CONTINUOUS MAGNETIC CELL SORTING

根据细胞的功能或表面特性分离细胞是一种 细胞生物学、癌症治疗和其他领域使用的许多技术的基础 生物技术。虽然免疫磁性细胞分离已被用于 所有这些领域，都受到相对较大规模的阻碍。 顺磁标记与靶细胞相比并通过相对 用于分离的粗装置。这项研究将有两个主要目标： 新型磁性细胞标签的开发以及分析和 使用该标签设计连续磁性细胞分选仪。这个新的 磁性标记由铁蛋白与单克隆抗体结合组成 针对靶细胞上的特定表面抗原。铁蛋白是一种 哺乳动物体内的铁储存蛋白。 铁蛋白标签的特性将使用许多特征来表征 技术包括：核磁共振波谱、原子 吸收光谱和透射电子显微镜。一个独特的 结合荧光和磁性标记的三明治标记技术将 与流式细胞术结合使用来表征细胞标记 绑定。人外周淋巴细胞和人乳腺癌来源 细胞将在本研究中与单克隆抗体结合使用 针对特定标记的抗体：CD3、CD4、CD8、CD16、CD34 和 Ber-EP4。这些标记物识别对骨髓重要的细胞 移植疗法：T细胞、辅助/诱导剂、细胞毒性/抑制剂、 分别是自然杀伤细胞、干细胞和乳腺癌细胞。 由于涉及的复杂性，需要连续的分析和设计， 磁性细胞分选仪需要使用 3 维颗粒图像 测速（3-D PIV）。 3-D PIV，由俄亥俄州立大学开发， 本研究的拟议分包地点将允许确定 高磁场中标记和未标记细胞的 3-D 速度矢量 字段。实验分析，结合相关方程 运动，将提供微米级系统的信息。与 根据这些信息，将开发出优化的细胞分选仪。此外， 将有可能设计更先进的分拣机，例如多级分拣机 系统，可以获得非常高的纯度或非常高的选择性 在相对较短的时间内。 最终目标是开发一种连续的磁性细胞分选机 大规模分离用于骨髓移植治疗。这样一个 分选机将适用于快速、大规模 T 细胞耗竭、癌症 细胞净化和造血干细胞富集。

Separation of cells based on their function or surface properties is a basis of many techniques used in cell biology, cancer therapy and in biotechnology. While immunomagnetic cell separation has been utilized in all these areas, it has been hampered by the relatively large size of the paramagnetic label as compared to the target cell and by the relatively crude devices used for separation. This study will have two primary goals: the development of a novel magnetic cell label, and the analysis and design of a continuous, magnetic cell sorter using this label. This new magnetic label consists of ferritin combined to monoclonal antibodies against a particular surface antigen on the target cell. Ferritin is an iron storage protein in mammals. The properties of ferritin labels will be characterized using a number of techniques including: nuclear magnetic resonance spectroscopy, atomic absorption spectroscopy, and transmission electron microscopy. A unique sandwich labeling technique combining fluorescent and magnetic label will be used in combination with flow cytometry to characterize the cell-label binding. Human peripheral lymphocytes and human breast carcinoma derived cells will be used in this study, in combination with monoclonal antibodies against the specific markers: CD3, CD4, CD8, CD16, CD34 and Ber-EP4. These markers identify cells important for the bone marrow transplantation therapy: T cell, helper/inducer, cytotoxic/suppressor, natural killer, stem and breast carcinoma cells, respectively. Due to the complexity involved, the analysis and design of a continuous, magnetic cell sorter will require the use of 3-Dimensional Particle Image Velocimetry (3-D PIV). 3-D PIV, developed at the Ohio State University, the proposed subcontract site of this study, will allow the determination of 3-D velocity vectors of labeled and unlabeled cells in high magnetic fields. The experimental analysis, combined with the relevant equations of motion, will provide information on the system at a micron scale. With this information an optimized cell sorter will be developed. In addition, it will be possible to design more advanced sorters, such as multistage system, in which very high purity or very high selectivity can be obtained in a relatively short time. The ultimate goal is to develop a continuous, magnetic cell sorter for large-scale separation for bone marrow transplantation therapies. Such a sorter will be applicable to rapid, large-scale T cell depletion, cancer cell purging and hematopoietic stem cell enrichment.