Direct immunomagnetic method for CD34+cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols

BACKGROUND: Cord blood is a useful source of HPCs for allogeneic transplant ation. HPC ex vivo expansion of a cord blood graft has been proposed as a w ay to increase the speed of engraftment and thus to reduce the occurrence o f transplantation-related complications. OBJECTIVE: The purpose of this study was to optimize a method for CD34+ cel l selection of thawed cord blood grafts under clinical grade conditions, in tended for application in a static, serum-free expansion culture. MATERIAL AND METHODS: Twelve samples were thawed and washed with dextran, a lbumin, and rHu-deoxyribonuclease I (RHu-DNase) to avoid clumping. CD34+ ce lls were selected by using a sensitized immunomagnetic bead and 9C5 MoAb co mplex. A buffer containing rHu-DNase, citrate, albumin, and immunoglobulin in PBS was used during the procedure. CD34+ cells were eluted and detached by using an immunomagnetic cell selection device. Cells from the enriched f raction were cultured for 6 days in serum-free medium supplemented with rHu -SCF, rHu-IL-3, rHu fetal liver tyrosine kinase 3 ligand, and rHu thrombopo ietin (50 ng/ml each). Cells were expanded in well plates and in two semipe rmeable bags. RESULTS: A mean of 1.94 x 10(6) (+/- 1.55) CD34+ cells was obtained, yieldi ng a CD34+ cell recovery of 52 +/- 12 percent. Nonspecific loss of CD34+ ce lls was 32 +/- 10 percent. CFU-GM and BFU-E/CFU-Mixed recoveries were 33 +/ - 15 percent and 27 +/- 12 percent, respectively. CD34+ cells obtained were functionally comparable with fresh CD34+ cells selected for clonogenic pot ential. The capacity for expansion was not significantly different in the t wo types of bags studied. HPCs in wells were expanded 33 +/- 14-fold for CD 34+ cells and 42 +/- Ig-fold for overall colonies. The expansion rates obse rved in wells were significantly superior to those obtained in bags. CONCLUSION: The feasibility of a clinical-scale cord blood selection proced ure based on a direct immunomagnetic method after thawing, followed by an e x vivo expansion culture using semipermeable bags, is shown. After 6 days o f expansion, it was possible to generate a 9-fold increase in CD34+ cells, a 6-fold increase in CFU-GM and a 13-fold increase in BFU-E/CFU-Mixed colon ies.