牛角膜内皮细胞玻璃化冷冻保护剂及组织低温断裂消除方法的研究
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摘要
深低温保存是实现角膜长期保存的最有效方法。角膜的慢速冻存法和冷冻干燥法本身存在着很多问题,这使得玻璃化法保存角膜成为一种重要的途径。
在玻璃化法保存中,冷冻保护剂能够促进冷冻体系向玻璃态的转变,减少对细胞或组织的低温损伤。由保护剂和载体溶液构成的玻璃化溶液是组织玻璃化法保存的研究热点之一。然而,对于不同的细胞或组织,同一种保护剂会显示出不同程度的毒性及渗透能力,使得最终的保护效果也有所不同。到目前为止,保护剂对细胞或组织的作用机理还不清楚。在为一种既定的细胞类型选择保护剂时,很大程度上依赖于实验的方法。
内皮细胞是角膜组织中最重要的细胞类型。本论文首先以牛角膜内皮细胞为保存对象,筛选合适的保护剂和玻璃化溶液。首先,系统地研究了渗透性保护剂,在考察其玻璃化能力和毒性的基础上,以冷冻保存的效果为判断标准,获得了一种较好的渗透性保护剂组分。然后,分别研究了糖类非渗透性保护剂和大分子非渗透性保护剂。同样以冷冻保存的效果为判断标准,在确定保存效果较好的糖和大分子的种类后,确定各自的最优浓度。本论文中,渗透性保护剂的筛选范围包括二甲基亚砜、乙二醇(EG)、1,2-丙二醇、2,3-丁二醇、乙酰胺和乙二醇单甲醚;糖类非渗透性保护剂包括木糖、果糖、甘露糖、葡萄糖、麦芽糖、蔗糖和海藻糖;大分子则包括聚蔗糖(MW 7,000)、葡聚糖(MW7,000)、硫酸软骨素(CS,MW 18,000～30,000)、牛血清白蛋白(MW 68,000)和聚乙二醇(MW6,000、10,000和20,000)。细胞活性评估的主要方法为台盼兰拒染法。通过实验测定和分析,结果发现:乙二醇、葡萄糖和硫酸软骨素的保护效果最好。由此获得的玻璃化溶液的组成是:载体溶液-乙二醇-葡萄糖-硫酸软骨素,其中,乙二醇、葡萄糖和硫酸软骨素的质量百分比浓度分别为52%、8%和3%。冷冻保存后角膜内皮细胞的活率高达89.4±2.1%,并且继续培养后细胞的活力能够得到恢复。
在组织的玻璃化法保存中,通常采用一步降温的方式,即将冻存对象直接投入液氮。这种降温方式的降温速率较快,会在组织内引起较大的热应力。当热应力超越组织的承受极限,组织就会发生低温断裂。断裂会影响组织的保存效果,而且有断裂存在的组织是绝不允许用于临床的。
本论文提出了两步降温即首先在冷氮气中降温然后再转移至液氮的冷冻方法。两步降温方法的降温速率较一步法降温要低,有利于削弱冻存组织内的热应力进而消除组织的断裂。本章首先研究了两步降温及复温过程中玻璃化溶液的断裂情况,并且确定了两步降温法的操作参数;然后分别以肉眼、光学显微镜及扫描电子显微镜考察了复苏后组织的断裂情况。结果表明,在两步降温的过程中,仅在纯玻璃化溶液的表面区域有宏观断裂,在溶液的主体部分没有断裂发生。也就是说,两步降温法能够消除玻璃化溶液主体部分的宏观断裂。在执行两步降温时,首先在降温的第一步,应将冻存对象置于氮气中-196～-135℃的温度区间内,在-80℃以上温度的降温速率应大于10℃/min:当其温度降到-100℃以下,即可将冻存对象浸入液氮,降温速率应小于165℃/min。在经历两步降温冷冻后的牛角膜、牛血管和猪的软骨组织上找不到任何宏观和微观的裂纹。本方法不需要大型设备,操作简便,适合于大规模推广使用。
组织的玻璃化法保存中,玻璃化溶液的导入和导出方案极大地影响着保存的效果。合适的方案有利于减弱对细胞的毒性损伤和渗透损伤。对于分步法导入和导出保护剂,保护剂与细胞的平衡时间是导入和导出方案中的重要因素。本论文以正交实验的方法确定分步导入和导出保护剂过程的平衡时间。结果表明:导入过程中,与10%EG、25%EG、50%EG和52%EG-8%Glucose-3%CS的最佳平衡时间分别为4 min、5 min、6 min和3 min:导出过程中,与50%EG-5%glucose、25%EG-5%glucose、10%EG-5%glucose和5%glucose的最佳平衡时间分别为4 min、6 min、6 min和6 min。
最后,论文应用筛选获得的玻璃化溶液和最佳的平衡时间以及两步降温方法保存牛角膜,发现角膜内皮细胞的活率为41.0±5.9%,显著高于目前文献报道的玻璃化法角膜保存的最好结果10%。
Cryopreservation is an effective method for the long-term storage of corneas.Lots of unsolved problems exist for corneal preservation by conventional slow freezing method and freeze-drying method.Therefore,it is necessary to explore the appropriate technique for the corneal vitrification.
