Density separation and cryopreservation of umbilical cord blood cells - Evaluation of recovery by means of short- and long-term culture

The clonogenic capacity of human umbilical cord blood (UCB) has been evaluated in several studies, showing high numbers of primitive hematopoietic progenitor cells. Recently, 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 considered an attractive source of hematopoietic stem cells for both research and clinical applications. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving UCB samples with minimal loss of progenitor cells. We compared separation over three different densities of Percoll (1069 g/ml, 1077 g/ml, 1084 g/ml), sedimentation over poligeline (Emagel), and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analysed for recovery of CD34(+) cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBC (hematocrit of the final cellular suspension 0.4 +/- 0.1%), while maintaining high recovery of CD34(+) cells (85.3%) and total recovery for CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming unit after five weeks of long-term culture (LTC). Moreover, the presence of SCF significantly increased CFU-GEMM (14 +/- 4 vs. 49 +/- 5, p less than or equal to 0.0005) and CFU-GM (112 +/- 18 vs. 178 +/- 19, p less than or equal to 0.025), but not BFU-E (42 +/- 7 vs. 53 +/- 7, p less than or equal to 0.375) growth. In conclusion, RBC depletion of UCB can be accomplished with minimal loss of committed and primitive hematopoietic progenitors. This procedure may have important implication in the large scale banking of UCB as well as ex vivo expansion/gene therapy protocols.