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Umbilical cord blood contains hematopoietic stem/progenitor cells that have proven useful clinically to reconstitute the hematopoietic system in children and some adults. Recent studies have suggested that cord blood contains mesenchymal stem/progenitor cells as well, which may have many additional uses on their own or in conjunction with their hematopoietic counterparts. In order to effectively utilize cord blood clinically, it must be frozen and banked. The protocols used for this have largely been adapted from those originally designed for bone marrow hematopoietic stem/progenitor cells, and there is no consensus on optimal procedures for cord blood cells. In this review, the considerations required to develop an ideal cryopreservation strategy are discussed, and an analysis of current literature related to these steps is presented. The closest consensus considering cord blood hematopoietic cells is presented as 5-10% concentrations of dimethyl sulfoxide (DMSO) using slow cooling and rapid thaw. New data involving a long-term culture-initiating cell (LTC-IC) assay testing such a protocol is also presented in which no statistical difference was observed for 5 vs. 10% DMSO at cooling rates of 1 °C/min. The ultimate impact of umbilical cord blood may be far more significant than is currently realized, and overall, clinical data justifies further development of umbilical cord blood banks worldwide. Cryopreservation processing that yields consistent high recovery of functionally viable cells is crucial for the further successful use of this important cell resource.

An optimal cooling rate is one of the critical factors influencing the survival of cells during cryopreservation. In this paper we describe a novel device, named the box-in-box, which was developed for optimal cryopreservation of human hematopoietic stem cells (HSC). This work presents the design of the device, a mathematical formulation describing the expected temperature histories of samples during the freezing process, along with actual experimental results of thermal profile tests. In experiments, when the box-in-box device was transferred from room temperature to a −80 °C freezer, a cooling rate of −1~−3.5 °C/min, which has been widely used for the cryopreservation of HSC, was achieved. In order to further evaluate this device, HSC cryopreservation was compared between the box-in-box device and a commercially available controlled rate freezer (CryoMed). The experimental data, including total cell population and CD34+ hematopoietic progenitor cell recovery rates, viability, and cell culture colony assays, showed that box-in-box worked as well as CryoMed instrument. There was no significant difference in either survival rate or the culture/colony outcome between the two devices. In conclusion, the box-in-box device can work as a cheap, durable, reliable and maintenance-free instrument for the cryopreservation of HSC. This concept of a box-in-box may also be adapted to other cooling rates to support cryopreservation in a wide variety of tissues and cells.

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