Comparison of decellularization methods for human corneal lenticules

Abstract

AbstractPurpose Human or animal thin corneal lenticules (L) have been used as biological carrier to experimentally reconstitute tissue‐engineered grafts. Decellularization of the stroma proved to reduce immune rejection after allografts in animals. Aim: to assess different decellularization methods of human corneal L.Methods Stromal L were prepared with a Moria microkeratome on human organ cultured corneas mounted on an artificial anterior chamber. After desepithelialization, an anterior L was obtained with a 90 μm head and a posterior one with a 110 μm head. Four physical methods (sonification at 37 kHz for 45 minutes (min), 3 freeze/thaw cycles in liquid nitrogen, heating at 50°C for 60 min, and hypoxia) and four chemical methods (75% ethanol for 2x10 min, 0,1% sodium Dodecyl sulphate for 24 hours (h), 4M urea for 4h, and 29% NaCl for 24h) were tested. Efficiency and safety were assessed by determining: 1/mortality of the keratocytes using the Hoechst‐Ethidium‐Calcein triple labelling (Pipparelli, IOVS2011;52:6018), 2/alteration of the stroma using transparency measurement (analysis of modulation and contrast transfer functions), and collagen fibres analysis by transmitted electron microscopy (TEM), and 3/elimination of the cell debris using a staining of cell cytoplasm (Dioc 6) and TEM.Results 75% ethanol and hypoxia triggered complete keratocytes mortality with minimal alterations of the stroma. None of the methods eliminated dead cells debris. Combination with the sonification helped fragmenting cellsConclusion Two simple and rapid methods allow complete decellularization of human corneal L. The ability of these Lt to promote endothelial cells adhesion and proliferation is under investigation.