兔髁状突软骨细胞藻酸盐凝胶三维培养体系的建立

目的:建立兔髁状突软骨细胞的藻酸盐凝胶三维培养体系.方法:利用机械与酶消化的方法获得均一性的兔髁状突软骨细胞,在二维贴壁培养条件下增殖至P2代;将P2代细胞高密度条件下转入藻酸盐凝胶培养介质,进行三维培养;分别对培养细胞凝胶微球行石蜡包埋与树脂包埋切片,对三维培养条件下的软骨细胞行组织化学、免疫组织化学检测和超微结构观察.藻酸盐凝胶三维体系培养6周后,以EDTA分解凝胶,收获培养的软骨细胞,免疫组化法检测三维体系中收获的软骨细胞的蛋白合成能力.结果:藻酸盐凝胶三维培养体系中的髁状突软骨细胞生长状态良好;培养6周后从凝胶体系中收获细胞的软骨特异性功能蛋白表达水平较转入三维体系前未见降低,培养细胞保持了良好的分化表型.结论:成功地建立了髁状突软骨细胞的藻酸盐凝胶三维培养体系,在该体系中,软骨细胞生长状态与功能蛋白分泌能力良好.

Establishment of alginate three-dimensional culture system for rabbit mandibular condylar chondrocytes

Objective: To establish the alginate three dimensional culture system for rabbit condylar chondrocytes. Methods: The cultured mandibular condylar chondrocytes of neonatal rabbits was released by mechanical dissection and enzyme digestion. The chondrocytes were first replicated under monolayer culture condition until the second generation, and then suspended in the aqueous sodium alginate beads with high density. The alginate beads were embedded in Epon and paraffin, sectioned, then studied histochemically, immunologically and ultra microscopically for the culture and growth characteristics of chondrocytes in alginate medium. After six weeks of culture in the alginate system, the chondrocytes encapsulated in alginate beads were removed from the culture medium with dissolving buffer (EDTA solution), then expanded on the coverslips for six to eight hours. Morphology and ability of proteoglycan and type II collagen synthesis were compared between the P2 chondrocytes in monolayer culture and the chondrocytes that had been just removed from three dimensional culture. Results: The chondrocytes cultured in the alginate medium grew well. Compared with the pre seeding state of condylar chondrocytes, the harvested cells from alginate medium showed an equivalent level of expression of the cartilage specific protein, type II collagen and aggrecan. So the chondrocytes cultured in alginate system maintained the excellent differentiated phenotype. Conclusion: The alginate three dimensional culture system for rabbit mandibular condylar chondrocytes was successfully established, in which the chondrocytes showed excellent differentiated phenotype.