Monolithic hydrogel nanowells-in-microwells enabling simultaneous single cell secretion and phenotype analysis,Lab on a Chip

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Monolithic hydrogel nanowells-in-microwells enabling simultaneous single cell secretion and phenotype analysis
Lab on a Chip ( IF 6.1 ) Pub Date : 2020-11-17 , DOI: 10.1039/d0lc00965b
Jane Ru Choi ₁ , Jeong Hyun Lee ₁ , Alec Xu ₁ , Kerryn Matthews ₁ , Shuyong Xie ₁ , Simon P Duffy ₂ , Hongshen Ma ₃

Affiliation

Cytokine secretion is a form of cellular communication that regulates a wide range of biological processes. A common approach for measuring cytokine secretion from single cells is to confine individual cells in arrays of nanoliter wells (nanowells) fabricated using polydimethylsiloxane. However, this approach cannot be easily integrated in standard microwell plates in order to take advantage of high-throughput infrastructure for automated and multiplexed analysis. Here, we used laser micropatterning to fabricate monolithic hydrogel nanowells inside wells in a microwell plate (microwells) using polyethylene glycol diacrylate (PEGDA). This approach produces high-aspect ratio nanowells that retain cells and beads during reagent exchange, enabling simultaneous profiling of single cell secretion and phenotyping via immunostaining. To limit contamination between nanowells, we used methylcellulose as a media additive to reduce diffusion distance. Patterning nanowells monolithically in microwells also dramatically increases density, providing ∼1200 nanowells per microwell in a microwell plate. Using this approach, we profiled IL-8 secretion from single MDA-MB-231 cells, which showed significant heterogeneity. We further profiled the polarization of THP-1 cells into M1 and M2 macrophages, along with their associated IL-1β and CCL-22 secretion profiles. These results demonstrate the potential to use this approach for high-throughput secretion and phenotype analysis on single cells.

中文翻译：

单块水凝胶纳米孔-微孔可同时进行单细胞分泌和表型分析

细胞因子的分泌是调节广泛的生物学过程的细胞通讯形式。测量单个细胞中细胞因子分泌的常用方法是将单个细胞限制在使用聚二甲基硅氧烷制成的纳升孔（纳孔）阵列中。但是，为了利用高通量基础设施进行自动化和多重分析，这种方法不能轻松地集成到标准微孔板中。在这里，我们使用激光微图案化技术，使用聚乙二醇二丙烯酸酯（PEGDA）在微孔板（微孔）中的孔内制造了整体式水凝胶纳米孔。这种方法可产生高纵横比的纳米孔，在试剂交换过程中可保留细胞和珠子，从而可同时分析单个细胞的分泌情况并通过免疫染色。为了限制纳米孔之间的污染，我们使用甲基纤维素作为介质添加剂来减少扩散距离。在微孔中单片化纳米孔的图案也显着增加了密度，在微孔板中每微孔提供约1200纳孔。使用这种方法，我们分析了单个MDA-MB-231细胞的IL-8分泌，显示出明显的异质性。我们进一步分析了THP-1细胞极化成M1和M2巨噬细胞，以及它们相关的IL-1β和CCL-22分泌特征。这些结果证明了使用这种方法进行单细胞高通量分泌和表型分析的潜力。

更新日期：2020-11-18

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