兔关节软骨细胞体外三维培养条件的优化

Optimization of Culture Conditions for Articular Chondrocytes in 3-Dimensional Alginate Hydrogel

实验使用海藻酸钠水凝胶作为细胞支架,模拟软骨细胞体内生长的三维环境,研究了体外三维培养条件下,不同浓度的胎牛血清(FBS)和硒酸复合液(ITS)体系对兔透明软骨细胞(hyaline cartilage)的生长、增殖和细胞外基质分泌活动的影响。结果表明,三维模式培养21天,透明软骨细胞仍然具有较好的增殖活性。在硒酸复合液及低浓度血清时,细胞未去分化,保持分泌Ⅱ型胶原(CollagenⅡ)和软骨聚集蛋白聚糖(Aggrecan)的能力,与之比较,高浓度胎牛血清(10%)培养条件下,在21天开始细胞去分化,即硒酸复合液在一定的血清浓度下有助于维持软骨细胞生长、增殖,避免了软骨细胞受高浓度血清影响而去分化。

Alginate hydrogels were used as scaffolds for chondrocyte cell culture in vitro in a three-dimensional environ- ment. Cells were cultured in DMEM medium with 10% FBS, 1% ITS plus 2% FBS, 1% ITS plus 4% FBS, and 1% ITS plus 8% FBS. At designated time points, the chondrocyte-seeded alginate hydrogels were harvested and evaluated with his- tological staining, immunohistochemistry, and semi-quantitative gene expression analysis. Viable cell density and cell divi- sion were also evaluated. Chondrocytes biosynthesis and cell division in 1% ITS with 2% FBS medium were similar to that in medium with 10% FBS. For a total culture of 21 days, phenotypic gene expression in chondrocyte-seeded hydrogels was maintained at high levels in medium with 1% ITS plus 2% FBS, while it was decreased to varying degrees in the other groups. In conclusion, media with 1% ITS plus 2% FBS could promote chondrocyte biosynthesis, cell division, and pre- vent cell dedifferentiation in three-dimensional alginate scaffolds.