title: Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty.
abstract: OBJECTIVE
  To describe a novel method of global cell viability assessment for Descemet membrane endothelial keratoplasty (DMEK) and the comparison of two contemporary methods of donor tissue preparation.
  
  
  METHODS
  DMEK transplants were prepared using two different methods: liquid bubble separation and manual peeling (n=8 each group). Samples were incubated with Hoechst, calcein-AM and ethidium homodimer prior to mounting on a curved imaging chamber. Z-stacked fluorescence microscopy images were combined to produce an in-focus global image capable of resolving all cell nuclei. Image processing software was used to define a calcein-positive live cell area, count all cell nuclei within this area and subtract ethidium-positive dead cells to derive the total viable endothelial cell count. Corrected global cell density was calculated by dividing the number of viable cells by the graft area, which had been corrected for imaging a curved surface.
  
  
  RESULTS
  Corrected global cell density was lower than the central endothelial cell density in both groups: 85.5% of the pre-preparation central endothelial cell density in the peel group and 75.8% in the bubble group. Corrected global cell density was significantly lower in the liquid bubble separation group than in the peel group (p=0.04).
  
  
  CONCLUSIONS
  Eye bank estimations of central endothelial cell density overestimate true cell density after graft preparation in DMEK. A peel method is less damaging and more consistent than a liquid bubble method. Cell loss correlated strongly with the degree of stromal hydration prior to bubble separation in the liquid bubble group.
authors.affiliation: Department of Corneal and External Disease, Moorfields Eye Hospital, London, UK Department of Cell Biology, University College London, Institute of Ophthalmology, London, UK.
authors.forename: Maninder
authors.initials: M
authors.lastname: Bhogal
authors.affiliation: Department of Cell Biology, University College London, Institute of Ophthalmology, London, UK.
authors.forename: Maria S
authors.initials: MS
authors.lastname: Balda
authors.affiliation: Department of Cell Biology, University College London, Institute of Ophthalmology, London, UK.
authors.forename: Karl
authors.initials: K
authors.lastname: Matter
authors.affiliation: Department of Corneal and External Disease, Moorfields Eye Hospital, London, UK Department of Cell Biology, University College London, Institute of Ophthalmology, London, UK.
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Allan
mesh_terms: D000368:Aged; D002452:Cell Count; D002470:Cell Survival; D055954:Corneal Endothelial Cell Loss / Q000175:diagnosis; D003470:Culture Media; D057111:Descemet Stripping Endothelial Keratoplasty; D004728:Endothelium, Corneal / Q000473:pathology*; D005125:Eye Banks; D006801:Humans; D008875:Middle Aged; D009924:Organ Culture Techniques; D014019:Tissue Donors; D020858:Tissue and Organ Harvesting / Q000379:methods*
chemical_list: D003470:Culture Media
keywords: Cornea; Eye (Tissue) Banking; Treatment Surgery