A rapid nylon-fiber syringe system to deplete CD14⁺ cells for positive selection of human blood CD34⁺ cells. Use of immunomagnetic microspheres

Abstract

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°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.