Obtaining of CD34+ cells from healthy blood donors: development of a rapid and efficient procedure using leukoreduction filters

Abstract

Human CD34+ cells are mandatory to study many aspects of human hematopoiesis. Their low frequency in blood or marrow and ethical reasons limit their obtainment in large quantities. Leukoreduction filters (LRFs) are discarded after preparation of red blood cells. The CD34+ cell concentration in healthy donor blood is low (1×10(3) -4×10(3) /mL), but their number trapped in one LRF after filtration of 400 to 450mL of blood is high (0.4×10(6) -1.6×10(6) ). To develop a procedure allowing obtainment of purified CD34+ cells from LRFs with a good yield, white blood cell (WBC) recoveries after a 500-mL continuous or after sequential elution (50- or 20-mL fractions) were compared. Different WBC and mononuclear cell (MNC) centrifugation methods were tested to minimize their PLT contamination before the CD34+ cell immunomagnetic selection. Cell functionality was finally analyzed under various culture conditions. The 20-mL back-flushing of LRFs allowed the most efficient WBC recovery. The next steps (110×g centrifugation, MNC separation on Ficoll, and washes) resulted in a cell suspension in which the lymphocyte recovery was approximately 76±10% and the PLT contamination below 1.6%. After immunomagnetic selection, 4×10(5) to 6×10(5) cells containing approximately 85% of functional CD34+ cells were obtained. This procedure allows the easy, rapid (<5hr), and efficient preparation of large quantities of CD34+ cells having functional activities similar to those of CD34+ cells from other sources. Therefore, easily available and virally safe, LRFs represent an important and regular WBC source to work with human CD34+ cells, but also with other WBC types.