Gosset Marjolaine, Berenbaum Francis, Thirion Sylvie, Jacques Claire
Paris Universitas-Université Pierre et Marie Curie Paris VI, Department of Physiology and Physiopathology, Centre National de la Recherche Scientifique (Unité Mixte de Recherche 7079), 7 quai St-Bernard, Paris 75252 Cedex 5, France.
Nat Protoc. 2008;3(8):1253-60. doi: 10.1038/nprot.2008.95.
The culture of chondrocytes is one of the most powerful tools for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate into fibroblasts when they are subcultured, which is a major problem. This protocol, involving primary cultures to limit dedifferentiation, describes two different methods for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes, both of which represent hyaline cartilage) from mice. Mice are of particular interest for cellular and molecular studies, as many tools suitable for use in mice are available. In addition, rapid development of transgenic and gene-targeted mice provides powerful instruments for biological studies. The protocol can be divided into four stages: isolation of cartilage (15 min per animal), isolation of chondrocytes (2 h extended overnight), seeding of chondrocytes (1 h 30 min) and growth in culture (6 d). To obtain confluency of chondrocytes using this protocol takes 7 d. Methods for phenotyping chondrocytes are also provided.
软骨细胞培养是探索软骨细胞分化和激活的细胞内及分子特征的最有力工具之一。然而，软骨细胞传代培养时容易去分化为成纤维细胞，这是一个主要问题。本方案涉及原代培养以限制去分化，描述了两种从小鼠不同解剖学来源（关节软骨细胞和肋软骨细胞，二者均代表透明软骨）培养软骨细胞的不同方法。小鼠对于细胞和分子研究尤为重要，因为有许多适用于小鼠的工具。此外，转基因和基因靶向小鼠的快速发展为生物学研究提供了强大的工具。该方案可分为四个阶段：软骨分离（每只动物15分钟）、软骨细胞分离（延长至过夜2小时）、软骨细胞接种（1小时30分钟）和培养生长（6天）。使用该方案使软骨细胞达到汇合需要7天。还提供了软骨细胞表型分析方法。
