Salari Alinaghi, Appak-Baskoy Sila, Coe Imogen R, Tsai Scott S H, Kolios Michael C
Institute for Biomedical Engineering, Science and Technology (iBEST) Toronto ON M5B 1T8 Canada.
Biomedical Engineering Graduate Program, Ryerson University Toronto ON M5B 2K3 Canada.
RSC Adv. 2021 Oct 5;11(52):32824-32829. doi: 10.1039/d1ra04875a. eCollection 2021 Oct 4.
Adherent cultured cells are widely used biological tools for a variety of biochemical and biotechnology applications, including drug screening and gene expression analysis. One critical step in culturing adherent cells is the dissociation of cell monolayers into single-cell suspensions. Different enzymatic and non-enzymatic methods have been proposed for this purpose. Trypsinization, the most common enzymatic method for dislodging adhered cells, can be detrimental to cells, as it can damage cell membranes and ultimately cause cell death. Additionally, all available techniques require a prolonged treatment duration, typically on the order of minutes (5-10 min). Dissociation of cells becomes even more challenging in microfluidic devices, where, due to the nature of low Reynolds number flow and reduced mixing efficiency, multiple washing steps and prolonged trypsinization may be necessary to treat all cells. Here, we report a novel acoustofluidic method for the detachment of cells adhered onto a microchannel surface without exposing the cells to any enzymatic or non-enzymatic chemicals. This method enables a rapid (, on the order of seconds), cost-effective, and easy-to-operate cell detachment strategy, yielding a detachment efficiency of ∼99% and cellular viability similar to that of the conventional trypsinization method. Also, as opposed to biochemical-based techniques (, enzymatic), in our approach, cells are exposed to the dissociating agent (, substrate-mediated acoustic excitation and microstreaming flow) only for as long as they remain attached to the substrate. After dissociation, the effect of acoustic excitation is reduced to microstreaming flow, therefore, minimizing unwanted effects of the dissociating agent on the cell phenotype. Additionally, our results suggest that cell excitation at acoustic powers lower than that required for complete cell detachment can potentially be employed for probing the adhesion strength of cell-substrate attachment. This novel approach can, therefore, be used for a wide range of lab-on-a-chip applications.
贴壁培养细胞是广泛应用于各种生物化学和生物技术领域的生物工具，包括药物筛选和基因表达分析。培养贴壁细胞的一个关键步骤是将细胞单层解离成单细胞悬液。为此已提出了不同的酶解和非酶解方法。胰蛋白酶消化法是最常用的使贴壁细胞脱离的酶解方法，但它可能对细胞有害，因为它会损伤细胞膜并最终导致细胞死亡。此外，所有现有技术都需要较长的处理时间，通常在几分钟（5 - 10分钟）左右。在微流控设备中，细胞解离变得更具挑战性，由于低雷诺数流动的性质和混合效率降低，可能需要多个洗涤步骤和长时间的胰蛋白酶消化才能处理所有细胞。在此，我们报告了一种新型的声流控方法，用于在不使细胞暴露于任何酶或非酶化学物质的情况下，使附着在微通道表面的细胞脱离。该方法实现了一种快速（约几秒）、经济高效且易于操作的细胞脱离策略，产生的脱离效率约为99%，细胞活力与传统胰蛋白酶消化法相似。而且，与基于生化的技术（如酶解技术）不同，在我们的方法中，细胞仅在附着于底物时才暴露于解离剂（即底物介导的声激发和微流）。解离后，声激发的影响降低为微流，因此，将解离剂对细胞表型的不良影响降至最低。此外，我们的结果表明，在低于完全细胞脱离所需声功率的条件下对细胞进行激发，有可能用于探测细胞 - 底物附着的粘附强度。因此，这种新方法可用于广泛的芯片实验室应用。
