Three-dimensional cell culture microarray for high-throughput studies of stem cell fate.
Three-dimensional cell culture microarray for high-throughput studies of stem cell fate.
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DOI：
10.1002/bit.22661
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发表时间：
2010-05-01
影响因子：
3.8
通讯作者：
Dordick JS
中科院分区：
文献类型：
--
作者：
Fernandes TG;Kwon SJ;Bale SS;Lee MY;Diogo MM;Clark DS;Cabral JM;Dordick JS
我们开发了一种新型的三维（3D）细胞微阵列平台，能够快速有效地跟踪干细胞的命运和定量特定的干细胞标志物。该平台由功能化载玻片上的小型化3D细胞培养阵列组成，用于空间可寻址的高通量筛选。使用微阵列点样器将细胞存款到改性的玻璃表面上，以产生由包封在藻酸盐凝胶点中的细胞组成的阵列，其体积低至60 nL。一种基于免疫荧光技术的方法也被用来量化特定的细胞标志物蛋白质水平在原位上缩小到细胞微阵列上的功能。我们的研究结果表明，该平台适合于研究小鼠胚胎干细胞（ES）的扩增，因为它们保留了其多能性和未分化状态。我们还研究了神经承诺的小鼠ES细胞的微阵列上，并观察到神经外胚层前体细胞的产生，其特征在于表达的神经标志物Sox-1，其水平也原位测量使用GFP报告系统。此外，使用双载玻片系统测试了平台的高通量能力，该系统允许快速筛选维甲酸和成纤维细胞生长因子-4（FGF-4）对小鼠ES细胞多能性的影响。这种高通量平台是研究干细胞扩增和分化中涉及的细胞机制的强大新工具，并为快速鉴定可用于指导细胞反应的信号和条件提供了基础。
We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses.