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以紫外光光刻及氢氟酸湿法蚀刻加工硅阳模,采用基于聚二甲基硅氧烷(PDMS)的软光刻技术制备9种不同结构尺寸的聚乳酸-羟基乙酸共聚物(PLGA)和PMDS三维微小凹图式。PLGA及PDMS三维微小凹图式经等离子氧蚀刻和多聚赖氨酸裱衬处理后进行C17.2神经干细胞培养。随着在图式上培养时细胞的增殖,C17.2神经干细胞逐渐在微小凹中聚集,表现出明显的三维生长行为;通过羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFDA-SE)染色后进行激光共聚焦显微扫描与三维重构,显示大部分细胞生长于微小凹中离底面30～90μm的区间内;免疫荧光结果显示C17.2神经干细胞在三维微结构中复合培养2d后呈现均一的巢蛋白(Nestin)阳性。结论:本文设计的微小凹图式适用于C17.2神经干细胞的三维培养及后续的分化研究,细胞于微小凹图式培养过程中可以保持均一的干细胞特性。
Nine kinds of polylactic acid-glycolic acid copolymers (PLGA) were prepared by soft lithography based on polydimethylsiloxane (PDMS) using ultraviolet lithography and hydrofluoric acid wet etching. ) And PMDS three-dimensional micro-concave pattern. PLGA and PDMS three-dimensional micro-concave pattern after plasma oxygen etching and poly-lysine lined with C17.2 neural stem cell culture. C17.2 neural stem cells gradually clustered in the microvessels with obvious three-dimensional growth behavior with the proliferation of cells cultured on the graph. After staining with carboxyfluorescein acetoacetate succinimidyl ester (CFDA-SE) Laser scanning confocal microscopy and three-dimensional reconstruction showed that most of the cells grew in the micro-concave area 30 ~ 90μm away from the bottom. The results of immunofluorescence showed that the C17.2 neural stem cells were uniformly distributed in the three-dimensional microstructure after 2 days Of Nestin positive. CONCLUSION: The micro concave pattern designed in this paper is suitable for the three-dimensional culture of C17.2 neural stem cells and the subsequent differentiation research. The cells can maintain uniform stem cell characteristics in the micro-concave pattern culture.