犬关节软骨细胞的体外分离与培养

Isolation and culture of canine articular chondrocytes in vitro

背景:自1967年Manning等提出的通过胰蛋白酶和细菌胶原酶联合消化法分离软骨细胞以来,软骨细胞的体外分离培养研究较多,但目前尚无统一标准。目的:研究犬关节软骨细胞在体外分离以及培养的条件,寻求体外扩增软骨细胞较简便、可行、高效的实验方法。方法:取3周龄幼犬关节软骨组织,应用胰酶、胶原酶制定8种消化方法获取软骨细胞,对比不同方法所获取细胞数量及细胞成活率,分离细胞进行原代及传代培养,观察各代软骨细胞形态。结果与结论:在体外分离犬关节软骨细胞的各种方法中,单纯应用Ⅱ型胶原酶消化法获得的软骨细胞数最多,细胞成活率最高。软骨细胞可以通过体外培养获得扩增,并且维持良好的细胞形态及表型,但仅限于5代以内。

BACKGROUND：Since Manning et al reported to isolate chondrocytes using trypsin and bacterial collagenase digestion in 1967,in vitro isolation and culture of chondrocytes has been widely studied,but there is still no unified standard.OBJECTIVE：To study the conditions for in vitro culture and isolation of chondrocytes and to explore a simple,feasible,and efficient experimental method to isolate,culture and proliferate canine chondrocytes in vitro.METHODS：The articular cartilage of 3-week-old puppies were isolated and digested with trypsin and type Ⅱ collagenase for harvesting chondrocytes in different conditions.We compared the number of cells cell survival rate obtained by different methods.Isolated cells were passaged and morphology of chondrocytes was observed.RESULTS AND CONCLUSION：The number and survival rate of chondrocytes was highest by using simple type Ⅱ collagenase digestion.Chondrocytes can be amplified through in vitro culture and maintain good cell morphology and phenotype,but can only be passaged less than five passages.