A rapid nylon-fiber syringe system to deplete CD14+ cells for positive selection of human blood CD34+ cells. Use of immunomagnetic microspheres.
To achieve a rapid and an efficient purification of CD34+ cells, we devised a nylon-fiber syringe (NF-S) and we manipulated it to deplete adherent cells from normal human blood mononuclear cells (MN cells). The cells processed by NF-S were further purified as the CD34+ fraction, using CD34 monoclonal antibody, immunomagnetic microspheres and chymopapain treatment to detach the microspheres. When steady-state human peripheral blood MN cells were processed by NF-S at 24 degrees C for 5 min, the frequency of monocytes (CD14+ cells) significantly decreased from 22.0 +/- 5.2% to 2.5 +/- 0.4%, with a 73% recovery of CD34+ cells. The subsequent immunomagnetic positive selection achieved preparations of 91 +/- 8% pure CD34+ cells with a 86 +/- 23% yield. The overall yield of CD34+ cells was 44 +/- 11%, and the time required for all procedures was 5 h. There was a tight and an inverse correlation (P < 0.0001) between the frequency of CD14+ cells in the initial cell population and the purity of CD34+ cells in the final preparation. A recommended frequency of CD14+ cells for achieving preparations of over 90% pure CD34+ cells was less than 4.4%. When combining NF-S and immunomagnetic microspheres, efficient bench-top separation of CD34+ cells in steady-state peripheral blood can be done in any laboratory, and fluorescence-activated cell-sorting is not required.
Yamaguchi
M
M
Department of Internal Medicine II, Hokkaido University School of Medicine, Japan.
Sawada
K
K
Sato
N
N
Koizumi
K
K
Sekiguchi
S
S
Koike
T
T
D018952:Antigens, CD34 / Q000032:analysis*; D001781:Blood Component Removal / Q000295:instrumentation / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods*; D018950:Lipopolysaccharide Receptors / Q000032:analysis*; D008863:Microspheres
D018952:Antigens, CD34; D018950:Lipopolysaccharide Receptors