A human corneal endothelium equivalent constructed with acellular porcine corneal matrix

Background & objectives: Artificial corneal endothelium equivalents can not only be used as in vitro model for biomedical research including toxicological screening of drugs and investigation of pathological corneal endothelium conditions, but also as potential sources of grafts for corneal keratoplasty. This study was aimed to demonstrate the feasibility of constructing human corneal endothelium equivalents using human corneal endothelial cells and acellular porcine corneal matrix. Methods: Porcine corneas were decellularized with sodium dodecyl sulphate (SDS) solution. Human corneal endothelial cells B4G12 were cultured with leaching liquid extracted from the acellular porcine corneal matrix, and then cell proliferative ability was evaluated by MTT assay. B4G12 cells were transplanted to a rat corneal endothelial deficiency model to analyze their in vivo bio-safety and pump function, and then seeded and cultured on acellular porcine corneal matrix for two wk. Corneal endothelium equivalents were analyzed using HE staining, trypan blue and alizarin red S co-staining, immunofluorescence and corneal swelling assay. Results: The leaching liquid from acellular porcine corneal matrix had little influence on the proliferation ability of B4G12 cells. Animal transplantation of B4G12 cells showed that these cells had similar function to the native cells without causing a detectable immunological reaction and neoplasm in vivo. These formed a monolayer covering the surface of the acellular porcine corneal matrix. Trypan blue and alizarin red S co-staining showed that B4G12 cells were alive after two wk in organ culture and cell boundaries were clearly delineated. Proper localizations of ZO-1 and Na+/K+ ATPase were detected by immunofluorescence assay. Functional experiments were conducted to show that the Na+/K+ ATPase inhibitor ouabain could block the ionic-pumping function of this protein, leading to persistent swelling of 51.7 per cent as compared to the control. Interpretation & conclusions: Our findings showed that B4G12 cells served as a good model for native corneal endothelial cells in vivo. Corneal endothelium equivalents had properties similar to those of native corneal endothelium and could serve as a good model for in vitro study of human corneal endothelium.

[1]  Xinyi Wu,et al.  Development and characterization of a full-thickness acellular porcine cornea matrix for tissue engineering. , 2011, Artificial organs.

[2]  Traian V Chirila,et al.  Human corneal endothelial cell growth on a silk fibroin membrane. , 2011, Biomaterials.

[3]  L. Germain,et al.  Corneal endothelial toxicity of air and SF6. , 2011, Investigative ophthalmology & visual science.

[4]  T. Levin-Harrus,et al.  Protective effect of different ophthalmic viscosurgical devices on corneal endothelium during severe phacoemulsification model in rabbits. , 2011, Ophthalmic surgery, lasers & imaging : the official journal of the International Society for Imaging in the Eye.

[5]  Hyun Seung Kim,et al.  The effect of C3F8 gas on corneal endothelial cells in rabbits , 2010, Japanese Journal of Ophthalmology.

[6]  Yong-mei Yang,et al.  Histological evaluation and biomechanical characterisation of an acellular porcine cornea scaffold , 2010, British Journal of Ophthalmology.

[7]  Xinyi Wu,et al.  A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes. , 2010, Biomaterials.

[8]  Shay Soker,et al.  Bioengineering endothelialized neo-corneas using donor-derived corneal endothelial cells and decellularized corneal stroma. , 2010, Biomaterials.

[9]  Xianqun Fan,et al.  Reconstruction of a Tissue-Engineered Cornea with Porcine Corneal Acellular Matrix as the Scaffold , 2009, Cells Tissues Organs.

[10]  L. Germain,et al.  Tissue engineering of feline corneal endothelium using a devitalized human cornea as carrier. , 2009, Tissue engineering. Part A.

[11]  Noriko Koizumi,et al.  [Cultivated corneal endothelial cell sheet transplantation in a primate model]. , 2009, Nippon Ganka Gakkai zasshi.

[12]  C. Werner,et al.  Cultivation of an immortalized human corneal endothelial cell population and two distinct clonal subpopulations on thermo-responsive carriers , 2008, Graefe's Archive for Clinical and Experimental Ophthalmology.

[13]  B. Han,et al.  Preparation and properties of a chitosan-based carrier of corneal endothelial cells , 2008, Journal of materials science. Materials in medicine.

[14]  R. Funk,et al.  Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics , 2008, Cells Tissues Organs.

[15]  John Fisher,et al.  Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering. , 2007, Biomaterials.

[16]  Qu Jia,et al.  Using Basement Membrane of Human Amniotic Membrane as a Cell Carrier for Cultivated Cat Corneal Endothelial Cell Transplantation , 2007, Current eye research.

[17]  K. Woodhouse,et al.  Decellularized placental matrices for adipose tissue engineering. , 2006, Journal of biomedical materials research. Part A.

[18]  B. Ratner,et al.  Development of an esophagus acellular matrix tissue scaffold. , 2006, Tissue engineering.

[19]  Koki Abe,et al.  Development of the Human Umbilical Vein Scaffold for Cardiovascular Tissue Engineering Applications , 2005, ASAIO journal.

[20]  K. Tsubota,et al.  Transplantation of corneal endothelium with Descemet’s membrane using a hyroxyethyl methacrylate polymer as a carrier , 2005, British Journal of Ophthalmology.

[21]  Michael Horrocks,et al.  Endothelial and Smooth Muscle Cell Seeding onto Processed Ex Vivo Arterial Scaffolds Using 3D Vascular Bioreactors , 2004, ASAIO journal.

[22]  N. Joyce,et al.  Proliferative response of corneal endothelial cells from young and older donors. , 2004, Investigative ophthalmology & visual science.

[23]  N. Fullwood,et al.  Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. , 2004, Investigative ophthalmology & visual science.

[24]  K. Engelmann,et al.  Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells , 2001, The British journal of ophthalmology.

[25]  K. Engelmann,et al.  Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism , 2001, Graefe's Archive for Clinical and Experimental Ophthalmology.

[26]  R. Birngruber,et al.  Thermal and Biomechanical Parameters of Porcine Cornea , 2000, Cornea.

[27]  P. Friedl,et al.  Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells. , 2000, Acta ophthalmologica Scandinavica.

[28]  T. Wood,et al.  Corneal endothelial cell transplantation using Descemet's membrane as a carrier , 1993, Journal of cataract and refractive surgery.

[29]  F. Delori,et al.  Corneal endothelial function and structure following cryo-injury in the rabbit. , 1984, Investigative ophthalmology & visual science.

[30]  M. Taylor,et al.  Dual staining of corneal endothelium with trypan blue and alizarin red S: importance of pH for the dye-lake reaction. , 1981, The British journal of ophthalmology.

[31]  H. Edelhauser,et al.  Effects of ionophores X537a and A23187 and calcium-free medium on corneal endothelial morphology. , 1981, Investigative ophthalmology & visual science.

[32]  D L Van Horn,et al.  Regenerative capacity of the corneal endothelium in rabbit and cat. , 1977, Investigative ophthalmology & visual science.