| 摘要: |
| 目的 联合单层培养和三维培养方法，探讨软骨细胞培养的理想方法。方法 第一步先将软骨细胞进行常规单层培养扩增,第二步再将单层培养第4代软骨细胞包裹于海藻酸钠水凝胶中进行三维培养。通过倒置显微镜定期观察软骨细胞的形态变化；应用锥虫蓝染色和LIVE/DEADR ○ Kit试剂盒检测软骨细胞的活力和细胞增殖；通过组织化学甲苯胺蓝特异性染色检测细胞外基质的形成变化。结果 单层培养3代之前软骨细胞能保持正常的组织学形态,三维培养能使去分化的4代软骨细胞逐渐恢复并持续维持正常的软骨组织表型。单层培养的软骨细胞的4代以前的细胞活力保持在90%左右,第4代以后开始下降；三维培养的软骨细胞活力都保持在90%以上。在细胞增殖率方面,单层培养6代软骨细胞(28 d)总的倍增率是同期三维培养的44倍。单层培养的软骨细胞4代之后,其细胞外基质平均染色强度降低(P<0.05)；三维培养21 d和28 d后的软骨细胞外基质表达强度与7 d时相比升高,差异有统计学意义(P<0.05)。结论 先通过单层培养使软骨细胞适宜扩增,然后将其转入水凝胶中三维培养恢复退变的软骨组织表型,可达到既扩增自体软骨细胞数目同时又保持正常组织表型的目的,两步培养法是一种较理想的软骨细胞培养方法。 |
| 关键词: 软骨细胞 细胞培养 表型 细胞增殖 海藻酸钠 |
| DOI：10.3724/SP.J.1008.2010.00 |
| 投稿时间：2010-04-23修订日期：2010-07-30 |
| 基金项目:上海市卫生局青年科研项目基金(2007Y19), 卫生部公益性卫生行业科研专项项目(200802029). |
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| Monolayer culture followed by three-dimensional culture of chondrocytes: a two-step method |
| GUO Shang-chun1, YUAN Ting2, RUI Bi-yu2, CHEN Xin2, SUN Hui2, ZENG Bing-fang2,ZHANG Chang-qing2* |
| (1. Shanghai Institute for Microsurgery of Extremities, Sixth People’s Hospital of Shanghai, Shanghai Jiaotong University, Shanghai 200233, China; 2. Department of Orthopaedics, Sixth People’s Hospital of Shanghai, Shanghai Jiaotong University, Shanghai 200233, China) |
| Abstract: |
| Objective To explore an ideal method for culturing autologous chondrocytes while maintaining their cartilaginous phenotype by combining monolayer culture with three-dimensional culture. Methods Chondrocytes were cultured by routine monolayer culture method, then the chondrocytes of fourth passage were seeded into alginate beads for three-dimensional culture following standard protocols. The cell morphology was observed by inverted microscope at predefined time points. The cell viability and proliferation were tested by trypan blue and LIVE/DEADR ○ Kit, respectively. And extracellular matrix formation was examined by toluidine blue staining. Results The cartilaginous phenotype of chondrocytes could only be maintained by passage 2 and 3 after monolayer culture. Three-dimensional culture could gradually regain and maintain the cartilaginous phenotype of de-differentiated chondrocytes (P4). Cells of P1-3 by monolayer culture could maintain 90% activity, which decreased from P4.The cytoactivity of cells by three-dimensional culture method was always above 90%. The total doubling rate till P6 by monolayer culture (28 days) was 44 times that by three-dimensional culture. The staining intensity of cartilaginous extracellular matrix was significantly decreased after P4 by monolayer culture (P<0.05). For three-dimensional method, the staining intensities of cartilaginous extracellular matrix at day 21 and 28 were significantly higher than that at day 7(P<0.05). Conclusion Monolayer culture allows proper proliferation of chondrocytes, and following three-dimensional culture in alginate beads can regain the cartilaginous phenotype of chondrocytes, thus achieving the aim of both amplification and maintenance of the cartilaginous phenotype. |
| Key words: chondrocytes cell culture techniques phenotype cell proliferation alginate |
