Use of photolithography to encode cell adhesive domains into protein microarrays
Use of photolithography to encode cell adhesive domains into protein microarrays
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DOI：
10.1021/la702883d
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发表时间：
2008-03-04
期刊：
影响因子：
3.9
通讯作者：
Revzin, Alexander
中科院分区：
文献类型：
--
作者：
Lee, Ji Youn;Shah, Sunny S.;Revzin, Alexander
蛋白质微阵列正在迅速成为创建组合细胞培养系统的有价值的工具，在组合细胞培养系统中，细胞分化的诱导剂可以以快速和多路复用的方式识别。在本研究中，蛋白质微阵列与光刻胶光刻技术相结合，可以打印细胞外基质（ECM）蛋白质阵列，同时精确控制“现场”细胞-细胞相互作用。在这种表面工程方法中，在玻璃基板上形成的微图案光刻胶层在微阵列打印期间充当临时模板，定义了打印蛋白质点内细胞粘附域的微米尺度尺寸和几何形状。去除光刻胶后，玻璃衬底含有微米级的细胞粘附区域，这些区域编码在300或500 μ m直径的蛋白质结构域内。采用荧光显微镜和原子力显微镜（AFM）表征蛋白质微模式。当与微图案表面孵育时，肝脏（HepG2）细胞附着在300或500 μ m直径的蛋白斑点上；然而，每个点内的细胞-细胞接触程度根据光刻胶模板的尺寸而变化，从单个细胞附着在30 μ m直径的特征上到驻留在100或200 μ m直径区域的多细胞团。重要的是，光刻胶去除过程被证明对几种ECM蛋白（胶原I、II、IV和层粘连蛋白）支持肝脏功能培养的能力没有不利影响。这里描述的微图型方法允许将小细胞群播种到单个细胞培养底物上，以并行地暴露于细胞-细胞和细胞-表面相互作用的多种情况下。这项技术对于高通量筛选干细胞组织规格或维持稀缺原代细胞分化表型所需的生物刺激特别有用。
Protein microarrays are rapidly, emerging as valuable tools in creating combinatorial cell culture systems where inducers of cellular differentiation can be identified in a rapid and multiplexed fashion. In the present study, protein microarraying was combined with photoresist lithography to enable printing of extracellular matrix (ECM) protein arrays while precisely controlling "on-the-spot" cell-cell interactions. In this, surface engineering approach, the micropatterned photoresist layer formed on a glass substrate served as a temporary stencil during the microarray printing, defining the micrometer-scale dimensions and the geometry of the cell-adhesion domains within the printed protein spots. After removal of the photoresist, the glass substrates contained micrometer-scale cell-adhesive regions that were encoded within 300 or 500 mu m diameter protein domains. Fluorescence microscopy and atomic force microscopy (AFM) were employed to characterize protein micropatterns. When incubated with micropatterned surfaces, hepatic (HepG2) cells attached on 300 or 500 mu m diameter protein spots; however, the extent of cell-cell contacts within each spot varied in accordance with dimensions of the photoresist stencil, from single cells attaching on 30 mu m diameter features to multicell clusters residing on 100 or 200 mu m diameter regions. Importantly, the photoresist removal process was shown to have no detrimental effects on the ability of several ECM proteins (collagens I, II, and IV and laminin) to support functional hepatic cultures. The micropatterning approach described here allows for a small cell population seeded onto a single cell culture substrate to be exposed to multiple scenarios of cell-cell and cell-surface interactions in parallel. This technology will be particularly useful for high-throughput screening of biological stimuli required for tissue specification of stem cells or for maintenance of differentiated phenotype in scarce primary cells.