Damage and protection of UC blood cells during cryopreservation

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.

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.

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.

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.