Specular microscopy revisited to allow endothelial controls of long-term stored corneas in an innovative bioreactor

Abstract

Purpose : Unlike short-term cold stored corneas that remain clear and thin, long-term organ cultured corneas cannot be assessed by specular microscopy (SM) because there have deep endothelial folds and maximal stromal swelling. Aim: To present non-contact SM of corneas stored in our innovative bioreactor (BR) in which they remain thin.

Methods : Fresh human corneas were stored in our BR, with a medium containing 2% fetal calf serum (CorneaMax, Eurobio) at 31°C. It restored the intraocular pressure while renewing the storage medium, preventing from edema and folding, in a totally closed system. Two transparent windows allowed multimodal corneal imaging. A custom made SM was developed with a CMOS camera, a x10 objective, a collimated external led source specially placed to retrieve specular reflection and a micrometric stage to move the BR. At least 5 images (center + 4 quadrants) were acquired at day (D) 2, 26 and 28. The endothelial cell density (ECD) was calculated using imageJ and a custom-made software. At D28 the reference ECD was measured with fluorescent microscopy after staining of endothelial cells nucleus with Hoechst 33342, and compared with SM done just before.

Results : Endothelial images were successfully obtained for all corneas in the BR at D2, 26 and 28 and never for OC corneas (n=41 pairs). Image quality varied depending on donors and day of control, but overall, 85% of images were of excellent and good quality, according to the scoring system of the Cornea Donor Study SM ancilliary study. The prototype SM had an extra wide field of view of 954 x738μm, 3 times greater than current eye banks SM available on the market. Because corneas were firmly blocked into the BR, the same endothelial areas could be observed at different time points. During in BR-storage, endothelial folds gradually decreased allowing counting more cells. There was not significant difference between ECD measured after Hoescht staining and SM ECD (p=0.189).

Conclusions : The bioreactor with its custom specular microscope combines the advantages of cold storage (closed system) and organ culture (long-term storage). It makes endothelial controls easier and safer without deconditioning corneas, with an increased reliability thanks to an extremely wide field of view and possibility to monitor changes over time.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.