Cryopreservation of umbilical cord blood with a novel freezing solution that mimics intracellular ionic composition.

BACKGROUND: Cryopreservation protocols have remained relatively unchanged since the first umbilical cord blood banking program was established. This study evaluated the preservation efficacy of a novel intracellular-like cryopreservation solution (CryoStor, BioLife Solutions, Inc.), the rate of addition of two cryopreservation solutions to cord blood units (CBUs), and reduced final dimethyl sulfoxide (DMSO) concentration of 5%. STUDY DESIGN AND METHODS: Split-sample CBUs were cryopreserved with either an in-house 20% DMSO-based cryopreservation solution or CryoStor CS10 at a rate of 1 mL/min (n = 10; i.e., slow addition) or as a bolus injection (n = 6; i.e., fast addition). Infrared images of exothermic effects of the cryopreservation solutions were monitored relative to the rate of addition. Prefreeze and postthaw colony-forming unit assays, total nucleated cells, and CD34+ cell counts were compared. RESULTS: Maximum temperature excursions observed were less than 6°C, regardless of the rate of solution addition. Fast addition resulted in peak excursions approximately twice that of slow addition but the magnitude and duration were minimal and transient. Slow addition of CryoStor CS10 (i.e., final concentration ≤ 5% DMSO) resulted in significantly better postthaw CD34+ cell recoveries; no other metrics were significantly different. Fast addition of CryoStor resulted in similar postthaw metrics compared to slow addition of the in-house solution. CONCLUSION: Slow and fast addition of cryopreservation solutions result in mean temperature changes of approximately 3.3 to 4.45°C. Postthaw recoveries with CryoStor were equivalent to or slightly better than with the in-house cryopreservation solution. CryoStor also provides several advantages including reduced processing time, formulation consistency, and reduced DMSO in the frozen product (≤ 5%).

Development of a novel assay to evaluate the functional potential of umbilical cord blood progenitors.

BACKGROUND: Although the colony-forming cell (CFC) assay provides the most relevant information regarding the functional potential of progenitors in a unit of umbilical cord blood (UCB), technical challenges associated with this assay have made it difficult to standardize the assay among testing laboratories. The purpose of this study was to assess the reproducibility of a newly developed functional assay (HALO SPC-QC [HALO], HemoGenix, Inc.). This test is based on the principle that cellular proliferation responses to cytokine stimuli are proportional to intracellular ATP levels from progeny cells generated in culture from progenitors. STUDY DESIGN AND METHODS: Results of the HALO assay were evaluated at two geographically distinct sites with matched aliquots from 12 different UCB units. RESULTS: A significant correlation between the two sites for total nucleated cell counts was observed (r = 0.98, p < 0.001). Similarly, a strong correlation between HALO results from both sites was observed (r = 0.94, p < 0.001). Also, despite using different methods at each site for the CFC assay, results from the two sites correlated (r = 0.79, p = 0.002). A good correlation between the CFC and HALO assays (r = 0.73, p < 0.005), however, was only observed at the site with the same cytokine cocktail for both the CFC and the HALO assays. CONCLUSION: These results support the notion that the HALO assay is a reasonable approach for measuring the functional potential of hematopoietic progenitors in UCB. Moreover, because the final readout for the HALO assay is instrument based, unlike the CFC assay, which requires a subjective enumeration of colonies, the HALO assay may be more amenable to standardization.