Magnetophoretic Cell Sorting and Analysis

磁泳细胞分选和分析

• 批准号: 8040868
• 负责人: Maciej Zborowski
• 金额: $ 36.86万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-18 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8040868
• 关键词: Appearance; Applications Grants; Biological Markers; Biomedical Research; Blood; Blood Cells; Cancer cell line; Cell Separation; Cells; Clinic; Collaborations; Complement; Computer Assisted; Computer software; Culture Media; Data; Detection; Diagnosis; Disease; Electrolytes; Electron Spin Resonance Spectroscopy; Electrons; Erythrocytes; Exhibits; Ferritin; Field Flow Fractionation; Funding; Hela Cells; Heme; Human; Image; Immunophenotyping; Iron; Iron Compounds; Label; Laboratories; Life; Link; Liquid substance; Magnetic Resonance Imaging; Magnetism; Malignant - descriptor; Malignant Neoplasms; Manganese; Mass Spectrum Analysis; Measures; Methods; Modeling; Molecular; Morphologic artifacts; Mossbauer Spectroscopy; Motion; Normal Cell; Ohio; Oncologist; Organic Iron Compounds; Particulate; Physiological; Predisposition; Primary Neoplasm; Property; Proteins; Protocols documentation; Reactive Oxygen Species; Research; Resolution; Rest; Site; Solid Neoplasm; Solutions; Spectrometry; Structure; Suspension substance; Suspensions; Technology; Testing; Tissues; Transferrin Receptor; Transport Process; Tumor Cell Line; Universities; Velocimetries; Whole Blood; absorption; analytical method; base; cancer cell; cancer therapy; cell analyzer; cell transformation; cytochrome c; energy density; improved; instrumentation; interest; magnetic beads; magnetic cell separation; magnetic field; magnetic sorter; neoplastic cell; outcome forecast; receptor expression; research facility; uptake

DESCRIPTION (provided by applicant): This is a competing renewal application that builds on the instrumentation and expertise developed in the course of the previous funding periods. We propose to investigate the feasibility of extending the current magnetic cell separation methods to applications relying on the cell's own, natural (intrinsic) magnetization. The application rests on the hypothesis that there is a link between malignant cell transformation and an increase in the cell magnetic susceptibility. The supporting evidence comes from a large body of data from other studies on the magnetic properties of tissues, in particular, from electron (spin) paramagnetic resonance, EPR, and studies of solid tumors. Additional supporting evidence comes from our own data on the magnetic field-induced motion (magnetophoresis) in physiologic electrolyte solutions of cancer cell lines (HeLa and Hep 3B), that demonstrate statistically greater velocity compared to controls (oxygenated red blood cells). We have shown an additional increase in the cell magnetophoretic mobility following the addition of soluble iron compounds to the culture media, suggesting an effect of intracellular iron uptake on the cell magnetization. The proposed effort is divided between: Specific Aims: SA1: To investigate the molecular mechanism for the observed increase in magnetophoretic mobility (MM) in selected cancer cell lines as compared to matched normal cells. In collaboration with experts in cell tracking velocimetry (Dr. Chalmers) and EPR spectrometry (Dr. Kuppusamy) at the subcontract site (The Ohio State University), the effect of iron in the medium on the cell magnetophoretic mobility and the increased intracellular paramagnetic content will be investigated on cancer cell lines. SA2: To improve the sensitivity and resolution of the cell magnetophoretic mobility analyzer, the Cell Tracking Velocimetry (CTV). The increase of magnetophoretic mobility resolution by a factor up to 10 fold will be accomplished by increasing the magnetic energy density and field gradient of the apparatus, by improving the imaging capability of the cell tracking hardware and software, and by including corrections for fluid dynamics artifacts in collaboration with an expert in field-flow fractionation (Dr. Williams). SA3: To measure the magnetic susceptibility of selected primary tumors at a single cell level against a baseline of normal blood cells. Primary tumor cell lines will be tested against normal controls for differences in the magnetophoretic mobility, in collaboration with an expert oncologist (Dr. Borden). SA4: To test the feasibility of the label-less, magnetic cancer cell separation on model blood cell suspensions. The proposed strategy is based on the soluble - rather than particulate - iron transport processes, and may therefore be more finely tuned to the pathobiology of the cancer cell than is practiced today. It could have a profound impact on how the magnetic cell separation is practiced and utilized and could complement and extend the applications of the already existing magnetic cell separation methods. PUBLIC HEALTH RELEVANCE: There is an intense interest in identifying cancer biomarkers for improved diagnosis and prognosis in the treatment of the disease. One particularly promising application is the detection, enumeration and analysis of circulating tumor cells (CTC's) in blood. There is evidence of abnormal paramagnetic contributions to the magnetic susceptibility of cells that have undergone malignant transformation coming from the laboratories collaborating on this grant application. We propose to systematically investigate this effect by using our state-of-the-art magnetic research facilities that include the magnetic cell separation laboratory and electron (spin) paramagnetic resonance (EPR) spectrometry facility available at the primary (Cleveland Clinic) and the subcontract (The Ohio State University) sites. The proposed research could have a profound impact on the magnetic separation methods in application to biomedical research and the treatment of cancer.

