Development and characterization of Lyophilized Transparized Decellularized stroma as a replacement for living cornea in
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Development and characterization of Lyophilized Transparized Decellularized stroma as a replacement for living cornea in deep anterior lamellar keratoplasty Marie-Rose Rovere . Coralie Ouilhon . Damien Salmon . Marek Haftek . Odile Damour . Ce´line Auxenfans
Received: 29 May 2018 / Accepted: 1 December 2018 Ó Springer Nature B.V. 2019
Abstract Corneal disease is the second cause of blindness in developing countries, where the number of corneal grafts needed by far exceeds the number available. In industrialized countries, although corneas are generally available for keratoplasty, onto inflamed and vascularized host beds they are often rejected despite immune-suppression. A non-immunogenic, transparent, cytocompatible stroma is therefore required, which can be lyophilized for long-term conservation. Decellularization methods were tested M.-R. Rovere O. Damour C. Auxenfans Banque de Tissus et de Cellules des Hospices Civils de Lyon, Hoˆpital Edouard Herriot, Place d’Arsonval, 69003 Lyon, France M.-R. Rovere O. Damour C. Auxenfans Laboratoire de Biologie Tissulaire et Inge´nierie The´rapeutique, UMR 5305, CNRS, SFRBioSciencesGerland-Lyon Sud, Universite´ Lyon 1, Lyon, France C. Ouilhon Service d’ophtalmologie, pavillon C, Hoˆpital Edouard Herriot, 5, place d’Arsonval, 69003 Lyon, France D. Salmon M. Haftek EA4169 ‘‘Fundamental, Clinical and Therapeutic Aspects of Skin Barrier Function’’, University of Lyon 1, Lyon, France C. Auxenfans (&) Banque de Tissus et Cellules, Pavillon I 1er e´tage, Hoˆpital Edouard Herriot, 69 437 Lyon Cedex 03, France e-mail: [email protected]
on porcine corneal stromas before validation on human corneas. Decellularization and lyophilization led to opacification of the stroma, which could be reversed by soaking in 100% glycerol. Cell-depleted transparized stromas were then lyophilized (LTDC) to allow their long-term conservation and water content was measured. The ultrastructure of LTDC corneas was examined by transmission electron microscopy (TEM). Histocompatibility antigens were undetectable on LTDC stromas by antibody staining. Finally, cytocompatibility of LTDC stromas was demonstrated on an ex vivo model of anterior lamellar keratoplasty. Differential staining was used to monitor colonization of LTDC stromas by cells from the receiving cornea. Only SDS-based decellularization produced acellular porcine stromas. The lowest SDS concentration tested (0.1%) was validated on human corneas. Unlike lyophilized corneas, LTDC stromas without residual water, express no histocompatibility markers, although TEM revealed the presence of cellular debris in an ultrastructural arrangement of collagen fibers very close to that of native corneas. This structure is compatible with colonization by cells from the receiver cornea in an ex vivo lamellar graft model. Our procedure produced non-immunogenic, transparent stromas with conserved ultrastructure compatible with long-term conservation. Keywords Decellularization Transparence Cyt
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