1Meltendorf C., 2Hincha D., 3Hoffmann F.

1Universitätsaugenklinik der Johann Wolfgang Goethe-Universität, Theodor- Stern-Kai 7, D-60590 Frankfurt/Main; 2Max-Planck-Institut für molekulare Pflanzenphysiologie, Am Muehlenberg 1, D-14476 Golm; 3Universitätsaugenklinik Benjamin Franklin der Freien Universität Berlin, Hindenburgdamm 30, D-12200 Berlin

Until now the frozen preservation of corneas has been unable to become established as a routine method. Unsatisfactory results of experiments using conventional techniques provided a reason to further examine the possibilities of vitrification.
Objective: Through the reduction in volume of the corneal tissue to be frozen and the use of suitable frozen product packaging, it was intended to achieve a more rapid heat exchange between the corneal tissue and the cooling medium. In addition, through the development of a freezing device it was intended to achieve flash freezing to -140°C so as to promote vitrification.
Methods: For the testing, posterior lamellae from pig corneas were used with a diameter of 7.5 mm. A very thin-walled (0.05 mm), transparent and Tefloncoated bag (Kapton/Teflon Peel Pouch) served as the frozen product packaging. Immersed in only 0.1 ml of the vitrification solution VS41a, the lamellae were cooled to -196°C. The flash freezing to -140°C was achieved by means of a newly developed freezing device using methylcyclopentane and propane as coolants. After reheating in a water bath (40°C) and a 24-hour organ culture, the endothel was stained with trypan blue and alizarin red. A computer-based endothel cell analysis was then conducted.
Results: In the work presented here it has been possible for the first time to carry out the vitrification of corneal lamellae without there being any ice formation or cracking of the frozen product. The use of Teflon-coated frozen product packaging enabled direct contact between the cornea and the frozen product packaging without the endothel cells being damaged. By using posterior corneal lamellae the volume of tissue to be frozen was able to be reduced by 88% in comparison to the previously used corneoscleral discs.
Conclusion: Since, despite the successful vitrification, only a maximum of 10% of the endothelial cells were vital after heating, the toxicity of the cryoprotectors which was determined to be the cause, and the devitrification which occurred during the heating process, should be further examined.

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