Studies On Vitrification Of Ovine Chondrocyte

In present study, adult ovine were used as experimental material for chondrocytes, which were cryopreserved by vitrification method. The experiment mainly divided into three parts. Firstly, the vitrification solutions were confected on the basis of the toxicity research of dimethyl sulfoxide (DMSO). Secondly, the suitable pre-equilibration time and temperature were selected due to analyzing the toxicity of vitrification solutions to chondrocytes depend on its survival rates. Lastly, the factors affecting chondrocytes were analyzed by examine the structure of cell membrane and vitality of succinate dehydrogenase (SDH) in mitochondria.(1) The toxicity of DMSO to chondrocytes: DMSO was a kind of permeable Cryoprotective agents (permeable CPAs) with high permeability ability and suitable for vitrifiation cryopreservation. The toxicity of DMSO to ovine chondrocytes depended on temperature and concentration. The concentration of DMSO for cryopreserving chondrocytes should be range from 10% to 45%. Moreover, the concentration of DMSO between 10% to 20% was suit for freezing chondrocytes when the temperature was from -20℃to 4℃.(2) The selection of pre-equilibration time and temperature: According to the detection of recovery rates, we found that pre-equilibration at 0℃was better than 20℃. In time selected experiment, the result showed that survival rates were reduced gradually by the time, so we choose 2 min as the best pre-equilibrium time.(3) The selection of the vitrification solution: VS1 and VSb selected according to the trend of recovery rates, survival rates and vitality of SDH. VS1, consisted of 10%DMSO, 16%EG, 9% glycerol, 12% acetamide, 3%PEG and 0.6M sucrose, as well as VSb which contain 14% DMSO,16% acetamide,2% PEG and 0.15M trehalose.(4) The effects of vitrification to chondrocytes: We have studied the recovery rates, the structure of the cell membrane and the vitality of SDH showed that in the process of cryopreservation the descent of the viability of SDH were remarkable before 20 days of cryopreservation. The recovery rates and damage of the membrane was increased with cryopreservation time. In conclusion, the optimum procedure of chondrocytes vitrification was suggested as follows: pre-equilibration at 0℃for 2 min, treating with VS1 or VSb, plunging directly into LN2, thawing in 38℃water.