How to make better, safer and easier endothelial controls of long‐term stored corneas with Specular Microscopy?

Purpose Unlike short term cold storage in which corneas remain clear and thin, long‐term organ cultured corneas cannot be rated by specular microscopy (SM) because of the storage‐induced stromal edema and deep endothelial folds. Aim: To present SM of corneas stored in our patented bioreactor (BR). Methods Human corneas were stored in our BR, with a commercial medium containing 2% foetal calf serum at 31°C. The BR restored the intraocular pressure while renewing the medium. By preventing from edema and folding, it improved endothelial cell (EC) viability in the long‐term. Two windows allowed easy multimodal corneal imaging. A new type of SM was developed (CMOS camera, ×10 objective, collimated LED, micrometric stage). For each cornea, 5 images (centre + 4 quadrants) were acquired at day 2 and 28 exactly in the same area, and treated with a custom‐made software and ImageJ to determine the EC density (ECD). Results All corneas remained thin and clear, allowing SM without deconditioning nor deswelling in specific media. The field of view of 935 × 748 μm was 3 times greater than those of both commercial eyebank SM. At D2 and 28, despite shallow residual endothelial folds, large areas of ECs were clearly visualized. ECD could be determined on most images easily. Epithelial cells could also be observed. Conclusions The BR combines the advantages of cold storage (closed system) and organ culture (long‐term). SM allowed endothelial assessment of long term‐stored corneas in our closed BR: it could make endothelial controls safer, easier and increase the cell count reliability especially thanks to a wider field of view.