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.
Showing the most recent 10 out of 88 publications
