Development and application of an integrated robotic imaging platform for cell culture monitoring and optimization

前言：细胞的特性和通过分化和表观遗传漂移发展的克隆谱系异质性在生物过程和组织工程的核心提出了许多根本性的挑战。迫切需要开发非破坏性的多参数分析技术，这种技术可以沿着跨越多代的轨迹评估细胞动力学。主要目标：我实验室的长期目标是开发生物工程策略，使生物过程和产品能够在单个细胞水平上进行优化和表征。在这种详细程度上，生物工程需要机器人显微镜、单细胞处理和操纵，以及数据分析的信息学方法。我们假设，使用长期高分辨率活细胞成像进行的细胞行为分析将提供足够的措施，使培养控制方案能够指导单个细胞沿着特定的分化轨迹发展。分类和控制方案将仅受成像的空间-时间-光谱分辨率以及动态行为和细胞外观识别的稳健性的限制。一种方法：该项目所开展的工作将涉及用于生物组装的单个细胞的机械和光子操纵的持续开发和优化；以及利用高精度显微镜机器人实现多维亮场和荧光时程成像；制定算法和软件来分析延长时间进程成像过程中产生的多变量数据。现在，这些组件中的每一个都将被集成到机器人成像生物反应器系统中，以实现对数百到数千个细胞的跟踪和分析，其中每个细胞的细节都可以随时间的推移而观察到。新奇的是：高内容多维活细胞成像与信息学和单细胞操纵机器人和光子学的集成代表着生物发现和先进生物过程工程方法发展的一个高度新颖的平台。此外，谱系信息学在细胞分析中的应用代表了培养分析中一个全新的细节水平-培养不是根据大体参数测量进行分析，而是在单细胞尺度上估计和跟踪多代人的参数。这项工作预计，随着细胞用于移植治疗和单细胞分析成为一种要求，将出现制造挑战。因此，拟议的研究计划有可能开发基本的方法和策略，将在细胞治疗和基于细胞的药物制造领域得到广泛应用。

Preamble: Characterization of cells and the development of clonal lineage heterogeneity through differentiation and epigenetic drift poses many fundamental challenges at the heart of bioprocess and tissue engineering. There is an urgent and specific need for the development of nondestructive multi-parameter analytical technologies that can assess cellular dynamics along trajectories that span multiple generations.      Objectives: The long term objective of my laboratory is to develop bioengineering strategies that enables the optimization and characterization of bioprocesses and products at the level of the individual cell.  Bioengineering at this level of detail requires robotic microscopy, single cell handling and manipulation, and an informatics approach to data analysis.  We hypothesized that cellular behavior analysis performed using long-term high resolution live-cell imaging will provide sufficient measures to enable culture control schemes that direct the development of individual cells along specific differentiation trajectories. Classification and control schemes will be limited only by the spatial-temporal-spectral resolution of imaging and the robustness of dynamic behavior and cell appearance identification.    Approach:  The work performed in this project will involve continued development and optimization of mechanical and photonic manipulation of individual cells for bio-assembly;  the implementation of multidimensional bright field and fluorescence time course imaging using high precision microscope robotics; the formulation of algorithms and software to analyze the multivariate data produced during extended time course imaging.  Each of these components will now be integrated into a robotic-imaging bioreactor system to enable the tracking and analysis of hundreds to thousands of cells at resolutions in which details are observable for each cell over time.Novelty:  The integration of high-content multidimensional live-cell imaging with informatics and single cell manipulation robotics and photonics represents a highly novel platform for biological discovery and the development of advanced bioprocess engineering methodologies.  Further, the application of lineage informatics for the analysis of cells represents an entirely new level of detail in culture analysis - cultures are not analyzed in terms of gross parameter measurements, but rather parameters are estimated and tracked at the single cell scale over multiple generations.  This work anticipates manufacturing challenges that will arise as cells are used for transplant therapy and single-cell analysis becomes a requirement.  As such, the proposed research program has the potential to develop fundamental methods and strategies that will find wide application in the areas of cell therapy and cell-based pharmaceutical manufacturing.