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Published on: July 16, 2011
Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion
S Querol1, G Capmany, C Azqueta
1Cell Therapy Center and Barcelona Cord Blood Bank, Cancer Research Institute, Llobregat Hospital, Barcelona, Spain. squerol@iro.es
Background:
Cord blood is a useful source of HPCs for allogeneic transplantation. HPC ex vivo expansion of a cord blood graft has been proposed as a way to increase the speed of engraftment and thus to reduce the occurrence of transplantation-related complications.
Objective:
The purpose of this study was to optimize a method for CD34+ cell selection of thawed cord blood grafts under clinical grade conditions, intended for application in a static, serum-free expansion culture.
Material And Methods:
Twelve samples were thawed and washed with dextran, albumin, and rHu-deoxyribo-nuclease I (RHu-DNase) to avoid clumping. CD34+ cells were selected by using a sensitized immunomagnetic bead and 9C5 MoAb complex. 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 fraction 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 thrombopoietin (50 ng/mL each). Cells were expanded in well plates and in two semipermeable bags.
Results:
A mean of 1.94 x 10(6) (+/- 1.55) CD34+ cells was obtained, yielding a CD34+ cell recovery of 52 +/- 12 percent. Nonspecific loss of CD34+ cells 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 potential. The capacity for expansion was not significantly different in the two types of bags studied. HPCs in wells were expanded 33 +/- 14-fold for CD34+ cells and 42 +/- 19-fold for overall colonies. The expansion rates observed in wells were significantly superior to those obtained in bags.
Conclusion:
The feasibility of a clinical-scale cord blood selection procedure based on a direct immunomagnetic method after thawing, followed by an ex vivo expansion culture using semipermeable bags, is shown. After 6 days of 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 colonies.
