Large scale purification of human blood CD34+ cells using a nylon-fiber syringe system and immunomagnetic microspheres

To establish an available, economical technique for the large-scale purification of CD34+ cells we used a nylon-fiber syringe (NF-S) for depletion of adherent cells and then selected CD 34+ cells from peripheral blood mobilized by G-CSF, using MAb and magnetic heads. PBSC were mobilized and collected from adult, healthy volunteers. With the effect of concentration of anti-CD34 MAb (9C5) on the purification of CD34+ cells from 1×108 NF-S treated cells (NF cells), the recovery and the purity of CD34+ cells was identical for 5, 10, 20, 40μg of 9C5. In this study, therefore, the concentration of 9C5 was maintained at >5μg/108 NF cells, with a cellbead ratio of 1:10. When half to one-third of leukapheresis product containing 121.5±26.0×108 mononuclear cells (mean±SD; n=6) was processed for CD34+ cell purification, 5.26±3.01×107 total purified cells were obtained with a purity of CD34+ cells at 94.9±8.5%. The numbers of CD34+ cells and total progenitor cells recovered in this process were 4.71±2.33×107 cells and 145.9+121.8×105 cells, respectively. The total recovery of CD34+ cells was 37.0±21.0%. The depletion of monocyte by NF-S reduced the 9C5 anti-human CD34 MAb needed for purifcation of CD34+ cells to one-third. Two patients were grafted with peripheral blood CD34+ cells selected by this procedure and achieved a rapid and consistent hematopoiesis. Our clinical data show that these cells are capable of rapid reconstitution of hematopoiesis after high-dose chemotherapy. The procedure contains an additional step; however, consistent high purity of CD34+ cells in the purified population and cost reduction were achieved, both critical issues in the better purging of malignant cells and for a wide clinical application.