THE EFFECT OF DIFFERENT THAWING METHODS, GROWTH-FACTOR COMBINATIONS AND MEDIA ON THE EX-VIVO EXPANSION OF UMBILICAL-CORD BLOOD PRIMITIVE AND COMMITTED PROGENITORS

Assuming a threshold of 2 x 10(7) nucleated cells (NC)/kg body weight required for transplantation and 10 +/- 5 x 10(8) NC per cord blood (C B) unit (n = 1828, July 1997), 100%, 65% and 25% of the CB units store d in the CB Bank Dusseldorf 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 overcom e by ex vivo expansion of cells important in the different phases of e ngraftment. Therefore, four combinations of SCF, Flt3-L, IL-3, erythro poietin and GM-CSF as well as three media were evaluated for their cap acity to amplify hematopoietic progenitors. A prerequisite for expansi on was the significantly higher recovery of CD34(+) cells, colony-form ing cells (CFC) and long-term culture-initiating cells (LTC-IC) by tha wing cryopreserved CB units with an isotonic albumin/dextran solution. When CD34(+) CB cells were cultured with the four cytokine combinatio ns in H5100 medium, all combinations promoted an expansion of total ce lls (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 pins IL-3 (94.3 +/- 62. 4-fold). H5100 medium or a serum-free medium supplemented with SCF, Fl t3-L plus IL-3 were superior to 20% FCS/RPMI-1640 medium in the expans ion of all progenitor cell types and were similarly effective in suppo rting the amplification of total cells, CFC, CFU-GM, BFU-E/CFU-E and L TC-IC (maximum at day 7: 6.7 +/- 3.4-fold and 5.5 +/- 0.5-fold, respec tively). However, the serum-free medium promoted a significantly highe r 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 demonst rate that committed progenitors as well. as the more immature CFU-GEMM and LTC-IC can be substantially amplified at the same time without ex hausting the proliferative potential.