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K.U. Loeffler, P. Seifert, J. Bednarz, C. Redbrake, K. Engelmann; Ultrastructural Features Of Corneal Buttons Cultured In Serum–free Versus Conventional Long–term Organ Culture Medium . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2933.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Optimum preservation of corneas for transplantation is of great importance in the success of penetrating keratoplasty. The need for serum–free organ cultures without potentially infectious material led to testing different formulations of serum–free organ culture media against the standard medium with minimal essential medium (MEM) and 2%fetal calf serum (FKS). In this study, we have investigated the ultrastructure of differently cultured corneas to see whether electron microscopy can add in the quality evaluation of specific culture conditions. Methods: Before entering the study, each cornea was checked by slit–lamp microscopy for pathologic changes, and only those with at least 2000 endothelial cells per mm2 were included. Donor age ranged from 40 to 70 years, and post mortem delay was less than 40 hours. Tissue from 28 different corneas cultured for 4 weeks in 4 different media (MI: serum–free medium (SFM), MII: MEM with 2% FKS (standard medium), MIII: MEM with selen, transferrin, lipids and insulin, and MIV: MEM with selen, transferrin, lipids, insulin, and albumin) was fixed in 2.5% glutaraldehyde solution and processed for electron microscopy. Morphological evaluation was performed in a double–blind fashion by 2 investigators and repeated twice. Results: Naturally, the tissue preservation of post–mortem material varies. Thus, it was difficult to evaluate and compare the respective specimens. Morphology ranged from well–preserved cellular organelles to advanced degeneration, and even within the same specimen, there was marked heterogeneity. There were also differences between stromal and endothelial preservation in different media. Intracellular crystalline changes were seen in several corneas, mostly in otherwise healthy looking keratocytes. No specific features associated with a specific medium could be identified but frequently the morphologic preservation of both corneas of the same donor was remarkably similar despite a different culture medium. Conclusions: There is a variety of pathologic changes that occur in organ culture irrespective of the medium that is used. None of the media tested here revealed significantly more cell damage than any of the others, while the original quality of the cultured cornea appeared extremely important. From our findings we also conclude that even fairly small islands of reasonably well–preserved tissue are sufficient for prolonged (4 weeks) corneal organ survival especially in serum–free media.
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