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.
This is a preview of subscription content, log in to check access.
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.
Bartels MC, Doxiadis IIN, Colen TP, Beekhuis WH (2003) Long-term outcome in high-risk corneal transplantation and the influence of HLA-A and HLA-B matching. Cornea 22:552–556CrossRefPubMedGoogle Scholar
Baylis O, Rooney P, Figueiredo F, Lako M, Ahmad S (2013) An investigation of donor and culture parameters which influence epithelial outgrowths from cultured human cadaveric limbal explants. J Cell Physiol 228:1025–1030CrossRefPubMedGoogle Scholar
Bayyoud T, Thaler S, Hofmann J, Maurus C, Spitzer MS, Bartz-Schmidt K-U, Szurman P, Yoeruek E (2012) Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells. Curr Eye Res 37:179–186CrossRefPubMedGoogle Scholar
Du L, Wu X (2011) Development and characterization of a full-thickness acellular porcine cornea matrix for tissue engineering. Artif Organs 35:691–705CrossRefPubMedGoogle Scholar
Du L, Wu X, Pang K, Yang Y (2011) Histological evaluation and biomechanical characterisation of an acellular porcine cornea scaffold. Br J Ophthalmol 95:410–414CrossRefPubMedGoogle Scholar
Farias RJM, Sousa LB, Lima Filho AAS, Lourenço ACS, Tanakai MH, Freymuller E (2008) Light and transmission electronic microscopy evaluation of lyophilized corneas. Cornea 27:791–794CrossRefPubMedGoogle Scholar
Frueh BE, Cadez R, Böhnke M (1998) In vivo confocal microscopy after photorefractive keratectomy in humans. A prospective, long-term study. Arch Ophthalmol 116:1425–1431CrossRefPubMedGoogle Scholar
Gilbert TW, Sellaro TL, Badylak SF (2006) Decellularization of tissues and organs. Biomaterials 27:3675–3683PubMedGoogle Scholar
James SE, Rowe A, Ilari L, Daya S, Martin R (2001) The potential for eye bank limbal rings to generate cultured corneal epithelial allografts. Cornea 20:488–494CrossRefPubMedGoogle Scholar
Ju C, Gao L, Wu X, Pang K (2012) A human corneal endothelium equivalent constructed with acellular porcine corneal matrix. Indian J Med Res 135:887–894PubMedPubMedCentralGoogle Scholar
Khaireddin R, Wachtlin J, Hopfenmüller W, Hoffmann F (2003) HLA-A, HLA-B and HLA-DR matching reduces the rate of corneal allograft rejection. Graefes Arch Clin Exp Ophthalmol 241:1020–1028CrossRefPubMedGoogle Scholar
Krahn KN, Bouten CVC, van Tuijl S, van Zandvoort MAMJ, Merkx M (2006) Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture. Anal Biochem 350:177–185CrossRefPubMedGoogle Scholar
Lin X-C, Hui Y-N, Wang Y-S, Meng H, Zhang Y-J, Jin Y (2008) Lamellar keratoplasty with a graft of lyophilized acellular porcine corneal stroma in the rabbit. Vet Ophthalmol 11:61–66CrossRefPubMedGoogle Scholar
Pang K, Du L, Wu X (2010) A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes. Biomaterials 31:7257–7265CrossRefPubMedGoogle Scholar
Ponce Márquez S, Martínez VS, McIntosh Ambrose W, Wang J, Gantxegui NG, Schein O, Elisseeff J (2009) Decellularization of bovine corneas for tissue engineering applications. Acta Biomater 5:1839–1847CrossRefPubMedGoogle Scholar
Reinhard T, Böhringer D, Enczmann J, Kögler G, Mayweg S, Wernet P, Sundmacher R (2004) Improvement of graft prognosis in penetrating normal-risk keratoplasty by HLA class I and II matching. Eye 18:269–277CrossRefPubMedGoogle Scholar
Sanchez I, Martin R, Ussa F, Fernandez-Bueno I (2011) The parameters of the porcine eyeball. Graefes Arch Clin Exp Ophthalmol 249:475–482CrossRefPubMedGoogle Scholar
Sanfilippo F, MacQueen JM, Vaughn WK, Foulks GN (1986) Reduced graft rejection with good HLA-A and B matching in high-risk corneal transplantation. N Engl J Med 315:29–35CrossRefPubMedGoogle Scholar
Sasaki S, Funamoto S, Hashimoto Y, Kimura T, Honda T, Hattori S, Kobayashi H, Kishida A, Mochizuki M (2009) In vivo evaluation of a novel scaffold for artificial corneas prepared by using ultrahigh hydrostatic pressure to decellularize porcine corneas. Mol Vis 15:2022–2028PubMedPubMedCentralGoogle Scholar
Shafiq MA, Gemeinhart RA, Yue BYJT, Djalilian AR (2012) Decellularized human cornea for reconstructing the corneal epithelium and anterior stroma. Tissue Eng Part C Methods 18:340–348CrossRefPubMedGoogle Scholar
Zhou Y, Wu Z, Ge J, Wan P, Li N, Xiang P, Gao Q, Wang Z (2011) Development and characterization of acellular porcine corneal matrix using sodium dodecylsulfate. Cornea 30:73–82CrossRefPubMedGoogle Scholar