In vitrification process,the selection of suitable cryoprotective agents(CPAs) is important, as the cryoprotectants facilitate the transition to a vitreous state and reduce cryo-damage to cells and extracellular matrix in tissue.The components of vitrification solution consisting of various CPAs and the carrier solution is currenetly one of key research topics.However,for different cells or tissues,the same CPAs may show toxicity and permeability to different extents,and thus the preservative efficacies are also different for different cells or tissues.So far,the protecting mechanisms of CPAs are not clear yet.It is not possible to predict how different CPAs and their combinations affect a given cell type,and hence the selection of cryoprotectants for a given type of cells is still largely dependent on experimental approach.
The corneal endothelial cell(CEC) is the most important cell type in the cornea tissue.The objective of the first part of this thesis was to select and test systematically possible CPAs and to obtain a suitable formula for vitrification of CECs.Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components,i.e.the penetrating CPAs,sugars and macromolecular compounds,were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide,ethylene glycol(EG),1,2-propanediol,2,3-butanediol,acetamide and ethylene glycol mono-methyl ether were chosen as the penetrating CPA components.Sugars including xylose,fructose,mannose,glucose,maltose,sucrose and trehalose were tested. Ficoll(MW 7 KD),dextran(MW 7 KD),chondroitin sulfate(CS,MW 18-30 KD),bovine serum albumin(MW 68 KD),and polyethylene glycol(MW 6 KD,10 KD and 20 KD) were chosen as the macromolecular compounds.The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar,and CS the most suitable macromolecule.The optimized concentrations for each component in the vitrification solution were 52%(w/w) EG,8%(w/w) glucose and 3%(w/w) CS.The CEC survival rate of 89.4±2.1%(mean±S.D.) was obtained using this formula and established vitrification protocol. Also,cell viability can recover after one passage post-culture.
For tissue vitrification,the commonly-used cooling method is to directly plunge the object into liquid nitrogen,i.e.one-step cooling.The cooling rate resulted from the method itself is high so that large thermal stress can be formed and accumulated within the tissues.The stress can potentially exceed the strength of the material,leading to its fracturing,which is a significant barrier to the cryopreservation of three-dimensional multicellular tissues.
In this thesis,a two-step cooling protocol was presented to eliminate the low temperature fracture of tissue,in which the object were placed in nitrogen vapor to be cooled first and then plunged into liquid nitrogen.Macroscopic phenomena about fracture during cooling and thawing were observed for vitrification solution alone.Then three tissue segments of bovine cornea,bovine blood vessel and pig cartilage were observed after preservation respectively by naked eyes,light microscopy and scanning electronic microscopy.In addition,operatingl parameters for this protocol were determined experimentally.The results showed that,using the two-step cooling protocol,fracture only occurred near the free-stream surface of solution. No fracture,whether macro- or micro-,can be found within the tissues.The operating procedure of this protocol is as follows,the object should be placed in vapor phase of above the liquid nitrogen with the temperature between -196～-135℃and the cooling rates higher than 10℃/min above -80℃.The object is immersed into liquid nitrogen when its temperature was decreased below -100℃and the initial cooling rate should be lower than 165℃/min.
For tissue preservation by vitrification,the protocols of CPA addition and removal can greatly influence the outcome of preservation.Suitable protocols help to weaken toxic and osmotic injuries.For CPA addition and removal by stepwise method,the equilibration time of CPA is an important factor.In this thesis,orthogonal tests were conducted in order to obtain the optimal equilibration time of CPA in each step.The results showed that,during CPA addition,the equilibration time was 4 min、5 min、6 min and 3 min respectively for 10%EG, 25%EG,50%EG and 52%EG - 8%Glucose - 3%CS,and4 min、6 min、6 min and 6 min for 50%EG-5%glucose,25%EG-5%glucose,10%EG-5%glucose and 5%glucose during CPA removal.
