(729g) High-Throughput Continuous Magnetic Sorting of Unlabeled Red Blood Cells (RBC) and Analysis by Cell Tracking Velocimetry (CTV)

We have designed and are developing a continuous magnetic sorter that can process  up to 10¹¹ RBCs in 24 hours from a bioreactor, producing these RBC.  This research is  part of a much larger project, sponsored by DARPA, to produce a portable, self-renewing supply of blood transfusions for battlefield casualties, i.e. “blood Pharming”.  These RBCs are produced from hematopoetic stem cells isolated from human placenta; allowed to grow in an undifferentiated state for many generations, and finally induced to produce mature RBC. Since cells grow at different rates, and do not necessarily differentiate all at the same time, mature RBCs must be continually removed from culture.

Since hemoglobin contains iron, mature RBCs are inherently magnetic and no additional labeling is required. Two primary technical challenges confront magnetic sorting of transfusable RBCs: gas exchange and scale-up.

The net electron spin of hemoglobin’s heme complex is positive, thereby inducing a magnetic susceptibility, only in the absence of oxygen. Therefore, in order to sort RBCs, degassing membranes are used to quickly and efficiently remove oxygen from the cell suspension. Immature, nonmagnetic progenitor cells are quickly returned to the oxygenated bioreactor.

      A continuous separation channel with one inlet and two outlets has been designed to meet the capacity of continuous processing 10¹¹ RBCs in 24 hours. Using 2 by 2 four-channel parallel system design, we were able running the separation at concentration at 10⁸ cells/ml, and flow rate at 0.1 ml/min. This would give the capacity of up to 10¹¹ RBCs in 24 hours with two systems.

      Over the last decade we have been developing and using an instrument, referred to as CTV, which allows the velocity of hundreds of cells and particles to be simultaneously determined, using microscopic imaging and computer analysis, in a well defined and nearly constant magnetic energy gradient. By plotting the settling velocity and magnetic velocity at the same time, we are able to further analyze not only the cell population, but further characterize the various fractions that are separated.