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Published on: July 16, 2011
Large scale purification of human blood CD34(+) cells using a nylon-fiber syringe system and immunomagnetic
1Department of Internal Medicine II, Hokkaido University School of Medicine, Sapporo, Japan.
Background:
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.
Methods:
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 2 10(8) NF-S treated cells (NF cells), the recovery and the purity of CD34(+) cells was identical for 5, 10, 20, 40 microg of 9C5. In this study, therefore, the concentration of 9C5 was maintained at >5 microg/10(8) NF cells, with a cellhead ratio of 1:10.
Results:
When half to one-third of leukapheresis product containing 121.5 + or - 26.0 2 10(8) mononuclear cells (mean + or - SD; n = 6) was processed for CD34(+) cell purification, 5.26 + or - 3.01 2 10(7) 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 + or - 2.33 2 10(7) cells and 145.9 + or - 121.8 2 10(5) cells, respectively. The total recovery of CD34(+) cells was 37.0 + or - 21.0%. The depletion of monocyte by NF-S reduced the 9C5 anti-human CD34 MAb needed for purification 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.
Discussion:
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.
