Cryopreservation is an essential step for autologous hematopoietic stem cell (HSC) transplantation and umbilical cord blood units (CBUs), and for allogeneic peripheral blood stem cells (PBSCs) or bone marrow (BM) when immediate infusion is not possible. However, the cryoprotectant dimethyl sulfoxide (DMSO) used for HSC cryopreservation can be toxic to cells post-thaw and to patients during infusion. The Rubinstein solution is validated to wash HSCs, but the unavailability of Dextran40 in Italy prompted a search for alternatives. This report discusses the use of Gelofusine, a 4% modified gelatin solution, as a substitute for Dextran40-based solutions in washing cryopreserved stem cell products.

The study includes: (1) validation of Gelofusine in 10 CBUs unsuitable for transplantation; (2) outcomes of the first 93 transplanted units washed with Gelofusine; (3) comparisons of recovery and viability in five paired autologous PBSC products washed with Gelofusine and Rubinstein-solution; and (4) comparisons of engraftment times in patients receiving units washed with Gelofusine and Rubinstein-solution.

For 10 CBUs washed with Gelofusine, CD34+ and TNC viability and recovery were 96%, 87%, 71%, and 75% respectively, higher than our reference values. In transplanted products, CD34+ and TNC viability and recovery were 96%, 89%, 82%, and 91% respectively. Comparisons with Rubinstein solution revealed similar TNC and CD34+ recovery but significantly higher TNC (89% vs. 68%) and CD34+ (97% vs. 89%) viability with Gelofusine. Engraftment times for both solutions were similar. These findings support Gelofusine as an effective and valid alternative to Rubinstein-solution for washing cryopreserved HSCs.

Transplantation of hematopoietic stem cells (HSCs) has been successfully developed as a part of treatment protocols for a large number of clinical indications, and cryopreservation of both autologous and allogeneic sources of HSC grafts is increasingly being used to facilitate logistical challenges in coordinating the collection, processing, preparation, quality control testing and release of the final HSC product with delivery to the patient. Direct infusion of cryopreserved cell products into patients has been associated with the development of adverse reactions, ranging from relatively mild symptoms to much more serious, life-threatening complications, including allergic/gastrointestinal/cardiovascular/neurological complications, renal/hepatic dysfunctions, and so on. In many cases, the cryoprotective agent (CPA) used-which is typically dimethyl sulfoxide (DMSO)-is believed to be the main causal agent of these adverse reactions and thus many studies recommend depletion of DMSO before cell infusion. In this paper, we will briefly review the history of HSC cryopreservation, the side effects reported after transplantation, along with advances in strategies for reducing the adverse reactions, including methods and devices for removal of DMSO. Strategies to minimize adverse effects include medication before and after transplantation, optimizing the infusion procedure, reducing the DMSO concentration or using alternative CPAs for cryopreservation and removing DMSO before infusion. For DMSO removal, besides the traditional and widely applied method of centrifugation, new approaches have been explored in the past decade, such as filtration by spinning membrane, stepwise dilution-centrifugation using rotating syringe, diffusion-based DMSO extraction in microfluidic channels, dialysis and dilution-filtration through hollow-fiber dialyzers and some instruments (CytoMate, Sepax S-100, Cobe 2991, microfluidic channels, dilution-filtration system, etc.) as well. However, challenges still remain: development of the optimal (fast, safe, simple, automated, controllable, effective and low cost) methods and devices for CPA removal with minimum cell loss and damage remains an unfilled need.