Novel cryoprotectant significantly improves the post-thaw recovery and quality of HSC from CB
Introduction
Cord blood (CB) is a rich source of hematopoietic stem cells (HSC) and has been used in the treatment of children and adults with a range of hematologic, genetic and malignant disorders. Nucleated cell (NC) content of a CB donation is approximately 1 log less than that of a BM donation, and consequently cell dose for transplant is lower. There is a direct correlation between cell dose and both speed of engraftment and survival [1., 2., 3.]. Maintaining the integrity of the stem cells, from the time of collection, through processing and freezing, to when they are administered to the patient, is critical.
HSC are routinely cryopreserved in nitrogen at temperatures of −196°C; at these low temperatures almost all biologic functions are halted. The blood is kept at this temperature until it is ready to be used; this process involves rapidly thawing the blood in a waterbath at 37°C before being administered to the patient. The processes that cause the most damage to the cells are the freezing and thawing. In order to reduce the damaging effect of cryopreservation, DMSO is used. DMSO is an intracellular cryoprotectant that displaces water from within the cell and thus reduces the formation of ice crystals that would otherwise damage the cell. DMSO, however, is toxic to cells when exposed to temperatures above freezing. DMSO has also been associated with adverse effects to the transplant recipient.
HSC have been traditionally frozen using DMSO as cryoprotectant, diluted in dextran-40, saline or albumin, among others. Concentrations of 10% are widely used and there are also data showing 5% to be equally effective [4]. Worldwide, CB banks have been using cryopreservation protocols validated for other hematopoietic blood products [5] and validated for use with CB. However, there is a loss of cells put through the freeze–thaw cycle and new approaches that minimize this effect would be a step forward.
The product CryoStor™ (Biolife Solutions, Owego, NY, USA), is composed of dextran-40, sodium, potassium, calcium, magnesium, phosphate, HEPES, lactobionate, sucrose, mannitol, glucose, adenosine and glutathione. It has a pH of 7.6 and an osmolarity of 360. It is protein and serum free. The manufacturers have evaluated CryoStor™ with red blood, muscle, kidney and ovarian cells, amongst others. Results indicate CryoStor™ significantly improves post-thaw cell survival, provides more rapid recovery, higher yields and faster cell attachment and an enhanced cell survival at reduced cryoprotectant concentration [6, 7], by maintaining the viability and health of the cells during freezing, transportation and storage, and thereby reducing cryopreservation-induced cell damage.
The aim of this study was to establish first whether CryoStor™ is effective in cryopreserving HSC from CB, and secondly whether its use would be beneficial in increasing the quality and viability of the final stem cell product available for transplantation and/or stem cell expansion.
Section snippets
Methods
All procedures and evaluations (apart from CB collection) were carried out by one of the authors to avoid technician related variability.
Results
A total of 30 CB donations was processed for this study. Mean CB volume was 77 mL (range 49–129 mL), NC count was 19.4 × 106/mL (range 6.5–40.4 × 106/mL) and CD34+ viability was 99.3% (range 96.1–100%). The overall results are summarized in Table 1.
Discussion
Although cryopreservation of HSC has been performed for more than 30 years, new approaches to cryobiology need to be explored to optimize cell retrieval.
In this study we investigated the effects of a new extracellular cryoprotectant (CryoStor™) in freezing CB HSC. While CryoStor™ has been tested with a range of non-hematopoietic cells and tissues, CryoStor™ has not previously been tested on HSC, either from CB or BM. This study shows that CryoStor™, rather than dextran-40, results in
Disclaimer
Neither the Sydney Cord Blood Bank nor the authors have any monetary interests in the sales or production of CryoStor™. This research was funded by a research grant from Inner Wheel of Australia.
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