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

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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.