Clonogenic capacity and ex vivo expansion potential of umbilical cord blood progenitor cells are not impaired by cryopreservation

Umbilical cord blood (UCB) progenitor cells have been demonstrated to possess significant advantages over bone marrow (BM), in terms of proliferative capacity and immunologic reactivity. Therefore UCB has been recently been considered an attractive potential alternative to BM as a source of hematopoietic progenitor cells for clinical applications. Since several programs throughout the world are currently evaluating the feasibility of large-scale UCB banking for unrelated transplants, it was the aim of this study to evaluate whether cypropreservation procedures might heavily impair the clonogenic capacity, the feasibility of CD34⁺ selection and the ex vivo expansion potential of UCB progenitor cells. UCB samples were collected and cypropreserved as unseparated (n = 21) or mononuclear (MNC) cells (n = 15) within 12 h from delivery, and evaluated for viability, immunophenotype, cell and progenitor numbers after a minimum stay in liquid nitrogen of 6 months (range 6-14 months). Viability was always > 97% and no statistically significant difference was detected by flow cytometric analysis. Clonogenic recovery from unseparated cells was 80-87% for HPP-CFC, CFU-GEMM, BFU-E and CFU-GM, and from MNC cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, BFU-E and CFU-GM. CD34⁺ selection (n = 8) was performed on fresh and cypropreserved MNC cells using the MiniMACS immunomagnetic separation device, showing no difference in yield (68 ± 7%, vs 57 ± 4%, P ≤ 0.4) or in purity (89 ± 2% vs 81 ± 6%, ≤ 0.4), for fresh in comparison to cypropreserved MNC cells, After 14 days of liquid culture in the presence of different combinations of SCF, IL-3, IL-6 and G-CSF no statistically significant difference was detected in CFC fold-expansion for fresh or cypropreserved MNC cells and for CD34⁺ cells, either selected and cultured from fresh or cypropreserved MNC cells. In conclusion we can state that UCB is a potential source of primitive progenitor cells that can be cypropreserved unmanipulated or after physical separation without major losses in clonogenic capacity and immunophenotypic composition. More-over, CD34⁺ selection from cypropreserved MNC cells is feasible and ex vivo expansion is not impaired. These results have important implications in the large scale UCB banking, in view of the potential applications of ex vivo expanded hematopoietic progenitor cells for the engraftment of adult patients.