The Improvement of Cell Viability due to Dilution and Removal of DMSO in Thawing of Stem Cells

Lee, Hoon Kyung; Ryu, Kyung Ha; Whang, Il Tae; Kang, Eun Suk; Hong, Ki Sook; Kim, Kyung Hyo; Hong, Young Mi; Kim, Kyung Hee; Lee, Keun

Abstract

PURPOSE: Cryopreserved stem cells from cord blood are usually infused with Dimethyl Sulfoxide (DMSO) immediately after thawing. However, this process may cause cell damage due to osmotic shock, and the administration of DMSO may also cause toxic effects. We studied a new method of increasing cell viability by stabilizing osmolarity by adding dextran 40 and washing out DMSO.
METHODS
Thirty-five samples of cord blood were studied. RBCs were removed in 10% pentastarch. The cells were mixed with DMSO of 5, 10 and 20% each, and stored at -80 degree C. Cryopreserved cells were thawed and then diluted with dextran 40. DMSO was removed afterwards. The cell viability, osmolarity and colony forming capacity in this new thawing method were compared with the control group done by conventional methods.
RESULTS
The recovery rate of WBC after RBCs separation was 92.06% but the contamination rate of RBC was still high(29.90%). The concentration of DMSO significantly affected the survival of WBCs(P<0.05). The osmolarity was 330+/-17.7mOsm/L before freezing, 1,457+/-508.7mOsm/L after thawing prior to dilution and 811+/-199.6mOsm/L after dilution, suggesting that the dilution process was effective in reducing osmolarity. The number of viable cells increased from 6.01+/-1.61(x103/L) to 7.16+/-1.48(x103/L) after dilution but was not significant. The number of CFU-C was increased from 5.82+/-4.19(/105) to 7.58+/-3.16(/105) after dilution but was not significant.
CONCLUSION
Our method of removing DMSO during the thawing process yields a higher cell survival rate and less DMSO toxicities compared with the conventional method of direct injection with DMSO after thawing.