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 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.


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.


CONCLUSIONS
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.
Querol
S
S
Cell Therapy Center and Barcelona Cord Blood Bank, Cancer Research Institute, Llobregat Hospital, Barcelona, Spain. squerol@iro.es
Capmany
G
G
Azqueta
C
C
Gabarró
M
M
Fornas
O
O
Martín-Henao
G A
GA
García
J
J
D018952:Antigens, CD34 / Q000032:analysis*; D015415:Biomarkers; D001772:Blood Cell Count; D001793:Blood Preservation; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D015925:Cryopreservation; D005312:Fetal Blood / Q000166:cytology*; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D018189:Immunomagnetic Separation; D007231:Infant, Newborn; D008565:Membrane Proteins / Q000494:pharmacology
D018952:Antigens, CD34; D015415:Biomarkers; D016298:Hematopoietic Cell Growth Factors; D008565:Membrane Proteins; C084881:flt3 ligand protein