CONTINUOUS CELL SORTING BY MAGNETOPHORESIS

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仪来测量细胞的磁泳运动。基于自由悬浮细胞的磁泳的细胞分选具有优于依赖于细胞固定在磁性线和珠基质上的当前磁性分离方法的改进性能的潜力。通过优化流式分选器通道几何形状和施加的磁场（四极场中的环形流），我们实现了高分选速度（10[7]个细胞/s）和高细胞纯度（90%纯度的CD 34+，回收率为30%）。此外，由于分选期间经历的相对低的剪切应力，分离的细胞活力保持高。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州立大学的合作。基于当前努力的优势，我们建议进一步评估该技术，重点如下：目的1：将该技术与基于抗体缀合磁性纳米颗粒的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起始细胞与定向血祖细胞的阳性细胞分离。分级分离的细胞将在其表型和功能特征方面进行充分表征（与Taussig癌症中心的Macieplski博士合作）。其次，我们将测试阴性分离在从循环血液中分离罕见癌细胞中的应用。循环白色血细胞将被抗泛WBC抗体（如抗CD 45 Ab）靶向，并将与代表循环血液中可能候选癌细胞的未标记、非流动性非WBC分离（与OSU的查尔默斯博士合作）。目的2：研究新鲜、未处理（未标记）细胞的固有磁泳。磁泳细胞分选可以允许分离内在磁化不同的细胞（不应用外部磁性纳米颗粒）。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该技术，其次，在选定的因其升高的细胞内铁蛋白水平而闻名的原发性白血病样本上测试该技术。目的3：进一步研究磁泳过程，并完善细胞跟踪测速仪器应用于癌症治疗。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，以增强磁泳细胞分选并实现靶细胞的高纯度和回收（与Hafeli博士合作）。这项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。

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.