Cell Based Therapy

To date, clinical flow cytometric studies have been mainly limited to diagnostic, monitoring, and classification purposes. As the reliability of cell sorting improves, there has been an emergence of cell sorters as a means for cell-based therapy [1]. In this regard, high-speed flow cytometers may make the most impact, as these applications demand the greatest need for higher throughput and flexibility in measurement parameters; cells to be infused into a patient should be extracorporeal for the shortest time possible and isolated to the utmost level of purity. For instance, in the case of bone marrow transplantation, it was unknown which of the transplanted cells had the ma jor regenerative capacity. As a result, patients would receive transplants of whole HLA-identical sibling bone marrow and hope for the best outcome. HLA-mismatched, or unrelated grafts with concurrent immunosuppression raised issues of tolerance and graft vs. host disease (GVHD). In 1984, Martin et al. published a study reporting the use of "microfluorometry" to deplete T-cells from donor marrow in order to decrease GVHD [43]. In the 1990s, the discovery of the CD34+ phenotype led clinicians to believe that cells displaying this surface antigen contained the stem cell function of bone marrow, and it was soon shown that as few as 7 x 106 CD34+cells per kilogram would be sufficient for engraftment [44]. Cells could be labeled with a CD34-reactive antibody and purified in a cell sorter. Additional advances in hematology and immunophenotyping will undoubtedly lead to more successful transplantations with fewer complications [45,46].

Within the last few years, the rapidly expanding set of genes known to play a role in cellular differentiation has led to complex staining patterns [47-49], or "molecular signatures" [50] characteristic of the most primitive cells. So while the first applications of clinical flow cytometry relied on screening through large numbers of cells to acquire sufficient material for successful engraftment, more recent trials depend on the successful identification of a relatively rare population of uniquely labeled cells in the peripheral blood, marrow, or cord blood compartment. While fewer of these highly defined cells are needed, speed is of the essence, as by definition a more highly classified cell will be present in lesser quantities. As we better determine which cells are crucial to this and other processes, high-speed cell sorters will be at the forefront of many treatment regimes. A natural extension of the use of highspeed cell sorters in the setting of cancer treatment is for tumor cell purging. As our knowledge of the landscape of healthy cells increases, so too does the information regarding malignant cells [51,52]. For instance, if the phenotype of a known B- or T-cell leukemia is known, cells can be removed from the patient, treated ex vivo to remove cells bearing this combination of markers and reinfused into the patient [53].

As an additional consideration, high-speed cell sorting for clinical applications raises aspects of sorting not encountered with experimental protocols in the laboratory. Issues of absolute sterility, cleanliness, and reliability are of utmost importance, both to prevent sample contamination and to protect the sort operator from aerosolized particles. Further examples of cell sorters for cell-based therapy beyond hematologic disorders include gene therapy [52], pancreatic islet cell transplantation [54], and sperm sorting for gender preselection [55]. The latter is the area where sorting of mammalian cells for reintroduction to a living organism is most used and is established with the Food and Drug Administration (FDA). Originally devised for the selection of gender-specific livestock [55], the technique relies on the differential DNA contact of X and Y chromosome-bearing sperm. Because the X chromosome is significantly larger than the Y chromosome, when

Fig. 5 Scatter plot of bull sperm stained with a generic DNA dye. The X-bearing sperm contain more DNA, therefore appearing as a separate population from Y-bearing sperm

sperm are incubated with a fluorescent dye that generically binds to DNA, the fluorescence intensity is considerably higher from X chromosome-bearing sperm than that from those sperm containing a Y chromosome (Fig. 5). As such, X-bearing sperm can be separated in bulk and used for in vitro fertilization to greatly enhance the chances for female offspring. In humans, the difference in DNA content between X- and Y-bearing sperm accounts for an approximately 2% difference in overall DNA content, and hence fluorescence signal detected by flow cytometry. Indeed this method has been employed for the use of gender-selection in the case of families with known X-linked diseases, and to assist with "family balancing", sparking ongoing ethical debate.

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