Demonstration of repeated Positionally Assisted Negative particle Rejection for High-Speed Sorting

项目摘要 细胞和多细胞构建体的分选和富集对于生物医学研究的许多领域是有价值的 并且对于细胞疗法越来越重要。富集过程使用物理性质来执行 细胞的高速分离。然而，由于大多数细胞的物理相似性，这些方法不 提供高纯度产物。因此，需要基于多个参数的分选来获得纯细胞 人口。荧光激活细胞分选（FACS）可以基于> 20种细胞标志物分离细胞， 对于稀有细胞群体的分选非常有用。不幸的是，FACS是一个缓慢、低容量的过程。因此，在本发明中， 需要多种标记物来区分的稀有细胞群体或大量细胞的分离仍然是一个难题。 这是一个具有挑战性、耗时且昂贵的过程，需要多个步骤，每个步骤都容易导致细胞损失。 因此，我们设计了一种自动分拣过程，该过程结合了 流式细胞仪具有高速和高体积比的物理分离技术。我们的过程，称为 位置辅助负粒子排斥（PANR），使用声驻波来定位细胞 用于流式细胞仪分析和下游流体学来分选细胞。rPANR后的分选样本未稀释 通过鞘或其它流体，因此样品可以通过任何细胞浓缩技术快速收集。因为 声学流式细胞术提供的高体积速率和实施重复循环的简单性 PANR，rPANR提供了一种快速温和地分选细胞的途径。此外，rPANR系统将可用于 直径为1至1000 µ m的颗粒，这将使其适用于稀有细胞和大型细胞构建体。这 该项目将展示rPANR过程，以创建一个清晰的商业rPANR开发途径 在不到1小时内从109个细胞/ml中分选出少至10个细胞的仪器。此外，流速> 10 mL/min 将能够高速富集显示多个标记的细胞。最后，它将能够将粒子分类， 以超过10，000/s的速度将直径减小到1 mm。这些功能使rPANR过程的演示 这是创造一种新工具的第一步，这种工具将对生物医学研究、药物发现和 这是一个非常有用的细胞治疗工具。

Project Summary Sorting and enrichment of cells and multicellular constructs is valuable to many areas of biomedical research and is of growing importance for cellular therapies. Enrichment processes use physical properties to perform high-speed separation of cells. However, due to the physical similarity of most cells, these approaches do not provide a high purity product. Therefore, sorting based on multiple parameters is needed to obtain pure cell populations. Fluorescence activated cell sorting (FACS) can separate cells based on >20 cellular markers and is very useful for sorting of rare cell populations. Unfortunately, FACS is a slow, low volume process. Thus, isolation of rare cell populations or large numbers of cells that require multiple markers to distinguish remains a challenging, time consuming, and expensive process that requires multiple steps that are each prone to cell loss. Therefore, we have designed an automated sorting process that combines the sensitive multicolor detection of FACS with the high speed and high volumetric rate of physical separation technologies. Our process, termed Positionally Assisted Negative particle Rejection (PANR), uses an acoustic standing wave to position the cells for flow cytometry analysis and downstream fluidics to sortthe cells. The sorted sample after rPANR is not diluted by sheath or other fluids so sample can be rapidly collected via any cell concentration technology. Because of the high volumetric rate that acoustic flow cytometry offers and the simplicity of implementing repeated cycles of PANR, rPANR offers a pathway to rapidly and gently sort cells. In addition, the rPANR system will be usable with particles from 1 to 1000 µm in diameter, which will make it useful for rare cells and large cellular constructs. This project will demonstrate the rPANR process to create a clear development pathway to a commercial rPANR instrument that sorts as few as 10 cells from a 109 cells/ml in less < 1hr. Furthermore, flow rates of >10 mL/min will enable high-speed enrichment of cells displaying multiple markers. Finally, it will be able to sort particles up to 1 mm in diameter at rates of over 10,000/s. These features make demonstration of the rPANR process the first step in the creation of a new tool that will be of great value to biomedical research, pharma discovery, and, in time, a very useful tool for cellular therapeutics.