Recovery and Post-Thaw Assessment of Human Umbilical Cord Blood Cryopreserved as Quality Control Segments and Bulk Samples

Abstract

Quality control (QC) segments conjoined to a bulk sample container are used to evaluate the viability and quality of cryopreserved umbilical cord blood (UCB). Such QC segments are typically attached lengths of sealed tubing that are cooled concurrently with the bulk sample, both containing material from the same donor. QC segments are thawed independently of the bulk sample to assess the quality of the cryopreserved product. In current practice, there is typically post-thaw variation between the QC segment and the bulk sample which if suggestive of inadequate performance, could lead to material being needlessly discarded. In this study, these performance differences were quantified. Two cooling protocols in common use, 1 with and 1 without a "plunge" step to induce ice nucleation, gave equivalent results that maintained the QC segment versus bulk sample differences. Ice nucleated at significantly lower temperatures in the QC segments compared with the bulk samples, a consequence of their lower volume, thereby enhancing damaging osmotic stress. A reduction in total viable cells of approximately 10% was recorded in the QC segments compared with comparable bulk samples. It has been shown that CD45 + cells are more adversely impacted by this lower ice nucleation temperature than CD34 + cells, which can result in altered composition of the post-thaw cell population.

Chemical approaches to cryopreservation

“…Despite the obvious benefits, controlled nucleation is often overlooked in cryopreservation protocols, potentially because procedures that include nucleation control can be cumbersome and non-trivial. For instance, physical methods such as ice seeding 111 , 114 , electrofreezing 116 and shock cooling 117 require direct intervention with the material, accurate timing for their use during the cooling profile and are also difficult to automate and scale up. Chemical nucleants, which could be added to the culture media, offer a promising alternative as they do not require specific timing of application, can be applied to automated workflows and would be compatible with a range of cryopreservation vessels, such as plates, vials and cell bags.…”

Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender

“…Cryopreservation offers stable, extended storage and samples can be cryopreserved immediately after extraction e.g. for cord blood and then stored in a cell bank until required [ 5 ]. Additional processing of an initial sample can also take place before cryopreservation e.g.…”

Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies—A Review

“…During continued, slow cooling ice crystals will form by nucleation at a temperature below their melting point ( Figure 3 ), and aqueous solutions can cool significantly below this before nucleation and relatively rapid, crystal formation occurs ( 162 , 163 ). This effect is known as supercooling and the point at which it ends can be detected by a rise in temperature (the ice exotherm) as the first ice crystals form in the bulk solution.…”

“…At an appropriate point, close to the melting point of the suspending medium, a very rapid, limited cooling excursion is included in the protocol to lower the sample temperature by up to 10°C before returning to the original level. However, any beneficial impact of such a practice over conventional, linear cooling protocols has yet to be demonstrated ( 163 ).…”

“…During freezing, even when frozen together, there may be a significant difference in rate of heat loss and degree of supercooling before ice nucleation between the two volumes. This can produce differences in their post-thaw survival ( 133 , 163 ). Recovery of the smaller, QC sample may therefore not accurately reflect the outcome when the bulk sample is thawed and it is essential to be aware of, and compensate for, such issues.…”