Microcontact-Printed Hydrogel Microwell Arrays for Clonal Muscle Stem Cell Cultures.

Microcontact-Printed Hydrogel Microwell Arrays for Clonal Muscle Stem Cell Cultures.
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用于克隆肌肉干细胞培养的微接触印刷水凝胶微孔阵列。

DOI:
10.1007/978-1-4939-7283-8_6
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发表时间:
2017
期刊:
Methods in molecular biology (Clifton, N.J.)
影响因子:
--
通讯作者:
Cosgrove,BenjaminD
Cosgrove,BenjaminD
中科院分区:
--
文献类型:
--
作者:
Aguilar,VictorM;Cosgrove,BenjaminD

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成体肌肉干细胞(也称为卫星细胞)是解剖学上定义的一组细胞,对肌肉再生至关重要。在衰老和营养不良疾病中,肌肉干细胞表现出功能和分子的异质性,因此,单细胞检测技术对于阐明病理性干细胞功能障碍的机制至关重要。在这里,我们描述了使用微接触打印技术生产具有微孔特征的机械可调谐水凝胶的过程,用于克隆肌肉干细胞培养检测。微接触打印(μCP)是一种简单而通用的细胞培养底物生成方法,用于微尺度特征的空间限制单细胞及其后代的培养。我们解释了如何使用光刻和聚二甲基硅氧烷浇注来产生能够将纯化的细胞外基质蛋白打印到柔软的水合聚乙二醇水凝胶上的邮票,以产生以定义图案排列的微孔。我们总结了在水凝胶微孔培养中分析单个MUSC克隆的活性、迁移和分化的方法。
Adult muscle stem cells (also called satellite cells) are an anatomically defined population of cells that are essential for muscle regeneration. In aging and dystrophic diseases, muscle stem cells (MuSCs) exhibit functional and molecular heterogeneity; therefore, single-cell assay technologies are critical to illuminate the mechanisms of pathological stem cell dysfunction. Here, we describe the process of generating mechanically tunable hydrogels with micro-scale well features (“microwells”) using micro-contact printing for clonal muscle stem cell culture assays. Microcontact printing (μCP) is a simple and versatile method for generating cell culture substrates for micro-scale features for spatially restricting the cultures of single cells and their progeny. We explain how to use photolithography and polydimethylsiloxane casting to generate stamps capable of printing purified extracellular matrix proteins onto soft, hydrated poly(ethylene glycol) hydrogels to generate arrayed microwells in a defined pattern. We summarize methods to analyze the viability, migration, and differentiation of individual MuSC clones within hydrogel microwell cultures.
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影响因子: 2.9
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