Cord Blood Unit Cryopreservation: Positioning Segments for Potency Assessment

Introduction: Banked, unrelated umbilical cord blood units (CBU) are a rich source of hematopoietic stem cells for hematopoietic reconstitution. Potency assays on attached segments have been used for CBU release from the bank to the transplant center. CBUs have historically been manufactured and cryopreserved in bags with three segments (made from the tubing used to fill the bag) arranged across the top of the cassette. Prior to cryopreservation, the CBU is overwrapped, placed in a metal cassette and frozen in a control-rate freezer and submerged under liquid nitrogen for long-term storage. After cryopreservation, segments can be removed from the cassette, without disturbing the bag, for HLA typing, potency assays, or other testing. Last year, in an effort to increase the number of segments available for testing, we changed the configuration of the cryobag to create a fourth segment.  To fit in the cassette, the segments were arranged with two segments across the top and two segments bent at 90 degrees to fit between the 20% and 80% portions of the bag. When performing our annual stability testing, we realized that the two distal segments, positioned between the 20% and 80% compartments yielded lower potency results than the segments positioned across the top of the cassette.  We investigated this finding as described below.

Method: Three large fresh CBUs were processed separately on a Sepax cell processor (Biosafe, Geneva, Switzerland) and evenly divided into two cryopreservation bags. For each CBU, one bag was created with three segments and the other bag with four segments. The cryopreservative, dimethylsulfoxide in dextran, was added at a final concentration of 10% to both bags prior to cryopreservation. Segments were positioned either across the top (3 segments) or in the bent configuration (4 segments) for each of the paired CBUs. All units were cryopreserved and stored in a Thermogenesis Bioarchive.  After two weeks in storage, the CBUs were tested. The bags and segments were assayed for ALDH^br, viable CD34, CD45, glycophorin A and viability (7-AAD) along with colony forming units (CFUs), the potency assay used by our bank.

Results: The segments configured across the top of the bag correlated with results from the cryobag for ALDH^br (as a percent of viable CD45⁺, Table 1), CD34⁺ (as a percent of viable CD45⁺) and CFU. In contrast, segments placed between the 20% and 80% compartments were significantly lower than the bag for both ALDH^br (as a percent of viable CD45⁺) and CFU, although CD34⁺ (as a percent of viable CD45⁺) was equivalent.

Conclusions: Segments attached to CBUs can predict potency of the CBU by various parameters. However, proper placement of the segments within the cryopreservation cassette must be performed to ensure that potency measured on the segments reflects potency measured on the cord blood unit.