旋转系统下三维培养成纤维细胞-PGA复合物的实验研究

Experiment on fibroblast-PGA complexes cultured in rotary cell culture system

目的 观察微重力旋转培养系统对细胞种植于支架和细胞 支架复合物体外培养的影响。 方法 分离培养兔皮肤成纤维细胞 ,实验用第 2代细胞。 ( 1)分别用静止接种和动态接种方式 ,以 2× 10 6/cm3 的细胞密度接种于PGA网架 ,静止接种即滴加细胞悬液于PGA网架上 ,动态接种是把细胞悬液与PGA网架置于旋转细胞培养系统旋转接种 ,分别于 2、4、8、12、2 4h消化下网架上细胞后计数 ,通过计算细胞吸附率了解细胞吸附情况 ;( 2 )将相同细胞密度静止接种的细胞—PGA复合物分别置于旋转和静止条件下培养 ,于 1、3、5、7、14、2 1dMTT法检测细胞增殖 ;用倒置相差显微镜和扫描电镜观察细胞生长、基质形成及其细胞与PGA纤维结合状况的变化。 结果 细胞吸附量随时间延长而逐渐升高 ,动态组在接种后 8、12、2 4h细胞吸附率显著高于静止接种组 ,分别为 ( 46 70 %±2 16 %)比 ( 31 5 0 %± 3 5 4%) ;( 5 6 36 %± 3 18%)比 ( 34 2 8%± 3 16 %) ;和 ( 6 6 32 %± 4 6 0 %)比( 37 38%± 4 6 6 %)。在 3周培养中旋转培养组细胞增殖快于静止组 ,而且细胞在网架中分布比静止组均匀 ,并伴有活跃的细胞基质分泌。 结论 旋转培养系统具有促进细胞吸附增殖分化和在支架内均匀分布的特点 。

Objective To investigate the effects of RCCS on cell seeding onto 3-D scaffold and cell-scaffold composite culture in vitro. Methods Rabbit skin fibroblasts of passage 2 were seeded at 2×10 6 cell per cm 3 onto/into polyglycolic acid (PGA) foams by static seeding (dropping a cell suspension onto foams) or dynamic seeding (rotating PGA foams and a cell suspension in RCCS). Attachment of cells in foams was observed by cell-counting after trypsin digestion. The effects of culture condition were next studied by culturing cell-PGA complexes in RCCS versus static culture condition. Distribution and proliferation of cells in foams were investigated with MTT, stereomicroscope and scan electron microscope. Results Numbers of cells adhering to polymers increased gradually during an initial period of 24 hours. Eight, 12 and 24 hours after seeding, the rates of adhering cells were significantly higher in the dynamic seeding group than in the static seeding group (46.70%+2.16% vs.31.50%±3.54%; 56.36%±3.18% vs.34.28%±3.16%; 66.32%±4.60% vs.37.38%±4.66%; P<0.01). The dynamic culture method as compared to the static method resulted in new tissues with a higher cellularity and more uniform cell distribution during a 3 period of weeks. Conclusions RCCS has advantages of promoting cell attachment, uniform migration and proliferation in polymer scaffolds and can be used for construction of 3-D cell-polymer tissues in vitro.