title: Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells.
abstract: OBJECTIVE
  To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs).
  
  
  METHODS
  Posterior lamellae of ten bovine corneas were decellularized using ethylene diamin tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/mL) and 0.3% sodium dodecyl sulphate (SDS). Hematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining was done to confirm the absence of bovine cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using a DNA Purification Kit. HCECs were harvested from human donor eyes and seeded on the Descemet's membrane of the DBCs. Cell morphology was assessed after 6 h of incubation, and at days 1, 4, 7, 10 and 14. Expression of zonula occludens-1 (ZO-1), connexin-43 (CX-43), Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), natrium hydrogen carboanhydrase (Na(+)/HCO(3)(-)), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry.
  
  
  RESULTS
  HE staining and DAPI staining showed that bovine cells were substantially removed from the stroma and Descemet's membrane. A significant DNA reduction (mean before decelluraziation 365.3 ± 88.6 ng/mg, mean after decelluarization 23.2 ± 7.9 ng/mg, p < 0.001) was observed. HCECs formed a continuous, viable, predominantly polygonal monolayer with a mean cell density of 2380 ± 179 cells/mm(2) on DBCs. Immunohistochemistry analysis demonstrated positive staining for AE5, collagen type VIII, ZO-1, CX-43, Na(+)/HCO(3)(-), and Na(+)/K(+)-ATPase.
  
  
  CONCLUSIONS
  Phenotypical properties of HCECs on DBCs imply that the HCEC sheets are capable of maintaining an intact barrier and ionic pump function in vitro. DBCs might, therefore, be a promising scaffold for ex vivo expansion of HCECs. This xenogeneic substrate might be used for therapy of isolated corneal endothelial diseases.
authors.affiliation: Department of Ophthalmology, Eberhard-Karls University Tuebingen, Schleichstrasse 12, Tuebingen, Germany.
authors.forename: Tarek
authors.initials: T
authors.lastname: Bayyoud
authors.forename: Sebastian
authors.initials: S
authors.lastname: Thaler
authors.forename: Johanna
authors.initials: J
authors.lastname: Hofmann
authors.forename: Christine
authors.initials: C
authors.lastname: Maurus
authors.forename: Martin Stephan
authors.initials: MS
authors.lastname: Spitzer
authors.forename: Karl-Ulrich
authors.initials: KU
authors.lastname: Bartz-Schmidt
authors.forename: Peter
authors.initials: P
authors.lastname: Szurman
authors.forename: Efdal
authors.initials: E
authors.lastname: Yoeruek
mesh_terms: D000368:Aged; D000818:Animals; D001672:Biocompatible Materials; D015415:Biomarkers / Q000378:metabolism; D002417:Cattle; D002452:Cell Count; D018929:Cell Culture Techniques; D018031:Connexin 43 / Q000378:metabolism; D003315:Cornea; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D023822:Tissue Engineering; D054457:Tissue Scaffolds; D014183:Transplantation, Heterologous; D062826:Zonula Occludens-1 Protein
chemical_list: D001672:Biocompatible Materials; D015415:Biomarkers; D018031:Connexin 43; D016326:Extracellular Matrix Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D027982:Sodium-Bicarbonate Symporters; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D000254:Sodium-Potassium-Exchanging ATPase