The effect of different thawing methods, growth factor combinations and media on the ex vivo expansion of umbilical cord blood primitive and committed progenitors

Abstract

Assuming a threshold of 2 × 10⁷ nucleated cells (NC)/kg body weight required for transplantation and 10 ± 5 × 10⁸ NC per cord blood (CB) unit (n = 1828, July 1997), 100%, 65% and 25% of the CB units stored in the CB Bank Düsseldorf contain sufficient NC to engraft patients of 10 kg, 35 kg and 50–70 kg, respectively. Thus, there is a potential limitation for the use of CB in adults which, however, may be overcome by ex vivo expansion of cells important in the different phases of engraftment. Therefore, four combinations of SCF, Flt3-L, IL-3, erythropoietin and GM-CSF as well as three media were evaluated for their capacity to amplify hematopoietic progenitors. A prerequisite for expansion was the significantly higher recovery of CD34⁺ cells, colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) by thawing cryopreserved CB units with an isotonic albumin/dextran solution. When CD34⁺ CB cells were cultured with the four cytokine combinations in H5100 medium, all combinations promoted an expansion of total cells (43 to 356-fold) and CFC (49 to 462-fold) within 7 days, however, early progenitors as defined by mixed-colony formation (CFU-GEMM) were substantially amplified only with SCF, Flt3-L plus IL-3 (94.3 ± 62.4-fold). H5100 medium or a serum-free medium supplemented with SCF, Flt3-L plus IL-3 were superior to 20% FCS/RPMI-1640 medium in the expansion of all progenitor cell types and were similarly effective in supporting the amplification of total cells, CFC, CFU-GM, BFU-E/CFU-E and LTC-IC (maximum at day 7: 6.7 ± 3.4-fold and 5.5 ± 0.5-fold, respectively). However, the serum-free medium promoted a significantly higher expansion of CFU-GEMM (176.9 ± 81.7-fold) than H5100 medium (83.5 ± 26.2-fold) at day 7 and only under serum-free conditions, CFU-GEMM were maintained over 14 days in tissue culture. These results demonstrate that committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified at the same time without exhausting the proliferative potential.