Long-term Cryopreservation of Cord Blood Mononuclear Cells for Hematopoietic Stem Cell Transplantation

Lee, Kun Soo

Abstract

BACKGROUND: The use of human umbilical cord blood(CB) as a source of hematopoietic stem cell transplantation for the treatment of pediatric diseases has been well established. In previous study, we reported the method of improving the recovery rate of cryopreserved CB mononuclear cells after thawing and the possibility of long-term cryopreservation. In this study we compared the recovery rate of the cryopreserved mononuclear cells from large volume of CB according to various freezing conditions and stored CB mononuclear cells as a source of transplantation.
METHODS
Twenty five CB from Kyung-pook National University Hospital and the Hana Obstetric/Gynecology Hospital, Taegu, Korea were used. The mononuclear cell separation was done with Hypaque and CD34+ cells were collected by Fenwal with the monoclonal antibody and immunobeads within 6 hours after collection. The cell count and the viability rate was done by hemocytometer with trypan blue exclusion method. The mononuclear cells were cryopreserved with two different concentrations of fetal bovine serum and of dimethylsulfoxide and with two different methods of direct and programmed freezing in the liquid nitrogen. The recovery rate was compared after thawing. The differentiation ability of thawed mononuclear cell was performed with in vitro colony culture using methylcellulose medium and GM-CSF. The family history of inherited blood cell diseases, tests for viral(HIV, HBV, HCV and CMV) and bacterial (syphilis, etc) contamination and HLA and blood typing were done for the future transplantation source.
RESULTS
The mean volume of collected CB was 117(49-150)ml and the mean number of mononuclear cells was 5.0(2.8-7.5) x 10(6)/ml. The viability rate was 95.3-98.6%. The recovery rate of mononuclear cells was higher in the group of medium containing 10% of fetal bovine serum and 10% of dimethylsulfoxide after 30 days programmed freezing in liquid nitrogen(-196 degrees C). The recovery rate of long-term(180 days) cryopreserved mononuclear cells was 72.8%. The colony formations in culture of mononuclear cells were not different between before-and 14 days after-freezing. The seven total volume of CB mononuclear cells which had no family history of inherited blood cell diseases, no viral and bacterial contamination were cryopreserved after HLA and blood typing for the future transplantation.
CONCLUSION
In summary, because the long-term cryopreservation of CB mononuclear cells was possible with good recovery rate we expect the establishment of CB bank will overcome the limitation of donor in Korea in the near future.