人负重关节软骨细胞的体外分离与培养

In vitro isolation and cultivation of human chondrocytes from weight-loading cartilage

目的 研究人负重关节软骨细胞的分离培养技术。方法 对 10例手术切除的负重关节软骨进行0 2 %Ⅱ型胶原酶分离、10 %DMEM中培养 ,观察软骨细胞获得率、贴壁率、增殖状况及形态学改变。结果 ① 0 2 %Ⅱ型胶原酶消化尽 2 0 0～ 70 0mg软骨标本需 4～ 14h ,细胞获得率 (1 82± 0 5 7)× 10 6/g ,存活率(79 4± 9 9) %。②细胞可传 6～ 12代 ,前 4代培养时间平均 (33± 10 )d ,生长倍数平均 198倍 ,倍增时间(4 13± 1 34)d。③第 3、4代细胞呈多角形、部分梭形 ,8、9代后细胞梭状铺平。结论 30 0～ 5 0 0mg软骨标本 ,经该方法培养 3～ 4周后可达第 4代 ,细胞数可达 (0 6～ 1 0 )× 10 8个 ,可满足临床上修复 15～ 2 5cm2软骨缺损的需要。

Objective To establish the methods of isolating and cultivating chondrocytes from human weight-loading articulation for clinical application. Methods Cartilages from 10 cases after resection were digested by 0.2% collagenase Ⅱ and cultivated in 10% DMEM. The number of chondrocytes gained from wet cartilage, viability, multiplication and morphologic changes were observed. Results ① The time needed for completely digesting 200～700 mg cartilages by 0.2% collagenase Ⅱ varied from 4 h to 14 h, while collected additionally after 6h, the number of chondrocytes gained from wet cartilage was (1.82±0.57)×10 6/g with a viability of (79.4±9.9)%. ② The cells can be transferred by 6 to 10 times, the cultivation time of previous 4 passages was 33±10 days, developing times was198 on average, DT(doubling time) 4.13 d±1.34 d。③ At 3rd and 4th passage part of the cells begin to alter their shape to spindle and at 8th or 9th became flat, thin and fusiform。Conclusion 300～500 mg cartilage samples can be completely digested and after cultivated for 3～4 weeks, the cells were at 4th passage,the number of cells may reach (0.6～1.0)×10 8 which can fill cartilage defects of 15～25 cm 2 clinically.