Background: Current procedures for the cryopreservation of umbilical cord blood (UCB) progenitor cells, which are based on techniques used for BM, have had varying degrees of success (survival 9-118%). Improving the effectiveness of UCB cell therapies demands a more comprehensive understanding of freezing injury during cryopreservation.
Methods: Leukocyte concentrates from UCB, with or without 10% DMSO were cooled at 1 degrees C/min to different subzero temperatures (-5 to -50 degrees C), then either thawed directly (thaw) or plunged into liquid nitrogen before thawing (plunge). Single-platform flow cytometry with 7-amino-actinomycin D was used to directly quantify survival of CD34(+) cells. Fluorescent microscopy was used to examine plasma membrane integrity of nucleated cells.
Results: Without DMSO, recovery of nucleated cells was approximately 80% for both thaw and plunge. Survival was 9%, indicating damage to the plasma membrane. With 10% DMSO, nucleated cell recovery was also approximately 80%, indicating that DMSO does not improve recovery of nucleated cells. Survival, however, was much higher with DMSO, > 60% for nucleated cells thawed directly, and 30-55% for cells thawed from plunge, demonstrating cryoprotection conferred by DMSO. With DMSO, survival of CD34(+) cells was higher than that of nucleated cells, indicating that CD34(+) cells with 10% DMSO are more tolerant to cryopreservation than the total nucleated cell population.
Discussion: This study provides the necessary data on the low temperature response of UCB progenitor cells that are critical for the development of standards for the cryopreservation of UCB.