Unlocking the surgical potential of Descemet's membrane: a standardized decellularization protocol.

Descemet's membrane is the specialized extracellular matrix located between corneal stroma and endothelium. This basement membrane provides the biomechanical cues that sustain endothelium viability and function, making it an optimal cell scaffold. The present work describes a new decellularization protocol to remove the cellular components and obtain an acellular scaffold from Descemet's membrane. To induce cell lysis and eliminate all cytoplasmic and nuclear material, Descemet's membranes isolated from donor corneas were subjected to osmotic (hypotonic) shock. The efficiency of the decellularization process was evaluated by the quantification of total residual DNA and analysis by gel electrophoresis. Nuclei removal and extracellular matrix integrity after treatment were verified by histological analysis. In particular, the maintenance of collagen, glycoproteins, perlecan and elastin was analyzed in decellularized tissues compared to untreated controls. DNA quantification showed a 99% reduction of total DNA amount in decellularized Descemet's membranes compared to control ones, with only 28.9 ± 9.86 ng DNA/mg dry tissue residual. Furthermore, the agarose gel electrophoresis and the absence of visible nuclei after decellularization confirmed the efficiency of the process. Histological analyses showed that the composition of the extracellular matrix was not modified by the process. The decellularization protocol is effective in obtaining a Descemet's membrane that is depleted of donor DNA. Furthermore, the treatment preserves tissue matrix components. Descemet's membrane is already prestripped and provided by eye banks; therefore, a decellularized Descemet's membrane represents a valid candidate as a safe scaffold for intraocular surgery as, for example, in the treatment of refractory macular holes.