三维微小凹图式的制备及其与C17．2神经干细胞的复合

Fabrication of Three-dimensional Microwell Patterns and Their Integration with C17.2 Neural Stem Cells

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。

UV photolithography and hydrofluoric acid wet etching were used to produce silicon master molds and polydimethylsiloxane （PMDS）-based soft lithography was adopted to fabricate three-dimensional poly（lactic-co-glycolic acid） （PLGA） and PDMS microwell patterns with high aspect ratio and channel connection. Nine microwell patterns were thus obtained with different structural dimensions. Patterns were treated with oxygen plasma etching and polylysine coating to enhance hydrophilicity and cell compatibility for subsequent culture of CI？. 2 neural stem cells. With proliferation during the culture, C17.2 cells gradually distributed within the microwells, showing an ob- viously three-dimensional （3-D） growth behavior. The presence of channel structures greatly favored the 3-D growth of C17.2 neural stem cells on the microwell patterns. Multi-layered scanning with conIocal microscopy and 3-D ren- dering after carboxyfluorescein diacetate succinimidyl ester （CFDA-SE） staining showed that most C17.2 cells grew within a range of 30 to 90μm from the microwell bottom. Immunofluorescence staining indicated that C17.2 cells within 3-D microwell patterns were uniformly nestin-positive on day 2 after cell plating. It could well be concluded that the microwell patterns thus fabricated were suitable for the 3-D culture and subsequent differentiation of C17.2 neural stem cells. And the cells can be maintained with uniform stemness properties while cultured in these microwell patterns