Acoustic tweezing cytometry: technology development and stem cell applications

 描述（由申请人提供）：对细胞外机械信号的机械敏感性是许多发育、生理和病理过程的核心，影响细胞功能，包括生长、迁移、分化和凋亡。理解机械转导过程的分子机制依赖于能够向细胞施加受控机械力以引发和评估细胞反应的工具。 本研究的目标是开发一种新的基于超声的技术，声学镊子细胞术（ATC），作为一个强大的细胞力学和机械生物学工具。我们将进行系统和全面的研究，以开发创新的ATC平台，并表征ATC中用于细胞机械调节的亚细胞力产生，这将对许多实际应用和科学研究产生广泛的影响。 在这项研究中，我们将开发和 证明了ATC作为干细胞应用的新型实用策略的效用，特别是能够在人多能干细胞（hPSC）维持和理解hPSC的机械生物学方面取得新进展。hPSC能够自我复制，同时保留产生任何类型的特化细胞的能力，为疾病建模，药物筛选和未来基于细胞的治疗方法提供了有希望的来源，以治疗退行性疾病，如糖尿病和脊髓损伤。然而，控制hPSC生长仍然具有挑战性，因为目前克隆生长hPSC的方法效率低下，并且对于遗传操作和治疗目的定义不清。hPSC在细胞脱离和解离时易受凋亡影响，解离的单个hPSC的克隆效率通常<1%。因此，我们提出以下研究目标：1）开发一种创新的ATC技术平台，用于应用时空控制的亚细胞机械力; 2）确定ATC对hPSC存活和克隆效率的影响; 3）揭示ATC刺激提高hPSC存活和克隆效率的机制。

 DESCRIPTION (provided by applicant): Mechanosensitivity to extracellular mechanical signals is central to many developmental, physiological, and pathological processes, affecting cell functions including growth, migration, differentiation, and apoptosis. Understanding the molecular mechanisms underlying mechanotransduction process rely on tools capable of applying controlled mechanical forces to cells to elicit and assess cellular responses. The goal of this research is to develop a novel ultrasound-based technology, acoustic tweezing cytometry (ATC), as a powerful cell mechanics and mechanobiology tool. We will perform systematic and comprehensive studies to develop innovative ATC platform and characterize subcellular force generation in ATC for mechanical regulation of cells, which will have broad impact on many practical applications as well as scientific investigations. In this research, we will develop and demonstrate the utility of ATC as a novel and practical strategy for stem cell applications, specifically to enable novel advances in human pluripotent stem cell (hPSC) maintenance and understanding of mechanobiology of hPSCs. Capable of replicating themselves while retaining the ability to give rise to any type of specialized cells, hPSCs provide promising sources for disease modeling, drug screenings, and future cell-based therapeutics to treat degenerative diseases such as diabetes mellitus and spinal cord injury. However, controlling hPSC growth remains challenging because present methods to clonally grow hPSCs are inefficient and poorly defined for genetic manipulation and therapeutic purposes. hPSCs are vulnerable to apoptosis upon cellular detachment and dissociation, with a cloning efficiency of dissociated single hPSCs generally < 1%. Therefore, we propose the following specific aims in this research: 1) to develop an innovative ATC technology platform for applying spatiotemporally controlled subcellular mechanical forces; 2) to determine the effects of ATC on the survival and cloning efficiency of hPSCs; and 3) to reveal the mechanisms of ATC stimulation for improving survival and cloning efficiency of hPSCs.