The depletion of only the alloreactive T cells after human leukocytes culturing, which cause graft-versus-host disease (GVHD), yet conserves the other T-cells that provide graft-versus-leukemia (GVL) response, could significantly improve the chances of survival of transplant patients in allogeneic stem cell transplantation. One project in our lab consists of the immunomagnetic depletion of alloreactive T-cells for potential transplantation of patients that had previously had a total T-cell depleted, stem cell transplantation.

Immuno-magnetic separation is a popular large-scale cell separation method in both research and clinical application as a result of a number of attributes, including ease of use and relative low cost. However, there is always the potential of non-specific binding occurring during the cell labeling procedure, which would cause the un-expected lose of non-targeted cells, or the contamination of targeted cells (the positive fraction) with undesired cells This issue becomes more pronounced when one desires a high level of performance.

Using multiple surface markers, we have obtained depletions of unwanted alloreactive T-cells which are theoretically sufficient for a transplantation; however, the recovery of the desired, non-immunomagnetically labeled cells (i.e. the desirable T-cells) is significantly sub-optimum. Based on further studies/analysis to understand this sub-optimum performance, data from this project, as well as several other projects in our research group indicates that this problem can be attributed to a number of causes, all related to non-specific binding events.

We suggest that non-specific binding is an under appreciate phenomena in many cell separation processes, not just immunomagnetic systems, and the losses from non-specific events can become a significant problem when processes are scale-up and as higher and higher performance is expected. In this talk we will discuss our experimental methodologies and findings with respect to characterization of non-specific binding events in immunomagnetic cell separation process and suggest potential solutions.

Generally, MACS nanoparticles from Miltenyi Biotech were employed for the non-specific binding study and two separate technologies (CTV and ICP-OES) were used for cell magnetism analysis after incubation. The results of different cell types (PBLs from fresh blood, PBLs after MLR, KG1a cell line, MCF-7 cell line, etc.) were summarized and compared.

The study showed that nonspecific binding depends on a number of factors including: cell characteristics such as cell type, cell incubation time, cell incubation conditions, surface binding, internalization of particles into the cells, and cell binding to the separator itself. Implications and operating principles learned from these studies will be discussed.