转细胞培养系统：美国NASA发明的一款在体外进行三维细胞或组织动态培养的系统，微重力环境可以保护人体或动物脆弱的细胞，这种培养模拟了母体组织的结构和功能。

 

摘要
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Cultispher G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4 d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。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0001-7617-9156(孙恒)

关键词: 软骨细胞, 三维培养, 三维动态培养, 三维静止培养, 微载体, 旋转细胞培养系统, 软骨表型, 细胞增殖

Abstract:

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.

Key words: chondrocytes, three-dimensional culture, three-dimensional dynamic culture, three-dimensional static culture, microcarrier, rotary cell culture system, cartilage phenotype, cell proliferation

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