羊软骨细胞玻璃化冻存的研究
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摘要
本文以成年绵羊为实验材料,进行玻璃化冻存羊软骨细胞的研究。研究了二甲基亚砜(DMSO)对羊软骨细胞的应用浓度范围,并在此基础上设计了玻璃化溶液;根据不同预平衡时间和温度对羊软骨细胞存活率的影响,分析各组玻璃化溶液对软骨细胞的毒性,筛选出最佳预平衡方法;通过对冻存后羊软骨细胞的膜完整性和细胞内琥珀酸脱氢酶(SDH)活性变化的检测分析低温对羊软骨细胞的影响。
(1) DMSO对羊软骨细胞的毒性分析:DMSO是一种具有易玻璃化和高渗透性的渗透性冷冻保护剂,但对细胞的毒性受温度和浓度的影响比较大。在浅低温(-20℃~4℃)时,DMSO作为冷冻保护剂的最佳初始浓度为10%~20%。在深低温时,DMSO作为羊软骨细胞保护剂的最佳应用浓度范围为10%~45%。
(2)预平衡温度和时间的筛选:经过对预平衡时间和温度对细胞存活率的影响进行研究发现,玻璃化溶液在20℃下对羊软骨细胞的影响要比0℃大,因此预平衡温度设定在0℃;而细胞的存活率的变化趋势基本上是随预平衡时间的延长而降低,分析表明2 min为最佳平衡时间。
(3)玻璃化溶液的筛选:从羊软骨细胞的回收率、存活率和SDH的活性变化分析,认为玻璃化溶液VS1和VSb的冻存效果最好,其中VS1的成分:10% DMSO、16%乙二醇、9%丙三醇、12%乙酰胺、3%聚乙二醇和0.6 M蔗糖;VSb的成分:14% DMSO、16%乙酰胺、2%聚乙二醇和0.15 M海藻糖。
(4)玻璃化冻存后细胞活性的变化:对冻存后羊软骨细胞的回收率、膜完整性和SDH活性进行了初步研究,从各项指标来看,细胞的回收率和细胞膜完整性随着冻存时间的延长而逐渐下降,但下降速度不明显。SDH活性在玻璃化冻存1~20 d变化幅度较大,20~30 d下降趋势减弱,保持相对稳定。表明该玻璃化冻存方法适用于长期冻存羊软骨细胞。
上述实验结果表明,羊软骨细胞玻璃化冻存时,在0℃预平衡2 min,辅以适当配比的玻璃化溶液,采用直接投入液氮快速降温法和37℃快速复温的手段,可获得较好的冷冻效果。
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.
(1) DMSO对羊软骨细胞的毒性分析:DMSO是一种具有易玻璃化和高渗透性的渗透性冷冻保护剂,但对细胞的毒性受温度和浓度的影响比较大。在浅低温(-20℃~4℃)时,DMSO作为冷冻保护剂的最佳初始浓度为10%~20%。在深低温时,DMSO作为羊软骨细胞保护剂的最佳应用浓度范围为10%~45%。
(2)预平衡温度和时间的筛选:经过对预平衡时间和温度对细胞存活率的影响进行研究发现,玻璃化溶液在20℃下对羊软骨细胞的影响要比0℃大,因此预平衡温度设定在0℃;而细胞的存活率的变化趋势基本上是随预平衡时间的延长而降低,分析表明2 min为最佳平衡时间。
(3)玻璃化溶液的筛选:从羊软骨细胞的回收率、存活率和SDH的活性变化分析,认为玻璃化溶液VS1和VSb的冻存效果最好,其中VS1的成分:10% DMSO、16%乙二醇、9%丙三醇、12%乙酰胺、3%聚乙二醇和0.6 M蔗糖;VSb的成分:14% DMSO、16%乙酰胺、2%聚乙二醇和0.15 M海藻糖。
(4)玻璃化冻存后细胞活性的变化:对冻存后羊软骨细胞的回收率、膜完整性和SDH活性进行了初步研究,从各项指标来看,细胞的回收率和细胞膜完整性随着冻存时间的延长而逐渐下降,但下降速度不明显。SDH活性在玻璃化冻存1~20 d变化幅度较大,20~30 d下降趋势减弱,保持相对稳定。表明该玻璃化冻存方法适用于长期冻存羊软骨细胞。
上述实验结果表明,羊软骨细胞玻璃化冻存时,在0℃预平衡2 min,辅以适当配比的玻璃化溶液,采用直接投入液氮快速降温法和37℃快速复温的手段,可获得较好的冷冻效果。
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.
引文
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