Korean J Blood Transfus.  2011 Aug;22(2):89-98.

Cryopreservation of Collected Peripheral Blood Hematopoietic Stem Cell Product with 5% DMSO by Adding Nontoxic Natural Cryoprotectants

• Hamm JY
• Shon YH
• Suh JS

Abstract

BACKGROUND
Cryopreservation of hematopoietic stem cells has become an important process due to the therapeutic protocol, which includes stem cell transplantation after chemotherapy, for many hematological malignancies. The conventional medium contains 10% dimethyl sulfoxide (DMSO) as a cryoprotectant, but this has been reported to be related with many complications. We analyzed the usefulness of trehalose, catalase and zVAD-fmk for cryopreservation along with using a reduced concentration of DMSO to 5%.
METHODS
Peripheral blood stem cells were frozen in 10% DMSO as a control and also in 5% DMSO with trehalose and catalase. After 3 weeks of storage in a liquid nitrogen tank, the viability of the thawed hematopoietic stem cells was measured using Trypan blue staining and 7-AAD analysis via conducting flow cytometry. The colony forming potential was assessed using methylcellulose culture. We measured the viability of cells in 5% DMSO medium with or without addition of 30 uM zVAD-fmk right after thawing, and we also did this 6 and 24 hours after incubation.
RESULTS
Cryopreserved cells in 5% DMSO with trehalose and catalase showed similar survival (50.42%) compared with the control (49.78%). The viability of cells that were also treated with added zVAD-fmk showed a better result (13.12%) than without it (5.5%) after 24 hours of incubation. Colony forming assay showed similar colony formation in 5% DMSO with the natural cryoprotectants.
CONCLUSION
According to the results, lowering the DMSO concentration to 5% is significant and we can expect better cell viability and prevent many side effects of high dose DMSO when adding natural cryprotectants in the cryopreservation medium or by adding caspase-inhibitor right after thawing.

Keyword

Cryopreservation; Hematopoietic stem cell; Trehalose; Catalase; zVAD-fmk

MeSH Terms

Amino Acid Chloromethyl Ketones
Catalase
Cell Survival
Cryopreservation
Dimethyl Sulfoxide
Diminazene
Flow Cytometry
Hematologic Neoplasms
Hematopoietic Stem Cells
Methylcellulose
Nitrogen
Safrole
Stem Cell Transplantation
Stem Cells
Trehalose
Trypan Blue
Amino Acid Chloromethyl Ketones
Catalase
Dimethyl Sulfoxide
Diminazene
Methylcellulose
Nitrogen
Safrole
Trehalose
Trypan Blue