应用旋转灌注式生物反应系统体外构建组织工程神经的研究

Study on the construction of tissue engineering nerves in vitro by perfusion rotary cell culture system(RCCS)

目的:模拟微重力建立体外构建组织工程神经的方法,评价微重力环境对支持细胞生长的影响。方法:将皮肤前体细胞诱导分化的施万细胞作为支持细胞,联合壳聚糖神经导管、聚乳酸乙醇酸共聚物纤维支架,利用旋转灌注式生物反应系统体外培养,对照组为传统的静止培养。培养不同时间点取样,利用结晶紫染色,免疫荧光细胞化学染色,CCK-8细胞活力检测,扫描电子显微镜等方法观察支持细胞的生长状况。结果:体外培养7天后CCK-8细胞活力检测提示贴附在导管和纤维支架表面的细胞数量最多、活力最好,应用旋转灌注式生物反应系统体外培养较对照组高出约2倍,随后在14天、21天时略有下降;结晶紫染色及扫描电子显微镜观察显示7天时,与对照组相比,实验组支持细胞呈立体生长,分泌大量细胞外基质,生长状态更好;S100荧光免疫细胞化学染色显示微重力环境下施万细胞的标志物未发生改变。结论:模拟微重力环境较传统静止培养能更高效、优质地在体外构建组织工程神经。

Objective： To establish a method for construction of tissue engineered nerve in vitro mimicking micro-gravity. Methods： As seed cells, Schwann cells （SCs） differentiated from skin derived precursors （SKPs） were cultured with chitosan nerve conduit and polylactic glycolic acid （PLGA） copolymer fiber scaffold by using a perfusion rotary bioreactor systems in vitro. Traditional static culture was the control group. The crystal violet staining, immunofluorescence staining, CCK-8 cell viability testing and scanning electron microscopy were applied for evaluating the growth of seed cells at differ-ent time points. Results： 7 days after culture, the number and viability of seed cells approached the peak in both groups, while they were approximately twice as much in the perfusion RCCS as in the control group. Then they decreased slightly at 14 days and 21 days after culture in both groups. Crystal violet staining and scanning electron microscopy showed that the experimental group showed a three-dimensional growth of the seed cells at 7 days after culture, which secreted consid-erable extracellular matrix, compared with the control group. The immunofluorescence staining showed that it did not change the Schwann cell’s marker S100 cultured under a microgravity environment. Conclusions： The construction of tis-sue engineering nerves in vitro was more efficient with higher quality in the RCMW than the traditional static culture.