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

Summary: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 × 10 3 to 1 × 10 5 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 × 10 3 , 10 4 or 10 5 cells/ml and 45 ؎ 6% in cultured cells obtained from starting fractions containing 5 × 10 4 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 H 2 O 2 , 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.

Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells

“…This decrease in the cell counts was linked to the initiation of the apoptotic cascade. A priming period of 48 hrs was also shown to be essential by Giarratana et al [31] to evaluate the quality of expanded graft prior to its infusion into patient.In addition, such priming of the cells after revival also leads to the dilution of DMSO and FBS used during freezing, thus avoiding adversities associated with these two components. Therefore, it would be desirable to incubate revived cells for 48 hrs.…”

Large-Scale Ex Vivo Generation of Human Red Blood Cells from Cord Blood CD34+ Cells

“…Colony‐forming unit (CFU) assays of unexpanded human CB CD34 + cells and cells cultured for 5 days, 9 days, and 12 days were performed as described previously . Cells were incubated at 37°C in 5% CO 2 with >95% humidity for approximately 14 days.…”

Haematopoietic Culture Systems

“…Optimal seeding density for expansion of total cells, CD34 + cells, clonal progenitors, CFU-GM, BFU-E and LTC-ICs in this system has been noted at concentrations of 5 x 10 4 CD34 + BM cells/ml or 1 x 10 4 CB CD34 + cells/ml using serum-free media in a 14-day culture (Douay 2001). When the seeding density was increased, the production of terminally differentiated granulocytes also increased whilst overall cell expansion decreased (Giarratana 1998). Despite their widespread use, static cultures are limited to relatively low-density cultures with low total cell output.…”