Cryoprotectants For Vitrification Of Bovine Corneal Endothelial Cells And Elimination Technique Of Tissue Fracture At Low Temperature

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.