基于微阵列芯片的开放式多孔微组织构建

Fabrication of open porous microtissue based on micro-stencil array chip

目的探讨一种适合大鼠骨髓间充质干细胞生长的、具有开放式多孔结构的微组织及其最佳尺寸。方法采用激光打孔的方法构建1 500 μm和800 μm 2种孔径的聚甲基丙烯酸甲酯微阵列芯片。将明胶溶液经化学交联后均匀涂抹在微阵列芯片中,冷冻干燥16 h后,利用对应的顶针阵列顶出微阵列芯片中的微凝胶。扫描电镜观察微观结构,并对2种直径的微凝胶进行孔径和孔隙率的检测。随后,将第3代大鼠骨髓间充质干细胞以5×106/ml浓度接种至微凝胶上,体外培养7 d后,得到微组织,进行活/死细胞染色检测微组织上细胞存活状况,并用扫描电镜观察微组织上细胞生长状况和细胞外基质分泌状况。结果扫描电镜显示微阵列芯片能够成功制备表面具有开放式多孔结构的微凝胶,1 500 μm微凝胶的孔径和孔隙率是800 μm微凝胶的1.5倍左右。接种BMSCs 7 d后,活/死细胞染色显示1 500 μm微组织上细胞数量更多,荧光定量分析显示1 500 μm微组织上细胞活性约为800 μm明胶微组织的4倍左右;扫描电镜显示1 500 μm微组织上细胞形态良好,且周边有分泌的细胞外基质。结论通过设计并制作的微阵列芯片成功构建了开放式多孔微凝胶,并且利用此微凝胶成功构建出开放式多孔微组织;相比于800 μm微组织,1 500 μm微组织上细胞活性更好。

Objective To explore an open porous microtissue which is suitable for the growth of bone marrow mesenchymal stem cells (BMSCs), and the optimal size of this microtissue. Methods Polymethyl methacrylate (PMMA) micro-stencil array chips with pore diameters of 1500 μm and 800 μm were constructed by laser drilling technique. The gelatin solution was chemically crosslinked and uniformly spread on the micro-stencil array chips. After freeze-drying for 16 h, the microgels in the chip were ejected by the ejector-pin array. SEM images were taken to observe the microstructure of microgels. Pore size and porosity of two kinds of microgels were calculated using Image J Pro software. The P3 BMSCs were seeded on microgels at the concentration of 5×106/ml. After 7 days culture in vitro, the microtissues were obtained. Thereafter, the viability of seeded BMSCs was assessed using live/dead cell assay. The growth of the cells on the microtissues and the secretion of ECM were observed by scanning electron microscopy (SEM). Results The SEM images showed that the open porous structured microgels were successfully fabricated using the micro-stencil array chip. The pore size and porosity of 1 500 μm microgels were about 1.5 times that of the 800 μm microgels. After 7 days culture in vitro, the number of cells 1 500 μm microgels were more compared to 800 μm microgels, in the live/dead cell assay. Fluorescent quantitative analyses showed that the viability of BMSCs on 1 500 μm microtissues was 4 times that of 800 μm microtissues. SEM images showed that the cells on 1 500 μm microtissues grew well and surrounded by ECM. Conclusions The micro-stencil array chips were successfully used to construct microgels with open porous, which could be further used to fabricate open porous microtissues. The 1 500 μm microtissues were more favorable for BMSCs, compared to 800 μm microtissues.