A new density gradient for the separation of large quantities of rosette-positive and rosette-negative cells

Allogeneic transplantation of peripheral blood stem cells will not be feasible until new techniques are developed for large-scale depletion of T-lymphocytes. Small quantities of cells can be depleted of T-lymphocytes by sheep erythrocyte rosetting and Ficoll-diatrizoate discontinuous gradient fractionation. However, when processing 10(9)-10(10) cells, the fractionation step is inefficient because of the limited capacity of Ficoll-diatrizoate. We therefore developed a new discontinuous gradient for the separation of large numbers of sheep red blood cell (RBC) rosette-positive and negative cells. The gradient was designed so that sheep cells and rosettes would not interfere with the banding of rosette-negative cells. In that way, nonspecific entrapment was reduced, and high cell capacity and yield were achieved. The system fractionated rosetted cell suspensions into four populations: free sheep RBC, rosetted T cells, nonrosetted T cells, and low-density non-T cells. The 15-ml gradient routinely separated 3-24 X 10(8) cells. Progenitor yield ranged from 83% to 99%, with 95% depletion of T-lymphocytes. The method is rapid, reproducible, and inexpensive. This preparative technique could prove useful clinically when large-scale separation of E-rosette-positive and negative cells is required.