Abstract
Hematopoietic stem cells (HSCs) from umbilical cord blood have been successfully used to treat blood disorders but one major hurdle is the relatively low cell dose available. Double cord blood unit transplantation results in elevated engraftment failure because one unit predominates over the other. Various approaches are thus being undertaken to expand HSCs ex vivo from single cord blood units. We report here a protocol involving slow freezing (-1 A degrees C per minute to -120 A degrees C) + freezing medium containing DMSO + FBS + 24 h-50 % hWJSC-CM that enhances thaw-survival of CD34+ cells. Post-thawing, the fold, percentage and colony forming unit numbers of CD34+ cells were significantly increased (2.08 A +/- 0.3; 102 A +/- 1.17 %; 1.07 A +/- 0.02 respectively) while the percentages of apoptotic, necrotic, dead and sub-G1 phase cells (91.06 A +/- 3.63 %; 91.80 A +/- 5.01 %; 95.6 A +/- 3.61 %; 86.1 A +/- 16.26 % respectively) were significantly decreased compared to controls. Post-thaw culture in 24 h-50 % hWJSC-CM+FBS for 72 h showed further significant increases in CD34+ cells (fold: 2.28 A +/- 0.17; percentage: 153.3 A +/- 21.99 %, CFU: 1.6 A +/- 0.19) and significant decreases in apoptotic, necrotic, dead and sub-G1 cells (49.2 A +/- 3.59 %; 62.0 A +/- 4.30 %; 56.6 A +/- 5.06 %; 28.6 A +/- 5.74 % respectively) compared to controls. We hypothesize that these improvements are probably related to the high levels of cytokines, cell adhesion molecules and growth factors in hWJSC-CM that help to preserve cell membrane integrity during freezing and stimulate mitosis post-thaw. A 24 h-50 % hJWSC-CM may be a useful supplement for freezing CD34+ cells in cord blood banks.