Increase in post-thaw viability of cord blood HSC by adding rMnSOD: preliminary results

Umbilical cord blood (UCB) represents a source for hematopoietic stem cells (CB-HSC), and transplantation of cord blood has been part of clinical practice since more than 10 years (Lee et al., Blood 103, 2004). The UCB is usually collected by public or private cord blood banks, which store it for an unknown recipient for an undetermined time. The cryopreservation process, used for stem cell collection, is particularly critical for umbilical cord blood (Berz et al., Am J Hematol 82, 2007). The most used cryoprotectant agent (CPA) to avoid or reduce cell damage caused by ice crystals, is 10% dimethyl sulfoxide (Me2SO). Me2SO, while protecting the cells from membrane rupture, however has a toxic effect on them, depending on the temperature and the exposure time for both pre-freeze and post-thaw periods (Berz et al., Am J Hematol 82, 2007). Generation of oxygen-free radicals (ROS) is a major cause of cell damage during low-temperature storage (Katkova et al., Cryobiology 37 and Kearney, Burns 24, 1998). Cells counteract ROS accumulation through antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GPX). The addition of antioxidants to the conventional freezing medium improves the post-thaw cell recovery (Limaye et al., Stem Cell Res. 10, 2001). The purpose of this study is the improvement of cord blood HSC’s (CB-HSC) cryopreservation method by addition of a new isoform of recombinant manganese superoxide dismutase (rMnSOD), obtained from the native liposarcoma derived form (LSA-MnSOD) (Mancini et al., Int. J. Cancer 119, 2006). rMnSOD, while having the same enzymatic activity common to all SODs, differs for its higher molecular weight (30 kDa versus 24 kDa) due to the presence of an additional sequence, called leader peptide, not cleaved during its maturation. As demonstrated by Mancini et al. (2006), recombinant LSA-type MnSOD (rMnSOD) has a distinctive capacity to penetrate cells and converts ROS to H2O2. The latter is rapidly converted to O2 and H2O by catalase, thus resulting in a beneficial oxygenation of healthy cells. In this study, we isolated mononuclear cells by lymphoprep gradient tube (Pan Biotech, Germany) from cord blood after informed consent of healthy donors, received by the Cord blood Bank Ba.S.C.O of “Santobono Pausilipon” hospital of Naples. HSCs CD43+ were detected by flow cytometry and immunocytochemical methods at L.M., by using specific stem cell antibodies before and after the following experimental treatment. Cultured CB-HSCs were incubated with scalar concentrations from 1.5 µM to 0.015 µM of rMnSOD for 5 hours (with StemPro®-34 SFM, Life Technologies) and frozen using standard freezing protocol with 10% Me2SO. After thawing, CD34+ viable cells were counted by flow cytometry FACSCalibur II (Becton & Dickinson) by using BD Stem Cell Enumeration Kit (BD Biosciences). The results showed that the viability of CB-HSC CD34+ increased from 10% of the control to 45% by adding 0.15µM rMnSOD. Further experiments are progress to define the rMnSOD concentration able to obtain the greatest increase of post-thaw CB-HSC viability.