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

BACKGROUND: 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) ). STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Discarded leukoreduction filters: a new source of stem cells for research, cell engineering and therapy?

New adult stem cell sources, devoid of the technical/ethical/economical barriers of those presently available, would favor the ongoing development of in vitro cell engineering and transplantation. Hematopoietic transplantation opened the way to and remains the most successful cell transplantation procedure. CD34+ cells that include hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPs) are presently harvested from bone marrow (BM), cord blood or peripheral blood (after being mobilized from BM). The panel of potential donors, the quantities of collected cells and some other technical/medical problems still represent limiting factors to their transplantation in some patients. Steady state peripheral blood (SSPB) contains very low frequencies of CD34+ cells. They are trapped in leukoreduction filters (LRFs), which are discarded after the preparation of therapeutic red blood cell concentrates from individual blood donations. We recently developed a procedure allowing the easy and rapid elution of CD34+ cells from LRFs and we showed that they are functionally similar to those harvested from other sources. After providing an overview of the sources, interests and limitations of therapeutic HSCs presently available, we will provide arguments based on our and others' results suggesting that SSPB could become an attractive source of HSCs for hematopoietic transplantation and of other cell types for various research/development procedures.