Finally,with the vitrification solution and the optimal equilibration time,and the two-step cooling protocol obtained in this thesis,bovine cornea segments were vitrified and the survival rate of CECs was 41.0±5.9%,significantly higher than those reported in literature.
在玻璃化法保存中,冷冻保护剂能够促进冷冻体系向玻璃态的转变,减少对细胞或组织的低温损伤。由保护剂和载体溶液构成的玻璃化溶液是组织玻璃化法保存的研究热点之一。然而,对于不同的细胞或组织,同一种保护剂会显示出不同程度的毒性及渗透能力,使得最终的保护效果也有所不同。到目前为止,保护剂对细胞或组织的作用机理还不清楚。在为一种既定的细胞类型选择保护剂时,很大程度上依赖于实验的方法。
内皮细胞是角膜组织中最重要的细胞类型。本论文首先以牛角膜内皮细胞为保存对象,筛选合适的保护剂和玻璃化溶液。首先,系统地研究了渗透性保护剂,在考察其玻璃化能力和毒性的基础上,以冷冻保存的效果为判断标准,获得了一种较好的渗透性保护剂组分。然后,分别研究了糖类非渗透性保护剂和大分子非渗透性保护剂。同样以冷冻保存的效果为判断标准,在确定保存效果较好的糖和大分子的种类后,确定各自的最优浓度。本论文中,渗透性保护剂的筛选范围包括二甲基亚砜、乙二醇(EG)、1,2-丙二醇、2,3-丁二醇、乙酰胺和乙二醇单甲醚;糖类非渗透性保护剂包括木糖、果糖、甘露糖、葡萄糖、麦芽糖、蔗糖和海藻糖;大分子则包括聚蔗糖(MW 7,000)、葡聚糖(MW7,000)、硫酸软骨素(CS,MW 18,000～30,000)、牛血清白蛋白(MW 68,000)和聚乙二醇(MW6,000、10,000和20,000)。细胞活性评估的主要方法为台盼兰拒染法。通过实验测定和分析,结果发现:乙二醇、葡萄糖和硫酸软骨素的保护效果最好。由此获得的玻璃化溶液的组成是:载体溶液-乙二醇-葡萄糖-硫酸软骨素,其中,乙二醇、葡萄糖和硫酸软骨素的质量百分比浓度分别为52%、8%和3%。冷冻保存后角膜内皮细胞的活率高达89.4±2.1%,并且继续培养后细胞的活力能够得到恢复。
在组织的玻璃化法保存中,通常采用一步降温的方式,即将冻存对象直接投入液氮。这种降温方式的降温速率较快,会在组织内引起较大的热应力。当热应力超越组织的承受极限,组织就会发生低温断裂。断裂会影响组织的保存效果,而且有断裂存在的组织是绝不允许用于临床的。
本论文提出了两步降温即首先在冷氮气中降温然后再转移至液氮的冷冻方法。两步降温方法的降温速率较一步法降温要低,有利于削弱冻存组织内的热应力进而消除组织的断裂。本章首先研究了两步降温及复温过程中玻璃化溶液的断裂情况,并且确定了两步降温法的操作参数;然后分别以肉眼、光学显微镜及扫描电子显微镜考察了复苏后组织的断裂情况。结果表明,在两步降温的过程中,仅在纯玻璃化溶液的表面区域有宏观断裂,在溶液的主体部分没有断裂发生。也就是说,两步降温法能够消除玻璃化溶液主体部分的宏观断裂。在执行两步降温时,首先在降温的第一步,应将冻存对象置于氮气中-196～-135℃的温度区间内,在-80℃以上温度的降温速率应大于10℃/min:当其温度降到-100℃以下,即可将冻存对象浸入液氮,降温速率应小于165℃/min。在经历两步降温冷冻后的牛角膜、牛血管和猪的软骨组织上找不到任何宏观和微观的裂纹。本方法不需要大型设备,操作简便,适合于大规模推广使用。
组织的玻璃化法保存中,玻璃化溶液的导入和导出方案极大地影响着保存的效果。合适的方案有利于减弱对细胞的毒性损伤和渗透损伤。对于分步法导入和导出保护剂,保护剂与细胞的平衡时间是导入和导出方案中的重要因素。本论文以正交实验的方法确定分步导入和导出保护剂过程的平衡时间。结果表明:导入过程中,与10%EG、25%EG、50%EG和52%EG-8%Glucose-3%CS的最佳平衡时间分别为4 min、5 min、6 min和3 min:导出过程中,与50%EG-5%glucose、25%EG-5%glucose、10%EG-5%glucose和5%glucose的最佳平衡时间分别为4 min、6 min、6 min和6 min。
最后,论文应用筛选获得的玻璃化溶液和最佳的平衡时间以及两步降温方法保存牛角膜,发现角膜内皮细胞的活率为41.0±5.9%,显著高于目前文献报道的玻璃化法角膜保存的最好结果10%。
Cryopreservation is an effective method for the long-term storage of corneas.Lots of unsolved problems exist for corneal preservation by conventional slow freezing method and freeze-drying method.Therefore,it is necessary to explore the appropriate technique for the corneal vitrification.
