Evaluations of bioantioxidants in cryopreservation of umbilical cord blood using natural cryoprotectants and low concentrations of dimethylsulfoxide.

Transplantation using hematopoietic stem cells from umbilical cord blood (UCB) is a life-saving treatment option for patients with select oncologic diseases, immunologic diseases, bone marrow failure, and others. Often this transplant modality requires cryopreservation and storage of hematopoietic stem cells (HSC), which need to remain cryopreserved in UCB banks for possible future use. The most widely used cryoprotectant is dimethylsulfoxide (Me(2)SO), but at 37 degrees C, it is toxic to cells and for patients, infusion of cryopreserved HSC with Me(2)SO has been associated with side effects. Freezing of cells leads to chemical change of cellular components, which results in physical disruption. Reactive oxygen species (ROS) generation also has been implicated as cause of damage to cells during freezing. We assessed the ability of two bioantioxidants and two disaccharides, to enhance the cryopreservation of UCB. UCB was processed and subjected to cryopreservation in solutions containing different concentrations of Me(2)SO, bioantioxidants and disaccharides. Samples were thawed, and then analysed by: flow cytometry analysis, CFU assay and MTT viability assay. In this study, our analyses showed that antioxidants, principally catalase, performed greater preservation of: CD34+ cells, CD123+ cells, colony-forming units and cell viability, all post-thawed, compared with the standard solution of cryopreservation. Our present studies show that the addition of catalase improved the cryopreservation outcome. Catalase may act on reducing levels of ROS, further indicating that accumulation of free radicals indeed leads to death in cryopreserved hematopoietic cells.

Evaluation of trehalose and sucrose as cryoprotectants for hematopoietic stem cells of umbilical cord blood.

Bone marrow transplantation (BMT) is a therapeutic procedure that involves transplantation of hematopoietic stem cells (HSC). To date, there are three sources of HSC for clinical use: bone marrow; mobilized peripheral blood; and umbilical cord blood (UCB). Depending on the stem cell source or type of transplantation, these cells are cryopreserved. The most widely used cryoprotectant is dimethylsulfoxide (Me(2)SO) 10% (v/v), but infusion of Me(2)SO-cryopreserved cells is frequently associated with serious side effects in patients. In this study, we assessed the use of trehalose and sucrose for cryopreservation of UCB cells in combination with reduced amounts of Me(2)SO. The post-thawed cells were counted and tested for viability with Trypan blue, the proportion of HSC was determined by flow cytometry, and the proportion of hematopoeitic progenitor cells was measured by a colony-forming unit (CFU) assay. A solution of 30mmol/L trehalose with 2.5% Me(2)SO (v/v) or 60mmol/L sucrose with 5% Me(2)SO (v/v) produced results similar to those for 10% (v/v) Me(2)SO in terms of the clonogenic potential of progenitor cells, cell viability, and numbers of CD45(+)/34(+) cells in post-thawed cord blood cryopreserved for a minimum of 2 weeks. Thus, cord blood, as other HSC, can be cryopreserved with 1/4 the standard Me(2)SO concentration with the addition of disaccharides. The use of Me(2)SO at low concentrations in the cryopreservation solution may improve the safety of hematopoietic cell transplantation by reducing the side effects on the patient.

Bone marrow stroma inhibits proliferation and apoptosis in leukemic cells through gap junction-mediated cell communication.

Normal and leukemic blood cell progenitors depend upon the bone marrow (BM) stroma with which they communicate through soluble and membrane-anchored mediators, adhesive interactions and gap junctions (GJ). Regarding hematopoiesis, it is believed that it can be influenced by connexin expression, but the exact role of GJ in cell death and proliferation is not clear. Using flow cytometry, we monitored the division rate of leukemic cell lines, communicating and not communicating with stromal cell line through GJ. We found that GJ-coupled cells (i) did not proliferate; (ii) were kept in G0; and (iii) were protected from drug-induced apoptosis when compared to either total or uncoupled cell population. We conclude that GJ coupling between stroma and leukemic lymphoblasts prevents proliferation, keeping cells in a quiescent state, thus increasing their resistance to antimitotic drugs. Since GJ are particularly abundant in the sub-endosteal environment, which harbors blood stem cells, we also asked which cells within the normal human BM communicate with the stroma. Using a primary BM stroma cell culture, our results show that 80% of CD34+ progenitors communicate through GJ. We propose that blood cell progenitors might be retained in the low-cycling state by GJ-mediated communication with the hematopoietic stroma.