Abstract
Purpose :
Traditional cryopreservants like trehalose or glycerol can disrupt the corneal architecture and affect surgical outcomes. We devised DionySol®, a novel cryoprotectant, and investigated its effectiveness in preserving corneal stroma for lamellar keratoplasty for keratoconus or corneal ectasia.
Methods :
We previously reported OdinSol®, a polysaccharide cocktail, for cryopreserving cells and tissues in standard freezers. As OdinSol® inherently lacks capability of decellularization, it was modified into DionySol® for specific preservation of corneal stroma. The efficacy of DionySol® in decellularization was tested via long-term cryopreservation of human corneas. In brief, each of 6 paired donor corneas (ages 40-65 yrs, from Mid-America Transplant in St. Louis) was divided into four pieces. Each piece of cornea was immersed separately into a standard sterile 15ml cryovials (NalgeneTM) containing 10ml of either DionySol®, 90% glycerol or 15% trehalose at -80°C, with one piece being used as the fresh and non-frozen control. Frozen corneal segments were thawed and analyzed using histochemistry, TUNEL assays, and transmission electron microcopy (TEM) after six months.
Results :
DionySol® showed notable efficacy in maintaining the structural integrity of the corneal stroma. TUNEL staining indicated complete destruction of keratocytes, yet the collagen lamellae and fibrils remained similar to fresh controls under TEM analysis. Conversely, corneas preserved in glycerol or trehalose displayed significant collagen disorganization. Morphometric analyses revealed glycerol-treated tissues exhibited an evident crowding of the laminar structure and a doubling in the cross-sectional area of collagen fibrils, when compared with fresh controls or DionySol® treatment. Trehalose preservation resulted in a less compact corneal stroma with severely disrupted fibers, likely due to large ice crystal formation.
Conclusions :
DionySol® effectively decellularizes the corneal stroma and preserves its mechanical properties. This decellularization and structural preservation offers a substantial improvement over conventional corneal preservations, thereby reducing risks of immune rejection and enhancing restoration of keratoectasia. DionySol® is compatible with the use of standard freezers and more cost-effective by eliminating the need for deep freezing in liquid nitrogen facilities.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.