Magnetophoretic Cell Sorting and Analysis

描述（由申请人提供）：细胞磁泳运动是由电解质溶液中的磁场引起的细胞运动。在目前的资助期内（截至2008年1月31日），我们已经使用细胞跟踪速度法（CTV）比较了各种商业免疫磁性试剂结合对细胞磁泳流动性（MM）的影响，并证明MM在造血祖细胞富集、T细胞耗尽和罕见癌细胞检测中是磁泳细胞分选性能的准确预测指标。我们还测量了在不结合免疫磁性标记试剂的情况下感染疟疾寄生虫的红细胞和球状芽孢杆菌（一种原核生物）孢子的固有细胞MM。在这里，我们建议将研究扩展到癌细胞系和原发癌细胞。工作假设是正常和转化癌细胞之间的差异，包括它们的代谢活性和物理特性，导致细胞MM的差异。筛选现有癌细胞系的细胞MM与基线正常外周白细胞MM的差异。筛选将使用目前可用的CTV和磁流细胞分选设备进行。测量将在典型的细胞培养基和铁（以及潜在的其他顺磁性元素，如锰）水平增加的修饰培养基中进行。细胞系将从美国组织培养收集处购买，并将最初选择铁含量高的可能性高的细胞系（如白血病白细胞）。目标2。利用现有的永磁材料重新设计CTV以获得最大的磁能梯度配置。高磁能梯度是诱导弱磁性细胞运动和解决癌细胞样本均值与基线对照（外周白细胞）之间差异所必需的。现有的专业知识、分析和加工能力将为实现这一目标提供必要的资源。关于超导磁体使用的初步讨论得出的结论是，技术复杂性超过了该资助机制的能力。另一方面，高磁能永久材料的商业可用性和创新设计证明了在小型实验室规模上实现3特斯拉(T)和1,000 T/m梯度的可行性，足以与细胞一起工作。目标3。证明利用细胞固有磁泳流动性分离癌细胞的可行性。基于Aim1的发现以及Aim2提高的分析和分离能力，选择的原发肿瘤样本将被制备成单细胞悬液，并在背景对照下分析其MM。在最后的分析中，我们将使用免疫细胞化学和其他癌症分子标记来确定从临床活检样本中磁电泳分离的癌细胞的回收率和纯度。在没有转移性疾病迹象的癌症患者的外周血和组织中检测肿瘤细胞具有至关重要的临床意义，因为它将导致早期干预治疗和修改治疗方法，以试图阻止或延缓疾病进展。过去NIH的支持导致了磁性细胞分选和细胞分析仪器的发展，并将其应用于罕见的癌细胞检测和使用免疫特异性磁颗粒标记的干细胞分离（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）。基于我们对红细胞的初步研究，我们建议将这项研究扩展到癌细胞的检测和分析，而不需要繁琐的磁颗粒标记步骤，而只基于自然发生的弱细胞磁化。

DESCRIPTION (provided by applicant): 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.