Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring
Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring
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DOI：
10.1073/pnas.1615375114
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发表时间：
2017-03-07
影响因子：
11.1
通讯作者：
Melosh, Nicholas A.
中科院分区：
文献类型：
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作者：
Cao, Yuhong;Hjort, Martin;Melosh, Nicholas A.
在这里，我们报告了一种对多种细胞类型的细胞内蛋白质和 mRNA 进行时间分辨、纵向提取和定量测量的方法。通过细胞培养基质，在规定的采样区域内加入直径为 150 nm 的中空纳米吸管 (NS)，在超过 5 天的时间内从同一细胞或细胞群中重复采样胞质内容物。提取后，用常规方法分析细胞内容物，包括荧光、酶测定 (ELISA) 和定量实时 PCR。该过程是无损的，采样后细胞活力 > 95%，可进行长期分析。值得注意的是，细胞提取物的测量数量被发现构成了细胞内实际内容物的统计显着代表。从人类诱导多能干细胞 (hiPSC-CM) 衍生的心肌细胞群中分析了 48 条 mRNA 序列，其中 41 条得到了准确定量。 NS 平台从较大培养物中选定的细胞亚群中进行采样，即使在心肌细胞跳动等剧烈活动期间，也可以实现天然细胞间的接触和通信。该平台适用于细胞系和原代细胞，包括 CHO 细胞、hiPSC-CM 和源自 3D 皮质球体的人星形胶质细胞。通过随着时间的推移跟踪相同的细胞或细胞群，该方法提供了了解动态细胞行为的途径，包括诱导多能性和分化等过程。
Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with > 95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stemcells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.