Validation of acoustic tweezers for single-cell analyses of purine metabolism

 描述：缺乏同时实现高吞吐量、高精度和高细胞完整性的单细胞处理技术是细胞间通信研究的主要障碍。最近，我们的跨学科团队开发了一种基于表面声波(SAW)的微流控平台，称为“声波镊子”，与现有的用于单细胞分析的细胞操纵技术相比，具有显著的优势。我们的声镊子平台能够以亚微米精度调节单个细胞之间的距离。此外，它具有高度可伸缩性，能够为高通量研究创建大量细胞排列。细胞不需要标记，可以在其自然培养基中培养。此外，用于操纵细胞的声功率和频率与用于超声成像的声功率和频率在相同的范围内，这已被证明具有高度的生物兼容性。最后，产生声表面波所需的部件体积小，价格便宜，而且设备本身易于操作。凭借这些优势，声波镊子具有开创性的能力，能够以高吞吐量的方式在单细胞水平上提供对细胞内通信的精确时空控制，同时保持细胞的完整性。通过可视化荧光染料在细胞间的转移，声波镊子在几个同型和异型细胞群体中通过缝隙连接介导的细胞间功能通讯的研究已经证明了其转化潜力。我们在这个项目中的目标是进行声波镊子的高级开发，并在细胞间通信对细胞内代谢途径的影响的研究中验证它们。因此，我们将追求以下具体目标：(1)先进的声波镊子的发展，用于在单细胞水平上高产量、高通量地表征细胞间通信和嘌呤小体组装；(2)使用声波镊子对初级细胞模型中的嘌呤小体组装进行多参数研究；以及(3)使用声波镊子对神经元模型中的嘌呤小体组装和嘌呤代谢进行单细胞分析。在拟议的项目完成后，我们希望以Lesch-Nyhan病(LND)为疾病模型，以嘌呤小体为代谢状态的指示器，揭示基因如何影响复杂表型的机制。由于其独特的能力，可以高通量地创建具有指定结构的多细胞组件，我们预计声波镊子将成为单细胞分析的无价工具，并将满足生物工程、生物医学和制药研究领域的许多未得到满足的需求。

 DESCRIPTION: The lack of a single-cell manipulation technique that can simultaneously achieve high throughput, high precision, and high cell integrity is a major roadblock for studies of intercellular communication. Recently, our interdisciplinary team has developed a surface acoustic wave (SAW)-based microfluidic platform called "acoustic tweezers" that possesses significant advantages over existing cell-manipulation techniques for single-cell analysis. Our acoustic tweezers platform is able to modulate the distances between individual cells with sub-micron precision. In addition, it is highly scalable and capable of creating a large array of celluar arrangements for high-throughput studies. Cells do not need to be labelled and can be cultured in their native media. Furthermore, the acoustic power and frequency used to manipulate cells are in the same range as those used in ultrasonic imaging, which has proven to be highly biocompatible. Finally, the components required for SAW generation are small and inexpensive, and the device itself is easy to operate. With these advantages, the acoustic tweezers are groundbreaking in their ability to provide precise spatiotemporal control of intracellular communication at the single-cell level in a high-throughput manner while preserving cell integrity. The transformative potential of acoustic tweezers has already been demonstrated in studies on gap junction-mediated functional intercellular communication in several homotypic and heterotypic cell populations by visualizing the transfer of fluorescent dyes between cells. Our objective in this project is to conduct advanced development of acoustic tweezers and validate them in studies on the effects of intercellular communication on metabolic pathways within the cell. We will, therefore, pursue the following specific aims: (1) advanced development of acoustic tweezers for high-yield, high-throughput characterization of intercellular communication and purinosome assembly at the single-cell level; (2) multi-parametric investigation of purinosome assembly in a primary cell model using acoustic tweezers; and (3) single-cell analyses of purinosome assembly and purine metabolism in a neuronal model using acoustic tweezers. At the completion of the proposed project, we hope to uncover the mechanism for how a genotype affects complex phenotype using Lesch-Nyhan disease (LND) as the disease model and purinosome as an indicator of metabolic state. Due to its unique ability to create multicellular assemblies with prescribed architectures in high throughput, we expect that the acoustic tweezers will become an invaluable tool for single-cell analysis and will fulfill many unmet needs in the bioengineering, biomedical, and pharmaceutical research communities.