In vitrification process,the selection of suitable cryoprotective agents(CPAs) is important, as the cryoprotectants facilitate the transition to a vitreous state and reduce cryo-damage to cells and extracellular matrix in tissue.The components of vitrification solution consisting of various CPAs and the carrier solution is currenetly one of key research topics.However,for different cells or tissues,the same CPAs may show toxicity and permeability to different extents,and thus the preservative efficacies are also different for different cells or tissues.So far,the protecting mechanisms of CPAs are not clear yet.It is not possible to predict how different CPAs and their combinations affect a given cell type,and hence the selection of cryoprotectants for a given type of cells is still largely dependent on experimental approach.
The corneal endothelial cell(CEC) is the most important cell type in the cornea tissue.The objective of the first part of this thesis was to select and test systematically possible CPAs and to obtain a suitable formula for vitrification of CECs.Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components,i.e.the penetrating CPAs,sugars and macromolecular compounds,were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide,ethylene glycol(EG),1,2-propanediol,2,3-butanediol,acetamide and ethylene glycol mono-methyl ether were chosen as the penetrating CPA components.Sugars including xylose,fructose,mannose,glucose,maltose,sucrose and trehalose were tested. Ficoll(MW 7 KD),dextran(MW 7 KD),chondroitin sulfate(CS,MW 18-30 KD),bovine serum albumin(MW 68 KD),and polyethylene glycol(MW 6 KD,10 KD and 20 KD) were chosen as the macromolecular compounds.The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar,and CS the most suitable macromolecule.The optimized concentrations for each component in the vitrification solution were 52%(w/w) EG,8%(w/w) glucose and 3%(w/w) CS.The CEC survival rate of 89.4±2.1%(mean±S.D.) was obtained using this formula and established vitrification protocol. Also,cell viability can recover after one passage post-culture.
For tissue vitrification,the commonly-used cooling method is to directly plunge the object into liquid nitrogen,i.e.one-step cooling.The cooling rate resulted from the method itself is high so that large thermal stress can be formed and accumulated within the tissues.The stress can potentially exceed the strength of the material,leading to its fracturing,which is a significant barrier to the cryopreservation of three-dimensional multicellular tissues.
In this thesis,a two-step cooling protocol was presented to eliminate the low temperature fracture of tissue,in which the object were placed in nitrogen vapor to be cooled first and then plunged into liquid nitrogen.Macroscopic phenomena about fracture during cooling and thawing were observed for vitrification solution alone.Then three tissue segments of bovine cornea,bovine blood vessel and pig cartilage were observed after preservation respectively by naked eyes,light microscopy and scanning electronic microscopy.In addition,operatingl parameters for this protocol were determined experimentally.The results showed that,using the two-step cooling protocol,fracture only occurred near the free-stream surface of solution. No fracture,whether macro- or micro-,can be found within the tissues.The operating procedure of this protocol is as follows,the object should be placed in vapor phase of above the liquid nitrogen with the temperature between -196～-135℃and the cooling rates higher than 10℃/min above -80℃.The object is immersed into liquid nitrogen when its temperature was decreased below -100℃and the initial cooling rate should be lower than 165℃/min.
For tissue preservation by vitrification,the protocols of CPA addition and removal can greatly influence the outcome of preservation.Suitable protocols help to weaken toxic and osmotic injuries.For CPA addition and removal by stepwise method,the equilibration time of CPA is an important factor.In this thesis,orthogonal tests were conducted in order to obtain the optimal equilibration time of CPA in each step.The results showed that,during CPA addition,the equilibration time was 4 min、5 min、6 min and 3 min respectively for 10%EG, 25%EG,50%EG and 52%EG - 8%Glucose - 3%CS,and4 min、6 min、6 min and 6 min for 50%EG-5%glucose,25%EG-5%glucose,10%EG-5%glucose and 5%glucose during CPA removal.
Finally,with the vitrification solution and the optimal equilibration time,and the two-step cooling protocol obtained in this thesis,bovine cornea segments were vitrified and the survival rate of CECs was 41.0±5.9%,significantly higher than those reported in literature.
引文
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