Magnetophoretic Cell Sorting and Analysis

细胞磁电泳性是指细胞在电解液中受磁场作用而产生的运动。在.期间 当前资助期(截至2008年1月31日)我们已使用细胞跟踪测速仪(CTV)比较效果 不同商品化免疫磁试剂与细胞磁电泳动(MM)的结合 证明MM可以准确地预测磁电性细胞的分选性能 在造血祖细胞浓缩、T细胞耗尽和罕见癌细胞检测方面的应用。我们 还在没有免疫磁标记剂与红细胞结合的情况下测量了固有细胞MM 感染疟疾寄生虫和原核生物球状芽孢杆菌的孢子。在这里，我们建议将 对癌细胞系和原代癌细胞的研究。工作假说是，差异 正常和转化的癌细胞之间的差异，包括它们的代谢活动和物理性质， 目的1.筛选现有的癌细胞系以寻找细胞间的差异 对照基线正常外周血白细胞MM。筛查将使用当前 可提供CTV和磁流细胞分选设备。测量将在典型单元格中进行 培养基和含有更多铁(以及潜在的其他顺磁性物质)的改良基料 元素，如锰)。这些细胞系将从美国组织培养收藏中心购买， 并将在铁含量升高的情况下(如白血病白细胞系)最初被高概率地选择。 目标2.重新设计CTV，使其具有最大磁能梯度配置 永磁体材料。较高的磁能梯度是诱导弱磁场运动所必需的。 并解决肿瘤细胞样本均值与基线对照之间的差异 (外周血白细胞)。现有的专业知识、分析和加工能力将提供 实现这一目标所需的资源。关于超导应用的初步探讨 磁铁导致的结论是，技术复杂性超出了这笔资金的能力 机制。另一方面，高磁能永磁材料的商业可获得性 创新的设计证明了实现3特斯拉(T)场和1000 T/m梯度的可行性 在小规模的实验室里，足够用来处理细胞。目的3.论证癌细胞的可行性 通过固有的细胞磁电迁移进行隔离。基于Aim1中的发现和改进 由于AIM2，选定的原发癌症样本将获得分析和分离能力 制备成单细胞悬液，以背景对照分析其MM。在决赛中 分析，我们将确定从临床分离的癌细胞的回收率和纯度 活检标本采用免疫细胞化学和其他癌症分子标志物。在癌症患者的外周血和组织中检测到肿瘤细胞，但没有证据表明 转移性疾病具有至关重要的临床意义，因为它将导致早期介入治疗和 改变治疗方法，试图阻止或延缓疾病的发展。NIH过去的支持结果是 在磁性细胞分选和细胞分析仪器的发展中，及其在稀有 使用免疫特异性磁粒子标记的癌细胞检测和干细胞分离(现在 由我们的机构、克利夫兰诊所和俄亥俄州立大学商业化)。基于我们的 对于红细胞的初步研究，我们建议将这项研究扩展到检测和分析 癌细胞不需要繁琐的磁粉标记步骤，但仅基于 自然发生的，微弱的细胞磁化。

Cell magnetophoresis is a cell motion induced by the magnetic field in electrolyte solutions. During the current funding period (ending 01/31/08) we have used Cell Tracking Velocimetry (CTV) to compare effects of binding of various commercial immunomagnetic reagents on cell magnetophoretic mobility (MM) and demonstrated that MM is an accurate predictor of the magnetophoretic cell sorting performance in applications to hematopoietic progenitor cell enrichment, T cell depletion, and rare cancer cell detection. We also measured intrinsic cell MM without binding of the immunomagnetic tagging reagents on red blood cells infected with malaria parasites, and spores of Bacillus globigii (a prokaryote). Here we propose to extend the investigations to cancer cell lines and primary cancer cells. The working hypothesis is that differences between normal and transformed cancer cells, including their metabolic activity and physical properties, lead to differences in cell MM. Aim 1. To screen the available cancer cell lines for differences in cell MM against baseline normal peripheral leukocyte MM. The screening will be performed using currently available CTV and magnetic flow cell sorting equipment. The measurements will be performed in typical cell culture media and modified media with the increased level of iron (and potentially other paramagnetic elements, such as manganese). The cell lines will be purchased from American Tissue Culture Collection, and will be initially selected for high probability in elevated iron content (such as leukemic leukocyte lines). Aim 2. To re-design CTV for the maximum magnetic energy gradient configuration using available permanent magnet materials. The high magnetic energy gradient is necessary to induce motion of weakly magnetic cells and to resolve differences between sample means of cancer cells and baseline control (peripheral leukocytes). The existing expertise, analytical and machining capabilities will provide the necessary resources to accomplish this aim. Preliminary discussions regarding use of superconducting magnets led to the conclusion that the technical complexities exceed the capacity of this funding mechanism. On the other hand, the commercial availability of high magnetic energy permanent materials and innovative designs demonstrated feasibility of achieving fields of 3 tesla (T) and gradients of 1,000 T/m on a small laboratory scale, sufficient for work with cells. Aim 3. To demonstrate feasibility of cancer cell isolation by intrinsic cell magnetophoretic mobility. Based on the findings in Aim1 and the improved analytical and separation capabilities achieved as a result of Aim2, selected primary cancer samples will be prepared as single cell suspensions and analyzed for their MM against the background control. In the final analysis, we will determine recovery and purity of the cancer cells isolated magnetophoretically from clinical biopsy samples using immunocytochemistry and other molecular markers of cancer. Detection of tumor cells in the peripheral blood and tissues of cancer patients exhibiting no evidence of metastatic disease is of critical clinical importance as it will lead to early intervention therapy and modification of treatment in an attempt to halt or retard disease progression. The past NIH support resulted in the development of magnetic cell sorting and cell analysis instrumentation, and their application to rare cancer cell detection and stem cell separation using immunospecific magnetic particle labeling (now commercialized by our institutions, Cleveland Clinic and The Ohio State University). Based on our preliminary studies with red blood cells, we propose to extend this research to the detection and analysis of cancer cells without the requirement of a cumbersome magnetic particle tagging step, but only based on naturally occurring, weak cell magnetization.