Cryopreservation of Human Hematopoietic Cells with Membrane Stabilizers and Bioantioxidants as Additives in the Conventional Freezing Medium

Abstract

Cord blood (CB) and fetal liver (FL) cells are two alternative sources of human hematopoietic stem cells. Optimization of cryopreservation protocols is an important aspect in the banking of these tissues. Out of the multiple factors responsible for cryodamage of cells, membrane leakage and oxygen free-radical generation have been shown to contribute substantially toward the process. We have studied the effect of certain additives, like membrane stabilizers and bioantioxidants, to the conventional freezing medium on viability, nucleated cell recovery, and clonogenic potential of frozen cells. Our results show that trehalose, a membrane stabilizer, when used in combination with 10% dimethyl sulfoxide (DMSO) affords better cryoprotection as evidenced by significantly increased colony formation as compared to 10% DMSO alone. The cryoprotection afforded by trehalose persists at least for 1.5 years and there is no bias toward protection of a particular lineage. We also found that this increased cryoprotective effect of trehalose is seen both at -196 degrees C and -80 degrees C storage temperatures. Addition of taurine, an amino acid, another membrane stabilizer, and a natural cryoprotectant to the traditional freezing medium also results in beneficial effect. Of the three bioantioxidants tested, i.e., ascorbic acid, alpha-tocopherol acetate, and catalase, catalase shows maximum cryoprotective effect both at -196 degrees C and at -80 degrees C. Because the mode of cryoprotective action of catalase and trehalose are totally different, we tried a combination of these two compounds along with 10% DMSO. At -196 degrees C the protection afforded by the combination was significantly better than that afforded by individual components. At -80 degrees C, however, the combination did not give any added protection as compared to the individual single additives, although it was significantly better than 10% DMSO alone. These results indicate that the addition of membrane stabilizers and antioxidants to the conventional freezing medium may help to improve post thaw recovery of hematopoietic cells.

Effect of trehalose on cryopreservation of pure peripheral blood stem cells

“…Furthermore, as it has been reported in previous studies (20,21) that freezing with trehalose in combination with an antioxidant (catalase) or with Me 2 SO could improve cell viability, CD34 + cells were cryopreserved for a short (20 days) and long (3 months) duration, with a medium containing different combinations of catalase, Me 2 SO and trehalose. The current data indicate that, after thawing, the number and viability of the CD34 + cells was higher in all the samples cryopreserved with only 1M trehalose (Fig.…”

Preservation of stem cells

“…Low levels of trehalose (25 mg/ml) and catalase (100 μg/ml) were shown to reduce post thaw apoptosis in a murine HSC model 25 and improve recovery of colony forming units for umbilical cord blood and HSCs isolated from fetal liver. 26 Cryopreservation solutions supplemented with membrane stabilizer taurine, ascorbic acid and α-tocopheryl acetate also demonstrated improved post thaw recovery. 26 These studies suggest that additives beyond what are classically considered cryoprotective agents can be used to protect cells from the stresses of freezing and thawing and improve post thaw recovery.…”

Current development of bioreactors for extracorporeal bioartificial liver (Review)

“…Currently the University of Wisconsin ͑UW͒ solution and dimethyl sulfoxide ͑DMSO͒ are still recognized as the best frozen liquid and the most important permeable cryoprotectant, respectively. 45 Making amendment to the cryopreservation medium by reducing the concentration of bovine serum, adding one or a combination of membrane stabilizers ͑such as trehalose and taurine͒, 46,47 biological antioxidants ͑such as catalase, N acetyl cysteine, glutathione, and fetal bovine serum͒, 48,49 apoptosis inhibitor ͑such as Z-DEVD-FMK and IDN-1965͒ 50,51 with appropriate concentration, can significantly improve the freezing effect. ͑3͒ Optimization of the store configuration of cryopreservation.…”