PFI-TT: Throughput Characterization of a Prototype Second Harmonic Generation Flow Cytometer for Stem Cell Derived Cardiomyocyte Purification

该PFI项目更广泛的影响/商业潜力是开发一种纯化干细胞衍生心肌细胞（SC-CMs）的新技术。SC-CMs是一种很有前途的心脏细胞来源，可以在实验室中从干细胞中培养出来，用于解决这个国家面临的重大医疗保健问题。这些细胞有可能用于改造组织，以治疗心脏病/心力衰竭患者，或筛查药物的心脏毒性副作用。然而，在这些细胞用于这些应用之前，必须对这些细胞进行纯化，以从可能影响正常心脏功能的细胞群中去除不需要的细胞。目前，没有成熟的技术可以可靠地进行这种净化。该项目建议开发一种新的光学技术，可以专门识别SC-CMs，并创建一个原型仪器，可以有效地执行这种分析。该项目的长期影响将是通过能够使用这些纯化细胞来治疗心脏病患者并改善药物发现和开发过程，从而改善患者的医疗保健和生活质量。该项目的重点是开发一种原型的二次谐波生成（SHG）流式细胞仪来检测和识别SC-CMs，而不使用外源标记。研究目标和目的是：1)设计并集成用于SHG信号检测的光学系统与流式细胞术中常用的微流控系统；2)优化系统并表征最大理论分析通量；3)评估分析后的细胞活力。基于扫描贝塞尔光束和细长高斯光束，结合微流控电池处理系统，将开发一种光片光学系统。将进行研究，以评估识别SC-CMs的准确性和表征系统的吞吐量。最大吞吐量的理论估计将通过信号强度和流量数据的外推来确定。分析后的细胞活力将使用标准细胞测定法进行评估。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact/commercial potential of this PFI project is the development of a new technology for purifying stem cell derived cardiomyocytes (SC-CMs). SC-CMs are a promising source of heart cells that can be grown in the laboratory from stem cells and used to address significant healthcare issues facing this country. These cells can potentially be used to engineer tissue to treat patients with heart disease/failure, or to screen for cardiotoxic side effects of drugs. Before these cells can be used for these applications, however, the cells must be purified to remove undesired cells from the cell population that may affect normal heart function. Currently, no established technique can reliably perform this purification. The project proposes to develop a new optical technique that can specifically identify SC-CMs and to create a prototype instrument that can efficiently perform this analysis. The long-term impact of this project will be to improve healthcare and quality of life of patients by being able to use these purified cells to treat patients with heart disease and to improve the drug discovery and development process. The proposed project focuses on developing a prototype second harmonic generation (SHG) flow cytometer to detect and identify SC-CMs without the use of exogenous labels. The research goals and objectives are to 1) design and integrate an optical system for SHG signal detection with microfluidic systems commonly used in flow cytometry, 2) optimize the system and characterize the maximum theoretical analytical throughput, and 3) assess cell viability after the analysis. A lightsheet optical system will be developed based on scanned Bessel and elongated Gaussian beams, and combined with microfluidic cell handling systems. Studies will be performed to evaluate the accuracy for identifying SC-CMs and characterizing the throughput of the system. Theoretical estimates of maximum throughput will be determined by extrapolation of signal strength and flow rate data. Cell viability post-analysis will be assessed using standard cell assays.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.