Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for large-scale clinical applications

Abstract

The aim of this study was to evaluate the ex vivo expansion of normal CD34⁺ cells in gas-permeable polypropylene bags suitable for clinical use. Cells were cultured for 14 days in serum-free medium supplemented with SCF, IL3, IL6, FLT3-l, G-CSF ± MGDF or Epo. The bags supported the expansion of hematopoietic cells in a similar manner to small scale well or flask systems, allowing mean expansions of up to 2193-fold for total nucleated cells, 140-fold for CFU-GM and 66-fold for LTC-IC. Increasing the initial cell concentration from 5 × 10³ to 1 × 10⁵ CD34⁺ cells/ml induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion of the white blood cells produced. We tested the phagocytic activity and oxidative metabolism of the white blood cells produced. The percentage of phagocytic cells was 39 ± 0.5% in expanded cultures derived from fractions initiated at 5 × 10³, 10⁴ or 10⁵ cells/ml and 45 ± 6% in cultured cells obtained from starting fractions containing 5 × 10⁴ cells/ml, as compared to 58 ± 4% in normal controls. A study of the potential for oxygen-dependent microbe killing showed that the expanded cells produced H₂O₂, although in lesser quantities than control cells. We subsequently investigated the possibility of freezing expanded cells. Total cell recovery after thawing was 45 ± 4%, while recoveries of progenitors and stem cells ranged from 65 to 90%, without any influence of the initial cell concentration. This new approach could be of major interest for clinical practice, as it would allow evaluation of the quality of a graft prior to its infusion and employs experimental conditions which meet the criteria for potential clinical use.