Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial–mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-βs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-β signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-β1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-β-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-β1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-β receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-β1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD.

The bioengineering of corneal endothelial grafts consists of seeding in vitro cultured corneal endothelial cells onto a thin, transparent, biocompatible, and sufficiently robust carrier which can withstand surgical manipulations. This is one of the most realistic alternatives to donor corneas, which are in chronic global shortage. The anterior capsule of the crystalline lens has already been identified as one of the best possible carriers, but its challenging manual preparation has limited its use. In this study, we describe a femtosecond laser cutting process of the anterior capsule of whole lenses in order to obtain capsule discs of 8 mm diameter, similar to conventional endothelial grafts. Circular marks made on the periphery of the disc indicate its orientation. Immersion in water for 3 days is sufficient to completely remove the lens epithelial cells and to enable the seeding of corneal endothelial cells, which remain viable after 27 days of culture. Therefore, this method provides a transparent, decellularized disc ready to form viable tissue engineered endothelial grafts.

Purpose: Unlike corneas stored in cold storage (CS) which remain transparent and thin, corneas stored in organoculture (OC) cannot be assessed by specular microscopy (SM), because edema and posterior folds occur during storage and prevent from specular reflection. We previously developed an active storage machine (ASM) which restores the intraocular pressure while renewing the storage medium, thus preventing major stromal edema. Its transparent windows allow multimodal corneal imaging in a closed system. Aim: to present SM of corneas stored in this ASM. Methods: Ancillary study of two preclinical studies on corneas stored for one and three months in the ASM. A prototype non-contact SM was developed (CMOS camera, ×10 objective, collimated LED source, micrometric stage). Five non-overlapping fields (935 × 748 μm) were acquired in exactly the same areas at regular intervals. Image quality was graded according to defined categories (American Cornea Donor Study). The endothelial cell density (ECD) was measured with a center method. Finally, _SMECD was also compared to Hoechst-stained cell nuclei count (_HoechstECD). Results: The 62 corneas remained thin during storage, allowing SM at all time points without corneal deconditioning. Image quality varied depending on donors and days of control but, overall, in the 1100 images, we observed 55% of excellent and 30% of good quality images. _SMECD did not differ from _HoechstECD (p = 0.084). Conclusions: The ASM combines the advantages of CS (closed system) and OC (long-term storage). Specular microscopy is possible at any time in the ASM with a large field of view, making endothelial controls easy and safe.