An efficient large-scale thawing procedure for cord blood cells destined for selection and ex vivo expansion of CD34+ cells.

Abstract

THE MAIN DISADVANTAGES of transplantation of cord blood (CB) hematopoietic stem or progenitor cells (HSPC) are the delayed neutrophil and platelet reconstitution coupled with the difficulty in obtaining a large enough graft for patients weighing more than 30 kg (1). Both problems could be overcome by ex vivo expansion of CB progenitors. In most CB banks, the whole CB samples are stored in individual or double bags. The ex vivo expansion of progenitors in whole blood samples is inefficient; thus, a CD341 selection before an expansion procedure is a prerequisite. However, this selection using frozen-thawed blood samples is impaired by aggregate formation (“clumping”). To overcome this problem, addition of recombinant human deoxyribonuclease (DNase) has been proposed during thawing of CB cells prior to transplantation (2) or expansion (3). We have extended this procedure to clinical-grade conditions incorporating subsequent CD341 cell selection and expansion in two-step culture conditions. Two bags [(Macopharma, Lille, France) GSR 7000 A), each containing 125 ml of CB and 125 ml of cryoprotecting solution 20% dimethylsulfoxide (DMSO), from a single CB and frozen by using a controlled-rate procedure and stored in liquid nitrogen, were thawed in a water bath at 37°C. DNase (Pulmozyme Roche, Neuilly, France) (3750 units) and the MgCl2 solution (Cooper, Mellin, France) (0.5 M, 250 ml) were added to each bag. Two samples were pooled and then rehydrated slowly (10 min at 20–22°C) by adding 250 ml of Dextran 40-sorbitol solution (Braun Medical, Boulogne, France) and 3.3 ml of Gamma IV (0.5 g LFB). The cell suspension was filtered, incubated with anti-CD34, and transferred into the separation chamber (system Isolex 300i, Baxter, Deerfield, IL). An additional 7500 Units of DNase and 500 ml of MgCl2 were added before the addition of immunomagnetic microbeads (Baxter kit). The suspension was processed on Isolex 300i. The CD341 cells were transferred into 50-ml conical tubes and centrifuged for 10 min at 1500 rpm. The supernatant was removed, and the cells were resuspended in 10 ml of serum-free culture medium (Irvine Scientific, Santa Ana, CA). The CD341 cells were expanded in the same serum-free medium (200 ml; 10,000 cells/ml) in gas-permeable bags (Opticyte 390 cm2, Baxter, Maurepas, France) in the presence of stem cell factor (SCF; Amgen, Thousand Oaks, CA), megakaryocyte growth and development factor (MGDF) (Amgen), Flt3 ligand (RD Median 3.333 106 n 5 5) CD341 cells per sample (Fig. 1A), i.e., a mean recovery of 48.5% with respect to the values found in fresh CB samples (Fig. 1B). The purity of these