Cryopreservation Process For Corneas

Cryopreservation is a technique that offers the prospect of long-term storage of corneas.Successful corneal cryopreservation would offer sufficient corneal grafts for penetratingkeratoplasty and would be a new method for the eye-bank. The maintenance of an intact,functional corneal endothelial monolayer plays an important role in the cryopreservation ofcorneas, which is vital for sustaining corneal thickness, transparency and dehydratingcondition. There are two kinds of cryopreservation: slow freezing and vitrification. Thecorneas are usually cryopreserved using the slow freezing method. At present, someresearchers attempt to adopt vitrification for corneal preservation. Vitrification is a promisingnovel and simple procedure that attempts to eliminate the formation of both intracellular andextracellular ice during the process of cooling and warming, and vitrification is morecompetitive and effective than slow freezing. However, vitrification needs a combination ofhigh concentrations of cryoprotectant agents (CPAs) which has very high toxic and osmoticinjury when the CPA is loaded and removed. This thesis focuses on the freezing procedures,the optimal method for loading and removing of CPAs and the process of vitrification ofcorneas respectively.Firstly, during the process of corneal cryopreservation by slow freezing, the optimumconcentration of different CPAs and the appropriate loading and removing method of CPAsare investigated experimentally. The results indicate that loading and removing of CPAs oflow concentration by stepwise method can not increase the viability of endothelial cells, and10％DMSO is the optimal CPA for freezing of corneas which makes the lowest cell loss andthe good integrity of the endothelial monolayer.Secondly, the process of loading and removing of vitrification CPA is optimized. Theeffects of operating temperature, protocols of loading and removing of CPA, different intervaltime during loading and removing of CPA by stepwise method and different operating time ofloading and removing of CPA by ramp method on the cell viability are investigated. It can beconcluded that the viability of cell is higher when the operating temperature is lower, and thestepwise method can enhance the viability of cells, and the optimal interval time duringloading period is 4 minutes and the optimal interval time during removing period is 3 minutes.Thirdly, a new combined vitrification CPA is developed. The CPA solution contains20％propane-1, 2-diol, 15％DMSO, 15％trehalose and 5％polyethylene glycol. The optimal loading and removing method of CPA is stepwise method when the loading interval time is 3minutes and removing interval time is 5 minutes.Finally, corneas have been vitrified by being plunged in liquid nitrogen after CPA loadedand removed in terms of the specified protocol. The results show that the viability ofendothelial cells of corneas vitrified using 39％1,2-PD+15％trehalose(PD) and20％1,2-PD+15％DMSO+15％trehalose+5％polyethylene glycol (PDTP) are 74.5±1.0％and77.6±1.4％respectively. And most of the endothelial cells keep the integrity aftercryopreservation. The PDTP is more competitive than the PD for the corneal vitrificationcryopreservation.