FACSGuard - An additive for improving flow cytometry cell yield

FACSGuard - 一种用于提高流式细胞术细胞产量的添加剂

• 批准号: 8249704
• 负责人: Robert G Van Buskirk
• 金额: $ 17万
• 依托单位: CELL PRESERVATION SERVICES, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8249704
• 关键词: Address; Applied Research; Area; Atrial Fibrillation; Basic Science; Biological Assay; Biological Sciences; Biology; Biomedical Engineering; Buffers; Cancer cell line; Cell Culture Techniques; Cell Separation; Cell Survival; Cell Therapy; Cell Viability Process; Cell physiology; Cell surface; Cells; Cellular Stress; Centrifugation; Cytolysis; Detection; Development; Devices; Diagnostic; Digestion; Disease; Dissociation; Dyes; Engineering; Flow Cytometry; Generations; Generic Drugs; Guava Fruit; Heart Atrium; Hepatocyte; Human; Investigation; Laboratories; Lead; Liquid substance; Liver; Medical Device; Methods; Microarray Analysis; Molecular; Molecular Analysis; Nitrogen; Outcome; Pathway Analysis; Pathway interactions; Personal Communication; Phase; Plant Roots; Population; Preparation; Procedures; Process; Protein Structure Initiative; Protocols documentation; Reagent; Research; Reverse Transcriptase Polymerase Chain Reaction; Scientist; Side; Solutions; Sorting - Cell Movement; Stem cells; Stress; System; Techniques; Temperature; Testing; Therapeutic; Tissues; Transfusion; Variant; base; bioprocess; cancer stem cell; cancer therapy; cell type; cold temperature; culture plates; design; experience; human embryonic stem cell; human embryonic stem cell line; human stem cells; improved; improved functioning; inhibitor/antagonist; instrument; neoplastic cell; prototype; research and development; sample collection; stem cell therapy; technology development; tumor

DESCRIPTION (provided by applicant): There is increasing utilization of flow cytometry in both basic and applied research to accomplish diagnostic assays. One such area of research is the investigation of circulating cells that have detached from tumors. Another very active and contemporary area is the generation of flow cytometry side populations that contain multipotent cancer stem cells that can be present in less than 1% of the cell population. Furthermore, it is well known that the flow cytometry characterization of nearly all types of stem cells, including human embryonic stem cells, is a challenge due to the high cell loss during the sorting process. This has led some to consider the inclusion of ROCK inhibitors in the cell sorting protocol to increase viability and improve the detection of stem cell surface markers. To address these challenges CPSI Biotech proposes to develop the first FACS compatible sorting reagent, referred to as FACSGuard, expressly designed to protect cells during the flow cytometry process. This media will be designed to be used at normothermic (37oC) temperatures to ameliorate several stress pathways that are activated during a typical cell dissociation, separation and flow procedure. If successful, this reagent can be used in a variety of flow cytometry applications from low stress Guava to the high PSI (70) often required in more traditional FACS systems to separate other cell types. The Specific Aims of the project are the following: (1) Test a variety of candidate cell stress pathway inhibitors in conditions that mimic those experienced by cells under flow cytometry conditions for possible inclusion in FACSGuard; (2) Develop several versions of FACSGuard; and (3) Test the versions of FACSGuard on two human embryonic stem cell lines as well as other more robust cell types. Phase 2 will be dedicated to using RT-PCR and other microarray techniques to determine if one generic FACSGuard reagent is sufficient for most flow cytometry applications or if several cell and or condition-specific variants should be developed. The successful completion of this project will lead to an improved method of identifying and understanding cancer stem cells found in FACS side populations as well as directly and indirectly improve stem cell therapy applications. PUBLIC HEALTH RELEVANCE: This project is designed to develop a protective solution for stem cells so that both their viability and function can be protected during their separation and analysis. This will have direct benefit to diverse areas ranging from understanding cancer stem cells to stem cell therapy.

描述（由申请人提供）：在基础和应用研究中越来越多地利用流式细胞术来完成诊断测定。其中一个研究领域是研究与肿瘤分离的循环细胞。另一个非常活跃的当代领域是流式细胞术侧群体的产生，其中包含多能癌症干细胞，这些干细胞可能存在于不到 1% 的细胞群体中。此外，众所周知，由于分选过程中细胞损失较多，几乎所有类型的干细胞（包括人胚胎干细胞）的流式细胞术表征都是一个挑战。这导致一些人考虑在细胞分选方案中加入 ROCK 抑制剂，以提高活力并改善干细胞表面标记物的检测。为了应对这些挑战，CPSI Biotech 建议开发第一个 FACS 兼容分选试剂，称为 FACSGuard，专门设计用于在流式细胞术过程中保护细胞。该介质被设计为在常温 (37oC) 温度下使用，以改善在典型细胞解离、分离和流动过程中激活的几种应激途径。如果成功，该试剂可用于各种流式细胞术应用，从低应力番石榴到传统 FACS 系统分离其他细胞类型时经常需要的高 PSI (70)。该项目的具体目标如下：（1）在模拟流式细胞术条件下细胞所经历的条件下测试多种候选细胞应激途径抑制剂，以可能纳入FACSGuard； (2) 开发FACSGuard的多个版本； (3) 在两种人类胚胎干细胞系以及其他更强大的细胞类型上测试 FACSGuard 版本。第 2 阶段将致力于使用 RT-PCR 和其他微阵列技术来确定一种通用 FACSGuard 试剂是否足以满足大多数流式细胞术应用，或者是否应该开发几种细胞和/或条件特异性变体。该项目的成功完成将改进识别和理解 FACS 侧群体中发现的癌症干细胞的方法，并直接和间接改进干细胞治疗应用。 公共健康相关性：该项目旨在开发干细胞保护解决方案，以便在分离和分析过程中保护它们的活力和功能。这将对从了解癌症干细胞到干细胞治疗等各个领域产生直接的好处。