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

Authors


  • Supported in part by grants 97/2120 and 99/0612 from the Spanish Fondo de Investigaciones Sanitarias, and the Fundación José Carreras para la lucha contra la Leucemia.

Address reprint requests to: Sergio Querol, MD, Institut de Recerca Oncològica, Autovia de Castelldefels km 2,7, L'Hospitalet de Llobregat, 08907 Barcelona, Spain; e-mail: squerol@iro.es.

Abstract

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 × 106 (± 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.

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