DENSITY SEPARATION AND CRYOPRESERVATION OF UMBILICAL-CORD BLOOD-CELLS- EVALUATION OF RECOVERY IN SHORT-TERM AND LONG-TERM CULTURES

The clonogenic capacity of human umbilical cord blood (UCB) has been e valuated in several studies, which have shown high numbers of primitiv e hematopoietic progenitor cells. Recently, UCB progenitor cells were demonstrated to possess significant advantages over bone marrow (BM) i n terms of proliferative capacity and immunologic reactivity. Therefor e, UCB has been considered an attractive source of hematopoietic stem cells for both research and clinical applications. Previous reports ha ve documented a significant loss of progenitor cells by any manipulati on other than cryopreservation. We have evaluated the feasibility of f ractionating and cryopreserving UCB samples with minimal loss of proge nitor cells. We compared separation over three different densities of Percoll (1.069, 1.077 and 1.084 g/ml), sedimentation over poligeline ( Emagel), and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for re covery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed th e highest depletion of RBCs (hematocrit of the final cellular suspensi on 0.4 +/- 0.1%), while maintaining a high recovery of CD34+ cells (85 .3%) and total recovery of CFU-GEMM, BFU-E and CFU-GM. After cryoprese rvation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units after 5 wee ks of long-term culture. Moreover, the presence of Stem cell factor si gnificantly 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 acc omplished with minimal loss of committed and primitive hematopoietic p rogenitors. This procedure may have important implications in the larg e-scale banking of UCB and in ex vivo expansion/gene therapy protocols .