Collaborative Research: Investigation of Wound-healing at the Single Cell Level using Microfluidics-based Microsurgery

伤口愈合是包括人类在内的所有生物生存的关键。这也是区分生物和非生物的关键特征。由于细胞的体积很小，一个关键的挑战是缺乏一种能够以系统和可扩展的方式缠绕细胞的工具。该项目的目标是采用一种新的微工程平台，并建立损伤细胞和测量愈合效率的标准化程序。这一平台使得研究伤口愈合的分子和机械机制成为可能。这个项目的更广泛的意义和重要性是理解细胞修复伤口和恢复其正常生存功能的方式。它将深入了解细胞如何知道自己受到了伤害，它使用什么机制来启动修复过程，以及它如何知道修复何时完成。这项工作的更广泛的影响包括研究生暑期课程，K-12学生的外展活动，以及旧金山湾区科学节上面向普通公众的外展活动。伤口愈合对于维持动态平衡至关重要，最终也是生存的关键。细胞，如骨骼肌，在生理条件下会定期受伤。在单细胞水平上了解创伤反应对于确定细胞修复和生存所需的基本细胞功能至关重要。回答这些问题的关键障碍是缺乏一种可以将伤口复制到大量细胞中的工具，以及衡量愈合效率的定量测试。该项目的目标是通过使用微流控平台，并以蓝斑肌作为模式生物，研究单细胞伤口愈合的机制。蓝柱肌是一种能够在24小时内从剧烈创伤中恢复的单细胞，由于其强大的伤口愈合能力和高通量执行基因敲除实验的能力而被选为模型。该项目的目标是：1)使用连续流动微流控分离器以可重复性的方式建立创伤细胞的标准检测方法，并量化细胞的愈合效率；2)通过检测愈合过程中的基因表达来研究创伤愈合的分子途径；3)描述愈合过程中细胞机械性能的变化。该项目的意义和智力价值在于阐明了单细胞创伤愈合的分子机制以及细胞物理和机械性能的相应变化。该项目的变革潜力是微流体的新应用，以可重复的方式在单细胞水平上产生可控伤口。拥有细胞生物学专业知识的马歇尔实验室和拥有微流体专业知识的唐实验室之间的合作创造了独特的机会，可以解决传统单一学科项目中无法解决的工程学和生命科学交界处的高影响力问题。这项研究的更广泛的影响是开发了一个强大的实验模型，用于了解单细胞如何修复和维持动态平衡。该项目将确定触发修复反应的因素，以及单个细胞用于修复和恢复正常细胞功能的分子和物理过程的级联。此外，这项工作将有助于阐明愈合如何与其他重要的细胞功能有关，如形态发生，以及单细胞水平和组织水平的愈合是如何联系和协调的。

Wound healing is critical for survival in all living things, including humans. It is also the key feature that distinguishes living from non-living matter. Due to the small size of a cell, a key challenge is the lack of a tool that can wound cells in a systematic and scalable manner. The goal of this project is to employ a new micro-engineering platform and to establish a standardized procedure for wounding cells and for measuring the healing efficiency. This platform enables the study of the molecular and mechanical mechanisms that underlie wound healing. The broader significance and importance of this project is the understanding of the way cell repairs wounds and restores its normal function for survival. It will provide insights into how a cell knows that it has been injured, what mechanisms it uses to start the healing process, and how it knows when healing is complete. The Broader Impacts of this work include a graduate summer course, outreach activities for K-12 students, and outreach activities for the general public at the Bay Area Science Festival.Wound healing is essential for maintaining homeostasis and, ultimately, for survival. Cells, such as skeletal muscles, are wounded regularly under physiological conditions. Understanding wound response at the single-cell level is critical for determining fundamental cellular functions needed for cell repair and survival. Key barriers to answering these questions are the lack of a tool that can introduce wounds reproducibly to a large number of cells and a quantitative assay to measure healing efficiency. The goal of this project is to investigate the mechanisms underlying single-cell wound healing, by employing a microfluidic platform and using Stentor coeruleus as a model organism. Stentor coeruleus, a single cell capable of recovering from drastic wounds within 24 hours, is selected as a model because of its robust wound healing capacity, and the ability to perform gene knockdown experiments in a high throughput manner. The project objectives are to: 1) establish standard assays to wound cells in a reproducible manner using a continuous-flow microfluidic splitter, and to quantify the healing efficiency of cells, 2) investigate the molecular pathway of wound healing by examining gene expression during the healing process, and 3) profile the changes in the mechanical properties of the cell during the healing process. The significance and intellectual merit of this project lies in the elucidation of the molecular mechanisms underlying single-cell wound healing and the corresponding changes in the physical and mechanical properties of the cell. The transformative potential of this project is the novel application of microfluidics to generate controllable wounds at the single-cell level in a reproducible manner. The collaboration between Marshall Lab with expertise in cell biology and Tang Lab with expertise in microfluidics creates unique opportunities to solve high-impact problems at the interface of engineering and life sciences not possible in a traditional single-discipline project. The broader impact of this research is development of a powerful experimental model for understanding how single cells repair and maintain homeostasis. The project will identify factors that trigger the initiation of the healing response, and the cascade of molecular and physical processes a single cell employs to heal and restore normal cell function. Furthermore, the work will enable the elucidation of how healing relates to other important cell functions such as morphogenesis, as well as how single cell-level and tissue-level healing are related and coordinated.