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

用于高速分选的重复位置辅助负粒子剔除演示

• 批准号: 10081332
• 负责人: STEVEN W GRAVES
• 金额: $ 24.74万
• 依托单位: BENNUBIO INC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10081332
• 关键词: 3D Print; Acoustics; Algorithms; Antibodies; Area; Biological Models; Biomedical Research; Caliber; Cell Separation; Cell Therapy; Cells; Cellular Spheroids; Centrifugation; Clinic; Collection; Computer software; Consumption; Detection; Development; Flow Cytometry; Fluorescence-Activated Cell Sorting; Information Systems; Liquid substance; Magnetism; Mechanics; Microspheres; Modeling; Modification; Optics; Pathway interactions; Population; Positioning Attribute; Process; Reaction Time; Sampling; Sorting - Cell Movement; Speed; Stream; System; Target Populations; Technology; Therapeutic; Time; base; data acquisition; design; instrument; interest; millisecond; novel strategies; particle; physical property; physical separation; tool

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.

项目概要 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都很有价值 并且对于细胞疗法越来越重要。富集过程利用物理特性来执行 细胞的高速分离。然而，由于大多数细胞的物理相似性，这些方法并不 提供高纯度的产品。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) 可以根据 >20 种细胞标记物分离细胞， 对于稀有细胞群的分选非常有用。不幸的是，FACS 是一个缓慢、低产量的过程。因此， 需要多种标记来区分的稀有细胞群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化分选流程，结合了敏感的多色检测 FACS具有高速、高容积率的物理分离技术。我们的流程称为 位置辅助负粒子抑制 (PANR)，使用声学驻波来定位细胞 用于流式细胞术分析和下游流体学以对细胞进行分类。 rPANR后分选的样品未稀释 通过鞘液或其他液体，可以通过任何细胞浓缩技术快速收集样品。由于 声学流式细胞术提供的高体积速率以及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 系统将可与 直径为 1 至 1000 µm 的颗粒，这使其可用于稀有细胞和大型细胞结构。这 项目将展示 rPANR 流程，为商业化 rPANR 创建清晰的开发路径 该仪器可在不到 1 小时内从 109 个细胞/ml 中分选少至 10 个细胞。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，它将能够对粒子进行排序 直径可达 1 毫米，速率超过 10,000/s。这些特征使得 rPANR 过程的演示成为 创建新工具的第一步，该工具将对生物医学研究、药物发现具有巨大价值， 随着时间的推移，它成为细胞治疗的一个非常有用的工具。