Use of Decellularized SMILE (Small-Incision Lenticule Extraction) Lenticules for Engineering the Corneal Endothelial Layer: A Proof-of-Concept.

PURPOSE
To demonstrate the suitability of using decellularized SMILE (Small-incision Lenticule Extraction) lenticules for culturing and transplanting the corneal endothelium (CE).


METHODS
The SMILE lenticules, obtained during refractive surgery, were decellularized by incubating in CE culture medium and fetal bovine serum. Decellularization was confirmed by hematoxylin and eosin staining, DAPI staining, and gel electrophoresis. The amount of DNA per milligram of dry tissue weight was calculated to quantify the residual nuclear content. The transparency of the decellularized lenticules was determined by calculating the modulation transfer function. Immunostaining for stromal collagens and glycosaminoglycan was performed using specific antibodies. Engineered tissue was constructed by culturing the CE cells on lenticules and staining for ZO-1, Na/K ATPase, and N-cadherin. The functionality of the engineered tissues was assessed by transplanting them onto edematous human donor corneas and perfusing for 10 days ex-vivo.


RESULTS
The residual DNA per milligram of dry tissue weight was found to be significantly reduced (p < 0.0001) in serum (0.255 µg/mg) and Opti-MEM (0.140 µg/mg) when compared to fresh lenticules (3.9 µg/mg). Decellularization did not alter the arrangement of the collagen fibers or the transparency of the lenticules. CE cells attached and matured to express ZO-1, Na/K ATPase, and N-cadherin at two weeks after seeding. The engineered tissue upon transplantation significantly reduced the corneal edema (p < 0.05) and the transplanted cells remained intact on the SMILE lenticule post-transplantation.


CONCLUSION
This study demonstrates the suitability of using SMILE lenticules decellularized using a simple, chemical-free method for engineering the corneal endothelium for transplantation.