描述（由申请人提供）：这是一项竞争性续展申请，建立在之前资助期间开发的工具和专业知识的基础上。我们建议研究将当前磁性细胞分离方法扩展到依赖于细胞自身自然（固有）磁化的应用的可行性。该申请基于这样的假设：恶性细胞转化与细胞磁化率增加之间存在联系。支持证据来自其他有关组织磁特性的研究的大量数据，特别是来自电子（自旋）顺磁共振、EPR 和实体瘤研究的数据。其他支持证据来自我们自己的癌细胞系（HeLa 和 Hep 3B）生理电解质溶液中磁场诱导运动（磁泳）的数据，这些数据在统计上证明与对照（氧化红细胞）相比速度更快。我们已经证明，在培养基中添加可溶性铁化合物后，细胞磁泳迁移率进一步增加，表明细胞内铁摄取对细胞磁化的影响。拟议的工作分为： 具体目标：SA1：研究与匹配的正常细胞相比，在选定的癌细胞系中观察到的磁泳迁移率（MM）增加的分子机制。与分包地点（俄亥俄州立大学）的细胞追踪测速专家（Chalmers 博士）和 EPR 光谱测定法（Kuppusamy 博士）合作，将研究培养基中铁对细胞磁泳迁移率和细胞内顺磁性含量增加对癌细胞系的影响。 SA2：提高细胞磁泳迁移率分析仪（细胞跟踪测速仪（CTV））的灵敏度和分辨率。通过增加设备的磁能密度和场梯度、提高细胞跟踪硬件和软件的成像能力以及与场流分级专家（Williams 博士）合作对流体动力学伪影进行校正，可以将磁泳迁移率分辨率提高多达 10 倍。 SA3：在单细胞水平上测量所选原发性肿瘤相对于正常血细胞基线的磁化率。将与肿瘤专家（Borden 博士）合作，对原代肿瘤细胞系与正常对照进行磁泳迁移率差异测试。 SA4：测试模型血细胞悬浮液上无标记磁性癌细胞分离的可行性。所提出的策略基于可溶性铁而不是颗粒铁转运过程，因此可能比目前的实践更适合癌细胞的病理学。它可能会对磁性细胞分离的实践和利用产生深远的影响，并且可以补充和扩展现有磁性细胞分离方法的应用。 公共卫生相关性：人们对识别癌症生物标志物以改善疾病治疗的诊断和预后有着浓厚的兴趣。一项特别有前景的应用是血液中循环肿瘤细胞 (CTC) 的检测、计数和分析。有证据表明，与本次拨款申请合作的实验室发现，异常顺磁对发生恶性转化的细胞的磁化率有贡献。我们建议通过使用我们最先进的磁性研究设施来系统地研究这种效应，这些设施包括在主基地（克利夫兰诊所）和分包合同基地（俄亥俄州立大学）提供的磁性细胞分离实验室和电子（自旋）顺磁共振（EPR）光谱测定设施。拟议的研究可能会对磁分离方法在生物医学研究和癌症治疗中的应用产生深远的影响。