| In the present study, pregnant SD rats (17-19 days) were used for the studies on the embryonic cerebral cells (ECC), 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 in mitochondria. The structures and functions of ECC after cryopreservation were examined, and the factors affecting the viability of ECC were analyzed. The vitrified ECC were cultured in vitro. The morphological characters and functions were also studied after the culture. The results of experiments showed that:(1) The procedure of vitrification: ECC were pre-equilibrated by two-step method. The cells were exposed to 50% vitrification solutions diluted with D-Hank's medium for 10min at room temperature ( 20 癈) and then exposed to chilled (4癈) 90% vitrification solution for 1 Omin. The cells were transferred into 2ml plastic straws. The straws were stored at - 196癈 from 3 to 60 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: Cells were exposed to 2.0 M glycerol + 0.5 M sucrose in D-Hank's medium for 10min at room temperature. Then the cells were transferred into 0.5 M sucrose in D-Hank's medium for lOmin. At the end of dilution treatment, cells were washed three times with fresh D-Hank's medium.(2) The selection of the vitrification solution: According to the experiment results, VS4 solution, consisted of 20.5% (W/V) dimethyl sulfoxide (DMSO), 15.5% (WAV) acetamide, 10% (W/V) 2,3-butylene glycol and 6% (W/V) sucrose in D-Hank's medium, was the best one for ECC. After being cryopreserved for 3 days, the recovery rate was 91.00 + 2.48%, the survival rate was 83.21 ?.15%, and the vitality of SDH was 0.494+0.005. Among the permeating cryoprotectants, the combination of DMSO, acetamide and butylene glycol was the best one, and the second was the combination of DMSO, acetamide, propane glycol and butylene glycol. The effects of the non-permeating cryoprotectants were compared, and sucrose was more suitable than polyethylene glycol (MW 8000) for the vitrified ECC.(3) The changes of the structures and the functions of the vitrified ECC: By means of scanning electron microscopy ( SEM ), the ultrastructure of the cell membrane was observed. The membrane of fresh cells were smooth and integrated, while the vitrifiedcells became smaller after the pre-equilibration of VS4 because of the dehydration. The membrane of some cells were damaged. During the cryopreservation, the recovery rates decreased with the prolongation of cryopreservation, but the changes were not significant (P>0.05). The survival rates of ECC also reduced. The survival rates between the 3rd day, the 7th day, the 14th day and the 21st day were significantly different, respectively (P<0.01). The survival rates after 30 days cryopreservation were steady relatively. The 3 0-day's survival rates were not different significantly when compared with the 60-day's. At the forepart of the cryopreservation (3~30 days), the contents of H2O2 increased obviously, and the vitalities of SDH and SOD depressed distinctly. At the evening of the cryopreservation (30-60 days), the contents of I-bCh, the vitalities of SDH and SOD went to steady status. The results indicated that the cryopreservation by vitrification method was applicable to ECC for long-term preservation.(4) The culture of ECC after the cryopreservation: The vitrified ECC were cultured in vitro. The results indicated that the grow status of the vitrified ECC were worse than that of the fresh cells. But they could develop and differentiate normally. The vitality of SDH increased and the content of H2O2 decreased obviously after being cultured. The results demonstrated that the functions of some ECC could be restored during the culture.During the cryopreservation, the ECC were affected by the chemical toxicity of cryoprotectants, the process of freezing-thawing and the dilution...
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