CONTINUOUS CELL SORTING BY MAGNETOPHORESIS

通过磁泳连续细胞分选

• 批准号: 7172985
• 负责人: Maciej Zborowski
• 金额: $ 34.78万
• 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 2008-04-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7172985
• 关键词: Acute Myelocytic Leukemia; Antibodies; Arts; Benchmarking; Binding; Biological Assay; Blood; Blood Cells; Blood Component Removal; Blood specimen; Bone Marrow; Breast Carcinoma; CD34 gene; Cancer Center; Carrier Proteins; Cell Line; Cell Mobility; Cell Separation; Cell Survival; Cell Volumes; Cell physiology; Cell surface; Cells; Characteristics; Chemicals; Chemistry; Clinic; Collaborations; Commit; Complex; Condition; Contrast Media; Detection; Development; Diagnosis; Equilibrium; Erythrocytes; Ferritin; Gadolinium; Goals; Grant; Hour; Human; Immobilization; Immunoassay; Ions; Iron; Israel; Kinetics; Label; Lanthanoid Series Elements; Large-Cell Lymphomas; Lead; Leukocytes; Liquid substance; Lymphocyte; MCF7 cell; Magnetic Resonance Imaging; Magnetism; Malignant Epithelial Cell; Measurement; Measures; Methemoglobin; Methods; Microspheres; Modification; Molecular; Molecular Diagnosis; Monitor; Motion; Numbers; Ohio; Otolaryngology; PTPRC gene; Pan Genus; Patients; Performance; Phase; Phenotype; Predisposition; Process; Property; Range; Reaction; Recovery; Reporting; Research; Research Personnel; Sampling; Solid; Sorting - Cell Movement; Spectrum Analysis; Speed; Standards of Weights and Measures; Stem cells; Surface; Techniques; Technology; Testing; Time; Travel; Umbilical Cord Blood; Universities; Velocimetries; Viscosity; Work; absorption; antibody conjugate; base; cancer cell; cancer diagnosis; cancer therapy; cell separator; circulating cancer cell; data acquisition; experience; fluorescence activated cell sorter device; improved; instrument; instrumentation; leukemia; magnetic field; nanomagnetic; nanoparticle; nanoscale; neoplastic cell; particle; physical property; programs; shear stress; sound; technology development; tool; uptake

DESCRIPTION (provided by applicant): In the previous support period, we developed tools for continuous magnetic flow cell sorting based on cell magnetophoresis. We also developed cell tracking velocimetry to measure cell magnetophoretic mobility. Cell sorting based on magnetophoresis of freely suspended cells has the potential for improved performance over current magnetic separation methods that rely on cell immobilization on magnetic wire and bead matrices. By optimizing both the flow sorter channel geometry and the applied magnetic field (annular flow in a quadrupole field), we achieved high sorting speed (10[7] cells/s) with high cell purity (90% purity of CD34+, at recovery of 30%). Furthermore, separated cell viability remained high due to relatively low shear stress experienced during sorting. The technology development was aided by collaboration with the Cleveland Clinic Taussig Cancer Center and the Ohio State University. Based on the strengths of the current effort, we propose to further evaluate the technology with the focus on the following: Aim 1: To compare the technology with a benchmark high-gradient magnetic separation (HGMS) method based on extrinsic cell magnetization with antibody-conjugated magnetic nanoparticles. First, we will test positive cell separation of long term colony-initiating cells from committed blood progenitor cells. The fractionated cells will be fully characterized in terms of their phenotypic and functional characteristics (in collaboration with Dr. Maciejewski at the Taussig Cancer Center). Second, we will test negative separation in application to isolation of rare cancer cells from circulating blood. Circulating white blood cells will be targeted by anti pan WBC antibody (such as anti CD45 Ab) and will be separated from the un-labeled, non-mobile non-WBCs, representing the likely candidates for the cancer cells in the circulating blood (in collaboration with Dr. Chalmers at the OSU). Aim 2: To investigate the intrinsic magnetophoresis of fresh, un-manipulated (unlabeled) cells. Magnetophoretic cell sorting may allow separation of cells differing in their intrinsic magnetization (without application of extrinsic magnetic nanoparticles). We will test the technology first on cell lines that can be induced to express high levels of ferritin, and second, on selected primary leukemia samples known for their elevated intracellular ferritin level. Aim 3: To further investigate the magnetophoretic process and refine the cell tracking velocimetry instrumentation for applications to cancer therapy. In particular, we will determine the applicability of MRI contrast agents to modification of the magnetic susceptibility of suspending medium to enhance magnetophoretic cell sorting and achieve high purity and recovery of the target cells (in collaboration with Dr. Hafeli). The proposed research will help to fully characterize the magnetophoretic cell sorting process, further improve the tools, and determine the applications relevant to the diagnosis and therapy of cancer.

描述（由申请人提供）：在之前的支持期内，我们开发了基于细胞磁泳的连续磁流细胞分选工具。我们还开发了细胞跟踪测速法来测量细胞的磁泳流动性。基于自由悬浮细胞的磁泳进的细胞分选与目前依赖于磁线和磁头基质的细胞固定的磁分离方法相比，具有改善性能的潜力。通过优化流动分选通道的几何形状和外加磁场（四极电场中的环形流），我们实现了高分选速度（10[7]cells/s）和高细胞纯度（CD34+纯度为90%，回收率为30%）。此外，由于在分选过程中经历了相对较低的剪切应力，分离的细胞活力保持较高。这项技术的开发得到了克利夫兰诊所陶西格癌症中心和俄亥俄州立大学的合作。基于目前研究的优势，我们建议进一步对该技术进行评估，重点关注以下方面：目的1：将该技术与基于抗体共轭磁性纳米颗粒外源细胞磁化的基准高梯度磁分离（HGMS）方法进行比较。首先，我们将测试长期集落启动细胞与固定的血液祖细胞的阳性细胞分离。分离的细胞将在表型和功能特征方面得到充分的表征（与Taussig癌症中心的Maciejewski博士合作）。第二，我们将测试阴性分离在循环血液中罕见癌细胞分离中的应用。循环白细胞将被抗泛白细胞抗体（如抗CD45抗体）靶向，并将从未标记的、非移动的非白细胞中分离出来，这些非白细胞代表了循环血液中癌细胞的可能候选者（与俄勒冈州立大学的Chalmers博士合作）。目的2：研究新鲜、未处理（未标记）细胞的固有磁泳。磁泳析细胞分选可以分离内在磁化强度不同的细胞（不需要外加磁性纳米颗粒）。我们将首先在可以诱导表达高水平铁蛋白的细胞系上测试这项技术，然后在选定的以细胞内铁蛋白水平升高而闻名的原发性白血病样本上测试这项技术。目的3：进一步研究磁电泳过程，完善细胞跟踪测速仪器，用于癌症治疗。特别是，我们将确定MRI造影剂对悬浮介质磁化率的修改的适用性，以增强磁泳细胞的分选，实现高纯度和目标细胞的回收率（与Hafeli博士合作）。本研究将有助于充分表征磁泳细胞分选过程，进一步改进工具，并确定与癌症诊断和治疗相关的应用。