Effect Of Vitrification Cryopreservation On The Biological Characteristics Of Mice Bone Marrow Nucleated Cells

In the present study, Adult ICR mice were used for the studies on the bone marrow nucleated cells which were cryopreserved by the vitrification method. The suitable vitrification solution and procedure were selected due to the recovery rates, the survival rates and the vitality of SDH, SOD in mitochondria. The structures and functions of bone marrow nucleated cells after cryopreservation were examined. The vitrified bone marrow nucleated cells were cultured in vitro. The morphological characters and induced functions of bone marrow mesenchymal stem cells were also studied after the culture.(1) The procedure of vitrification: bone marrow nucleated cells were pre-equilibrated by two-step method. The cells were exposed to 50% vitrification solutions diluted with D-Hanks medium for 5min at room temperature ( 20) and then exposed to chilled (4) 90% vitrification solution for 5min. The cells were transferred into 2ml plastic straws. The straws were stored at -196 from 1 to 30 days after cooled by being plunged directly into LN2. The cells were expelled from the straws immediately after warming in 38癈 water, and then washed in diluents At the end of dilution treatment, cells were washed three times with fresh D-Hanks medium.(2) The selection of the least days of vitrification cryopreservation which cells get to a stabilization state of survival rates and the vitality of SDH, SOD. According to the experiment results, vitrification solution, consisted of 18.5% (W/V) dimethyl sulfoxide (DMSO), 14.0% (W/W) acetamide, 9.0% (W/V) propanediol and 4.1% (W/V)PEG in D-Hanks medium, was effectual for bone marrow nucleated cells. After being cryopreserved for 20 days, cells get to a stabilization state of survival rates and the vitality of SDH, SOD, etc. The recovery rate was 86.57 1.33, survival rate was 85.88 1.98, the vitality of SDH was 0.598 0.011, H2O2 content was 0.327 0.010, the vitality of SOD was 61.36 1.53. The results suggest that cryoprotectants used in the paper was quite suitable for the bone marrow nucleated cells.(3) The culture of bone marrow nucleated cells after the cryopreservation. The vitrified bone marrow nucleated cells were cultured in vitro. The results indicated thatthe grow status of the vitrified bone marrow nucleated cells were worse than that of the fresh cells. But they could develop and differentiate normally, including induced into neural cells and chondrogenic. The results demonstrated that the functions of mice bone marrow nucleated cells were not affected heavily by vitrification cryopreservation.During the cryopreservation, the bone marrow nucleated cells were affected by the chemical toxicity of cryoprotectants, the process of freezing-thawing and the dilution of vitrification solutions, but this technique still maintained the viability of cells. So it was applicable to cryopreserved the bone marrow nucleated cells for long times. These results proved that this technology could be used to clinic research.