New Concepts in Fluidics and Cellular Mechanics for Controlled Microinjection

CBET-0828733 Fan显微注射是一种广泛接受的方法，用于将精子、细胞核或溶质等物质引入生物细胞，用于不育治疗、转基因和治疗性克隆或冷冻保存目的。仅在美国，每年就进行数百万次用于此类应用和相关生物医学研究的细胞显微注射操作。最常用的显微注射过程被称为卵胞浆内精子注射，作为体外受精操作的一部分。该过程包括微量吸管穿透卵细胞的外围，包括透明细胞和质膜，然后运送精子进行受精。迄今为止，即使在压电辅助细胞穿刺装置的帮助下，显微注射过程的产率也不理想地低，因为细胞在穿透和物质递送期间容易受损，并且随后引起异常生长。此外，手术的成功在很大程度上取决于操作者的技能。由于操作条件是以特定的方式选择的，因此在不同的实验室或不同的细胞中，结果通常是不可重现的。为了弥补这些不足，PiIs团队最近开发了一种称为旋转振荡钻的技术，该技术通过微型电机以非常小的角度冲程旋转移液管（例如，0.2度）和高频（例如，500 Hz）。在PI小组的初步研究中，该操作被证明是非常有效的。然而，如果不对涉及微量移液器和流体环境中的软生物细胞之间的小规模相互作用的复杂的显微注射过程有基本的了解，就无法实现这一总体目标。PI的目的是解决显微注射过程中的瞬态微机械界面动力学，并监测卵母细胞膜的流变学特性。这种基本的理解将为确定理想的操作条件奠定科学基础，而无需进行冗长的试错研究。 该研究有两个科学重点：（i）模拟生物细胞被高度灵活的微量移液管刺穿时的微观动力学。(ii)基于从（i）中获得的见解，为我们的Ros-Drill显微注射工艺开发最佳控制方案。PI将强调对小尺度和频率依赖的材料特性，流体-移液管-膜相互作用以及纳米级电容力传感设备的集成的基本理解。这些关键的知识将可能消除对人类经验的依赖。如果成功，这项拟议中的研究将为细胞生物学家引入一个革命性的工具。这种计算机控制的微注射技术（Ros-Drill）将具有很大的潜力，以减少人为因素造成的不确定性和误差。它将提高成功率（注射），减少对各种物种的试验次数（人工授精、细胞手术、治疗性克隆的基因或药物输送）。该系统还将集成微尺度力和位置传感装置，通过这些装置，实验生物学家将直接受益于新兴的微机电系统技术。除了这些基本的科学影响外，PI计划立即在专业期刊和会议上传播研究结果。PI还将扩大K-12教育的一些正在进行的外展活动，我们每年都积极参与这些活动。

CBET-0828733FanMicroinjection is a well-accepted method to introduce matter such as sperm, nucleus, or solutes into biological cells for infertility treatment, transgenic and therapeutic cloning, or cryopreservation purposes. Millions of cellular microinjection operations for such applications and relevant biomedical research are conducted in the United States alone every year. The most utilized microinjection process is known as intracytoplasmic sperm injection as a part of the in vitro fertilization operation. The procedure consists of micropipette penetration through the egg cell's periphery including the zona pellucida and the plasma membrane, followed by the delivery of sperm for fertilization. To date, even with the help of piezo-assisted cell piercing device, the yield of microinjection process is undesirably low because the cells are easily damaged during the penetration and substance delivery, and subsequently causing abnormal growth. Furthermore, the success of the operation strongly depends on the operator's skill. Since the operating conditions are selected in an ad-hoc fashion, the results are often irreproducible at different laboratories or for different cells. To remedy these shortfalls, the PiIs team has recently developed a technology called the Rotationally Oscillating Drill which rotates the pipette via a micro-motor at very small angular strokes (e.g., 0.2 degrees) and at high frequencies (e.g., 500 Hz.). This operation is proven to be quite effective in the preliminary studies of the PI's group. This overarching goal cannot be achieved, however, without basic understanding of the complex microinjection process involving small-scale interactions between micropipettes and the soft biological cells in a fluid environment. The PI's objective is to resolve the transient micro-mechanical interfacial dynamics of the microinjection process and to monitor the rheological properties of the membrane of the oocytes. This fundamental understanding will establish scientific basis in determining the ideal operating conditions without lengthy trial-and-error studies. The research has two scientific thrusts: (i) Modeling the micro-dynamics while biological cells are pierced by highly flexible micropipettes. (ii) Developing optimal control protocols for our Ros-Drill microinjection process based on the insight gained from (i). The PIs will emphasize the basic understanding of small-scale and frequency-dependent material properties, fluid-pipette-membrane interactions, and the integration of nanoscopic capacitive force sensing devices. These crucial knowledge will potentially remove the dependence on human experience.If successful, the proposed study will introduce a revolutionary tool for cell biologists. This computer controlled microinjection technology (Ros-Drill) will have great potential to reduce the uncertainty and errors caused by human factors. It will increase the success rate (in injection) and reduce the number of tests on various species involved (in artificial insemination, cell surgery, and gene or drug delivery for therapeutic cloning purposes). The system will also integrate microscale force and position sensing devices, through which the experimental biologists will directly benefit from the emerging microelectromechanical systems technology. In addition to such fundamental scientific impact, the PIs plan on immediate dissemination of the findings in professional journals and at conferences. The PIs will also expand on a number of on-going outreach activities in K-12 education, in which we actively participate every year.