Rapid and Effective Isolation Of (T) Cell Populations By a New Method Avoiding Magnetic Beads

In the last two decades, conventional isolation of therapeutic cells has been carried out using high affinity antibodies and magnetic bead technology. Although good purity and reasonable yields can be obtained in many cases, major disadvantages remain comprising biological interference of non-reversible selection reagents (e.g. stimulation, receptor blockade etc.), the difficulty to purify complex multi-parametric cell populations by positive selection, time consuming protocols as well as limitations in high-throughput processing. We report here on the development of a new non-magnetic and fast cell selection technology applying immune affinity chromatography. Therefore, a matrix consisting of beads coated with Streptactin and low-affinity recombinant Fab-fragments (Fab-Streptamers) directed against defined T cell surface antigens has been generated. Stable and specific target cell retention is achieved by passing whole blood or PBMCs over the affinity matrix. After binding and washing, target cells can be gently retrieved by D-biotin administration. Eluted cells are then passed over a second matrix removing D-biotin and free Fab-Streptamers, subsequently yielding in a label-free authentic cell population for further use. Most importantly, total cell processing times can be kept extremely short; depending on the sample size even down to several minutes. Sequential isolation steps are possible and allow positive selection of complex cell populations like regulatory T cells or central memory T cells defined by several markers in high yields and purities. We are currently integrating this approach into a fully closed separation device for clinical cell purification. In addition, high throughput cell selection for diagnostic or basic research applications can be achieved by embedding the matrix into pipette tips and the use of suitable pipetting robots.