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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
  • Céline AuxenfansEmail author
Article
  • 6 Downloads

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 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 Cytocompatible Non-immunogenic 

Notes

Compliance with ethical standards

Conflict of interest

None of the authors have any conflicts of interest to declare.

Human and animal rights

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Human corneas were provided by the cornea bank at the Hospices Civils de Lyon (Lyon, France), they were rejected for clinical use because of their low endothelial density and recolted with donor family informed consent and in accordance with the ethical standards of the institutional and/or national research committee (Declaration to research ministry No. DC-2008-162) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Banque de Tissus et de Cellules des Hospices Civils de LyonHôpital Edouard HerriotLyonFrance
  2. 2.Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, UMR 5305, CNRS, SFRBioSciencesGerland-Lyon SudUniversité Lyon 1LyonFrance
  3. 3.Service d’ophtalmologie, pavillon CHôpital Edouard HerriotLyonFrance
  4. 4.EA4169 “Fundamental, Clinical and Therapeutic Aspects of Skin Barrier Function”University of Lyon 1LyonFrance
  5. 5.Banque de Tissus et Cellules, Pavillon I 1er étageHôpital Edouard HerriotLyon Cedex 03France

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