基于细胞内压的精准显微操作与测量方法研究

Cell inner pressure has significant influences on cell manipulation and cell measurement results. This project is aimed to the cell inner pressure-based precise cell manipulation and measurement techniques research. We will develop an intracellular balanced pressure model based-inner pressure measurement method. In this method, the inner pressure is measured through precise estimation of capillary force and feedback of injection pressure when liquid in micropipette has entered into a balanced state. At present, cell injection is the most popular cell manipulation technique and the micropipette aspiration is the most widely used cell elastic modulus measurement method. The obtained cell inner pressure is utilized to perform inner pressure-based dynamic cell quantitative injection and cell elastic modulus measurement. Firstly, dynamic modeling of cell injection process is performed to decouple the influences of inner pressure and injection pressure on the injection volume. With online-measured cell inner pressure, the injection pressure is adjusted to according to the model to compensate the injection volume control error resulting from the inner pressure, and achieve quantitative injection volume control between cells with different inner pressures. Secondly, the cell deformation resulted from cell inner pressure and aspiration pressure are decoupled using the finite element modeling of cell in micropipette aspiration. The cell deformation generated by inner pressure is excluded and real cell elastic modulus is measured. Finally, the quantitative drug is injected to two types of cardiomyocytes with different inner pressures to evaluate their response to drugs. The inner pressure and ZP Young’s modulus of oocyte in different development stages are measured to study the influences of them on cell development. The above two applications are performed to validate the effectiveness and applicability of the research in this project.

细胞内压对细胞操作和测量影响很大，本项目旨在探索基于内压的精准细胞操作和测量方法。本项目将研究一种基于胞内平衡压模型的细胞内压测量方法。该方法在针管液位平衡状态下，通过对毛细力的精确测量和注射压的精确反馈实现细胞内压的精确测量。在此基础上，针对细胞定量注射和微管吸持两种典型操作和细胞弹性测量方法，建立内压条件下的细胞动态定量注射和弹性测量方法。首先在已知细胞内压的情况下，通过注射过程动态建模解耦注射压和内压对注射量的影响，调整注射压消除内压造成的注射量误差，实现不同内压的细胞定量注射。进一步的，在已知内压的情况下，通过微管吸持细胞的有限元仿真解耦内压和吸持压产生的形变量。进而将内压产生的形变量去除，实现真实细胞弹性的测量。最后，我们对不同内压的心肌细胞定量注射药物测量其药性，测量不同卵母细胞发育阶段内压和弹性变化趋势。通过这两项示范应用来验证本项目方法的有效性和潜在的应用价值。

细胞内压是细胞内物理环境的重要组成部分，对细胞弹性的测量结果和胞内显微注射的注射量具有显著影响。测量细胞内压并量化其对上述细胞显微操作与检测结果的影响，对提高细胞弹性测量和胞内定量显微注射的精度具有重要意义。然而当前的细胞内压测量方法需要依赖专门的昂贵仪器且对细胞的伤害较大，严重限制了其推广应用。本项目在常规的细胞显微操作与电阻检测系统上分别开发了基于胞内平衡压模型的细胞内压测量方法和基于注射电路模型的细胞内压测量方法，单个细胞的平均测量时间控制在20s和35s以内,实现了简易快速的自动化细胞内压测量。尤其后者通过高分辨率的电阻检测和注射压强控制可以实现Pa级分辨率的细胞内压检测。在此基础上通过对胞内注射过程进行受力分析，确定了注射压、细胞内压与注射量的对应关系，并基于油中注水法完成了细胞内压条件下的注射实验建模，根据细胞的平均内压基于实验模型调整细胞注射压，将内压释放前后的家猪卵母细胞的注射量平均相对误差控制在2.79%，优于5%的计划书研究目标，实现了不同内压细胞之间的定量注射。进一步的，本项目通过对微管吸持过程进行受力分析和有限元建模，量化了细胞-微管摩擦系数、管口尺寸以及细胞内压对细胞弹性测量结果的影响，并据此对测得细胞内压进行矫正，通过细胞内压释放前后的微管吸持测量结果进行对比，将内压条件下的细胞弹性测量误差降低到3.24%，优于5%的计划书研究目标。最后本项目对成熟前后的卵母细胞进行了内压测量，未检测到显著变化。本项目发表SCI源期刊论文10篇，包括IEEE Trans论文4篇，中科院院刊论文1篇。EI源期刊《机器人》杂志论文1篇；EI会议论文3篇，其中会议最佳论文决赛奖1篇（ROBIO2021）。授权中国发明专利5项，受理发明专利4项。协助培养博士研究生2名，硕士研究生6人。