Summary:We tested two positive selection techniques for separation of CD34 + cells from bone marrow and analyzed the yields of CD34 + cells, BFU-E, CFU-GM, CFU-MK and LTC-IC after selection and expansion. An immunoadsorption procedure (CellPro) and an immunomagnetic (Baxter) CD34 + cell separation method were employed to purify the same bone marrow samples from seven normal subjects. Mean yields of CFU-GM and CFU-MK and absolute numbers of LTC-ICs were not different in the two purified cell populations. In contrast, the mean recovery of BFU-E was significantly lower for the immunoadsorption (21 ؎ 14%) than for the immunomagnetic technique (44 ؎ 27%). After separation, CD34 + cells were evaluated in 10-day liquid cultures for their expansion capacity in terms of total cells and progenitors. The expansion capacity of progenitors such as CFU-GM, CFU-MK and especially BFU-E selected by immunoadsorption was higher than the capacity of progenitors obtained by immunomagnetism, although final total and progenitor cell numbers are similar. Our results suggest that the populations separated by the two techniques differ mainly in the expansion capacity of progenitors and in the recovery of BFU-E after the selection procedure. These differences between two methods, which already are widely employed in research and in clinical transplantation, should be taken into account when considering the aims of the experiments. Keywords: CD34 antigen; cell separation; ex vivo expansion; long-term culture-initiating cells Hematopoietic stem/progenitor cells specifically express the CD34 surface antigen and this property provides a basis for the purification of stem cells from different sources. The several techniques commercially available today to purify CD34 + cells, which have major applications both in fundamental research and in clinical stem cell transplantation, differ by the procedures and anti-CD34 antibodies employed. 1 Our objective was to compare the phenotypic profile and clonogeneic capacity before and after expansion of cells selected by immunoadsorption 2 or using an immunomagnetic system. 3 These two commercial systems are, to date, the most widely employed in a variety of clinical settings. Until now, all teams who compared different selection techniques used different samples for each technique. 4,5 Thus, in the present study, the same bone marrow sample was used simultaneously for both CD34 + cell separation methods while, in addition, the expansion capacity of the selected cells was investigated in liquid cultures.
Materials and methods
Bone marrow (BM) cell preparationBM cells were obtained from patients undergoing hip surgery, all of whom gave informed consent for BM cell donation. Light-density mononuclear cells (MNCs) were separated on a Ficoll-Isopaque density gradient (1.077 g/ml; Seromed, Biochrom, Berlin, Germany). MNCs were washed twice and the cell concentration was adjusted to 0.5-1 × 10 8 /ml in Ca-and Mg-free PBS (Gibco, Life Technologies, Paisley, UK) supplemented with 1% bovine serum albumin (BSA) pro...
