The research involved in the field of ophthalmology, especially when focused in testing and in the development of medical devices or procedures that may have an impact on the corneal endothelium health, necessitates a great number of investigations, which sometimes have to face some technical difficulties.

As an example, the use of donor corneas for research purpose is unethical, as they are needed for transplantation. Fortunately, some animal models, such as the porcine eye, have anatomical and physiological properties quite similar to the human eye and therefore are useful alternatives to donor corneal tissues for performing valuable research.

Another important matter is related to the method used for collecting and analysing all qualitative and quantitative image data. In Europe, the most common method to assess the viability of the corneal endothelium is based on the trypan blue staining and visual evaluation of the mortality zones using optical microscopy. However, this method is performed manually and, therefore, the operator-dependent variability of data analysis may be a problem, especially when considering the ever-increasing number of analyses to be performed.

To overcome this problem, the Alchimia R&D team developed a standardized method for the quantitative analysis of the endothelial cell mortality with a semi-automated microscopy technology through the Fiji ImageJ software, specifically designed to help researchers in the analyses of the biological pictures.

The first step of this method is the preparation of the corneal button from the porcine eye globe and the staining of the corneal endothelium with the trypan blue. Afterwards, the stained tissue is held under the optical microscope and analysed using the most appropriate magnification. At this point, a camera connected with the microscope can take a series of pictures of the corneal endothelium. These pictures are then “transferred” to the Fiji ImageJ software that, by using special techniques to optimise the corneal images (e.g., the corneal endothelium is not a homogeneous and flat tissue), can release clear images of the central 8-mm central area (which corresponds to the usually transplanted corneal button). Within this area, the software detects the presence of the blue pixel, corresponding to the dead cells, and calculates the percentage of endothelial cells mortality in comparison to the total amount of endothelial cells.

This method was validated using negative (health corneal tissues) and positive (corneal tissues that were heavily damaged by toxic substances) controls.

In conclusion, this semi-automated quantitative system for the measurement of the corneal EC mortality resulted effective, standardized, and reproducible.

For us, this means a straightforward tool to perform a first screening of corneal tissues to compare different corneal storage conditions and experimental research on corneal tissues. In addition, when used on already commercially available products, this method will also serve as a post-market safety evaluation.


  • Giurgola L. et al. Development of a quantitative method to determine corneal endothelial cell mortality. Presentation at the European Associations of Tissue Banks (EATB), 2018
  • Wenzel DA et al. A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons. J Vis Exp 2019;152.
  • Guduric-Fuchs J et al. Immunohistochemical study of pig retinal development. Mol Vis 2009; 15:1915:1928