Mechanical cues such as stiffness have been shown to influence cell gene expression, protein expression, and cell behaviors critical for tissue engineering. The mechanical cue of confinement is also a pervasive parameter affecting cells in vivo and in tissue-engineered constructs. Despite its prevalence, the mechanical cue of confinement lacks assays that provide precise control over the degree of confinement induced on cells, yield a large sample size, enable long-term culture, and enable easy visualization of cells over time. In this study, we developed a process to systematically confine cells using micropillar arrays. Using photolithography and polydimethylsiloxane (PDMS) molding, we created PDMS arrays of micropillars that were 5, 10, 20, or 50 μm in spacing and between 13 and 17 μm in height. The tops of micropillars were coated with Pluronic F127 to inhibit cell adhesion, and we observed that mesenchymal stem cells (MSCs) robustly infiltrated into the micropillar arrays. MSC and nucleus morphology were altered by narrowing the micropillar spacing, and cytoskeletal elements within MSCs appeared to become more diffuse with increasing confinement. Specifically, MSCs exhibited a ring of actin around their periphery and punctate focal adhesions. MSC migration speed was reduced by narrowing micropillar spacing, and distinct migration behaviors of MSCs emerged in the presence of micropillars. MSCs continued to proliferate within micropillar arrays after 3 weeks in culture, displaying our assay's capability for long-term studies. Our assay also has the capacity to provide adequate cell numbers for quantitative assays to investigate the effect of confinement on gene and protein expression. Through deeper understanding of cell mechanotransduction in the context of confinement, we can modify tissue-engineered constructs to be optimal for a given purpose. Impact Statement In this study, we developed a novel process to systematically confine cells using micropillar arrays. Our assay provides insight into cell behavior in response to mechanical confinement. Through deeper understanding of how cells sense and respond to confinement, we can fine tune tissue-engineered constructs to be optimal for a given purpose. By combining confinement with other physical cues, we can harness mechanical properties to encourage or inhibit cell migration, direct cells down a particular lineage, induce cell secretion of specific cytokines or extracellular vesicles, and ultimately direct cells to behave in a way conducive to tissue engineering.

中文翻译：

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间充质干细胞整合到一种新型的微柱限制试验中。

诸如刚度之类的机械暗示已显示出影响细胞基因表达，蛋白质表达以及对组织工程至关重要的细胞行为。限制的机械提示也是影响体内和组织工程构建体中细胞的普遍参数。尽管其局限性很普遍，但缺乏能够对细胞诱导的局限度进行精确控制，产生大量样本，能够进行长期培养并能够随时间轻松观察细胞的分析方法。在这项研究中，我们开发了一种使用微柱阵列系统限制细胞的方法。使用光刻和聚二甲基硅氧烷（PDMS）成型，我们创建了微柱的PDMS阵列，其间隔为5、10、20或50μm，高度为13至17μm。微柱的顶部涂有Pluronic F127以抑制细胞粘附，我们观察到间充质干细胞（MSC）牢固地渗透到微柱阵列中。通过缩小微柱间距来改变MSC和细胞核形态，并且随着限制的增加，MSC中的细胞骨架元素似乎变得更加分散。具体而言，MSC在其周围表现出肌动蛋白环并呈点状粘连。通过缩小微柱间距降低了MSC的迁移速度，并且在存在微柱的情况下出现了不同的MSC迁移行为。培养3周后，MSC继续在微柱阵列中增殖，这显示了我们测定的长期研究能力。我们的测定法还具有为定量测定法提供足够的细胞数量，以研究限制对基因和蛋白质表达的影响的能力。通过在限制条件下更深入地了解细胞机械转导，我们可以修改组织工程构建物以使其达到特定目的的最佳状态。影响陈述在这项研究中，我们开发了一种使用微柱阵列系统限制细胞的新方法。我们的检测方法可洞悉响应机械限制的细胞行为。通过更深入地了解细胞如何感知和响应限制，我们可以对组织工程构造进行微调，以使其达到特定目的的最佳状态。通过将限制与其他物理线索结合起来，我们可以利用机械特性来鼓励或抑制细胞迁移，将细胞引导至特定谱系，