Microfabricated platform for studying stem cell fates
Microfabricated platform for studying stem cell fates
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DOI：
10.1002/bit.20254
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发表时间：
2004-11-05
影响因子：
3.8
通讯作者：
Bhatia, SN
中科院分区：
文献类型：
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作者：
Chin, VI;Taupin, P;Bhatia, SN
允许对单细胞进行平行定量分析的平台将是实现后基因组生物学潜力不可或缺的一部分。在干细胞生物学中，多孔克隆干细胞的研究目前既低效又耗时。因此，为了调查低频事件，必须对大样本进行调查。我们报告了一种简单，通用，高效的微模式阵列系统，有利于干细胞增殖的培养和动态监测。该平台可以实现：1)对培养的单个细胞进行平行、自动化、长期（类似于今天的数周）的活细胞显微镜观察；2)随着时间的推移跟踪单个细胞的命运（增殖、凋亡）；3)分化后代与创始无性系的相关性。为了实现这些目标，我们使用微加工技术在玻璃盖上创建了类似10,000个微孔的阵列。井的尺寸是可调的，直径范围为20 - 500mm，高度范围为10 - 500mm。微阵列可以涂上粘附蛋白，并集成到培养室中，使用标准可编程显微镜台对每个孔进行快速（类似于min）可寻址监测。与多井格式的细胞不同，所有细胞共享相同的介质（包括旁分泌生存信号）。盖盖作为衬底的结合也使平台与传统的高倍率光和荧光显微镜兼容。我们通过分析异质成年大鼠神经干细胞群的增殖动力学来验证这种方法。利用这个平台，人们可以进一步询问不同干细胞亚群对控制其行为的微环境线索（有丝分裂原、细胞间相互作用和细胞-细胞外基质相互作用）的反应。在未来，该平台还可以通过定制表面涂层、微孔尺寸和培养环境来适应其他细胞类型的研究，从而能够并行研究许多不同的细胞反应。(C) 2004 Wiley期刊有限公司
Platforms that allow parallel, quantitative analysis of single cells will be integral to realizing the potential of postgenomic biology. In stem cell biology, the study of clonal stem cells in multiwell formats is currently both inefficient and time-consuming. Thus, to investigate low-frequency events of interest, large sample sizes must be interrogated. We report a simple, versatile, and efficient micropatterned arraying system conducive to the culture and dynamic monitoring of stem cell proliferation. This platform enables: 1) parallel, automated, long-term (similar todays to weeks), live-cell microscopy of single cells in culture; 2) tracking of individual cell fates over time (proliferation, apoptosis); and 3) correlation of differentiated progeny with founder clones. To achieve these goals, we used microfabrication techniques to create an array of similar to 10,000 microwells on a glass coverslip. The dimensions of the wells are tunable, ranging from 20 to > 500 mum in diameter and 10 - 500 mum in height. The microarray can be coated with adhesive proteins and is integrated into a culture chamber that permits rapid (similar to min), addressable monitoring of each well using a standard programmable microscope stage. All cells share the same media (including paracrine survival signals), as opposed to cells in multiwell formats. The incorporation of a coverslip as a substrate also renders the platform compatible with conventional, high-magnification light and fluorescent microscopy. We validated this approach by analyzing the proliferation dynamics of a heterogeneous adult rat neural stem cell population. Using this platform, one can further interrogate the response of distinct stem cell subpopulations to microenviron mental cues (mitogens, cell-cell interactions, and cell-extracellular matrix interactions) that govern their behavior. In the future, the platform may also be adapted for the study of other cell types by tailoring the surface coatings, microwell dimensions, and culture environment, thereby enabling parallel investigation of many distinct cellular responses. (C) 2004 Wiley Periodicals, Inc.