Continuous Sorting of Cells Based on Differential P Selectin Glycoprotein Ligand Expression Using Molecular Adhesion.
Continuous Sorting of Cells Based on Differential P Selectin Glycoprotein Ligand Expression Using Molecular Adhesion.
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基于差异 P 选择蛋白糖蛋白配体表达的细胞连续分选，利用分子粘附。
DOI：
10.1021/acs.analchem.7b02878
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发表时间：
2017
影响因子：
7.4
通讯作者：
Sulchek,Todd
中科院分区：
文献类型：
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作者：
Tasadduq,Bushra;McFarland,Brynn;Islam,Muhymin;Alexeev,Alexander;Sarioglu,AFatih;Sulchek,Todd
细胞表面分子粘附控制许多重要的生理过程，并用于识别细胞以进行分析和纯化。但最有效的细胞粘附分离技术在其应用中使用标签或长期附着物。虽然无标记分离微系统通常根据大小、硬度和形状来分离细胞，但它们通常不能为可从分子表达获得的细胞类型提供足够的特异性。我们展示了一种无标记微流体方法，能够基于表面分子粘附进行高通量细胞分离。细胞流过微通道，该微通道在通道顶部设计有成角度的脊，并涂有针对靶细胞受体的特异性粘附配体。脊稍微压缩经过的细胞，使得可以以足够的表面积进行粘合接触，而不会因为细胞刚度而不适当地影响细胞轨迹。因此，分选对细胞粘附敏感，但对硬度或细胞大小不敏感。细胞和脊之间的强制相互作用确保可以使用高流速，而不会出现升力淬灭粘附的情况。作为该方法原理的证明，我们通过使用涂有 P 选择素的脊状通道，根据 PSGL-1 配体的差异表达来分离 Jurkat 和 HL60 细胞系。我们证明了 PSGL-1 阳性的 Jurkat 和 HL60 细胞分别富集了 26 倍和 3.8 倍，PSGL-1 阴性的 Jurkat 和 HL60 细胞分别富集了 4.4 倍和 3.2 倍。将出口数量增加到 5 个可以提高 PSGL-1 选择的分辨率，从而将单一细胞类型分级为具有高、中和低 PSGL-1 表达的细胞亚群。细胞的流动速度高达 0.2 m/s，相当于设计几何结构下每分钟 0.45 万个细胞，这比之前基于粘合剂的分选方法高出 2 个数量级以上。由于细胞与粘附表面的相互作用时间短，分选方法不会因分子结合而进一步激活细胞。这种方法可用于无标记选择细胞以获得高表达的分子表型。
Cell surface molecular adhesions govern many important physiological processes and are used to identify cells for analysis and purifications. But most effective cell adhesion separation technologies use labels or long-term attachments in their application. While label-free separation microsystems typically separate cells by size, stiffness, and shape, they often do not provide sufficient specificity to cell type that can be obtained from molecular expression. We demonstrate a label-free microfluidic approach capable of high throughput separation of cells based upon surface molecule adhesion. Cells are flowed through a microchannel designed with angled ridges at the top of the channel and coated with adhesive ligands specific to target cell receptors. The ridges slightly compress passing cells such that adhesive contact can be made with sufficient surface area without unduly affecting cell trajectories because of cell stiffness. Thus, sorting is sensitive to cell adhesion but not to stiffness or cell size. The enforced interactions between the cells and the ridges ensure that a high flow rate can be used without lift forces quenching adhesion. As a proof of principle of the method, we separate both Jurkat and HL60 cell lines based on their differential expression of PSGL-1 ligand by using a ridged channel coated with P selectin. We demonstrate 26-fold and 3.8-fold enrichment of PSGL-1 positive and 4.4-fold and 3.2-fold enrichment of PSGL-1 negative Jurkat and HL60 cells, respectively. Increasing the number of outlets to five allows for greater resolution in PSGL-1 selection resulting in fractionation of a single cell type into subpopulations of cells with high, moderate, and low PSGL-1 expression. The cells can flow at a rate of up to 0.2 m/s, which corresponds to 0.045 million cells per minute at the designed geometry, which is over 2 orders of magnitude higher than previous adhesive-based sorting approaches. Because of the short interaction time of the cells with the adhesive surfaces, the sorting method does not further activate the cells due to molecular binding. Such an approach may find use in label-free selection of cells for a highly expressed molecular phenotype.