title: Considering 3D topography of endothelial folds to improve cell count of organ cultured corneas.
abstract: The posterior side of the cornea is covered by the endothelial monolayer, which governs corneal transparency but cannot proliferate. Determination of endothelial cell density (ECD) is therefore the minimal and mandatory quality control in all eye banks. It avoids primary graft failures caused by endothelial insufficiency, and allows allocation of corneas to surgical techniques requiring different numbers of endothelial cells (ECs). Corneas stored in organ culture (17% of grafts worldwide), are characterized by heavy stromal swelling and numerous deep endothelial folds, up to 200 µm high. During microscopic en face observation, flat surfaces are thus exceptional and EC counting is biased by parallax errors, resulting in overestimated eye bank ECD (ebECD). We used a motorized transmitted light microscope to acquire Z-stacks of images every 10 µm, and processed them to reconstruct the 3D surface of the folded endothelium. This method (3D-ECD) takes into account the local point-by-point slope in order to correct ECD. On a set of 30 corneas, we compared 3D-ECD and ebECD determined on five identical zones at the center of the cornea. 3D reconstruction allowed us to visualize twice as many cells, and ebECD was 8.1 ± 4.5% (95%CI 6.4-9.7) higher than 3D-ECD, with 1744 ± 488 versus 1606 ± 473 cells/mm2. 3D counting makes it possible to increase cell sampling and to correct overestimation by the conventional en face counting still routinely performed in eye banks.
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health Engineering, Jean Monnet University, 10, Rue de la Marandiere, 42023, Saint-Étienne Cedex 2, France.
authors.forename: Clotilde
authors.initials: C
authors.lastname: Jumelle
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health Engineering, Jean Monnet University, 10, Rue de la Marandiere, 42023, Saint-Étienne Cedex 2, France.
authors.forename: Thibaud
authors.initials: T
authors.lastname: Garcin
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health Engineering, Jean Monnet University, 10, Rue de la Marandiere, 42023, Saint-Étienne Cedex 2, France.
authors.forename: Anne Sophie
authors.initials: AS
authors.lastname: Gauthier
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health Engineering, Jean Monnet University, 10, Rue de la Marandiere, 42023, Saint-Étienne Cedex 2, France.
authors.forename: Yaël
authors.initials: Y
authors.lastname: Glasson
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health Engineering, Jean Monnet University, 10, Rue de la Marandiere, 42023, Saint-Étienne Cedex 2, France.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Bernard
authors.affiliation: Ecole Nationale Supérieure des Mines de Saint-Etienne, Saint-Etienne, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Gavet
authors.affiliation: Trybvn, Châtillon, France.
authors.forename: Jacques
authors.initials: J
authors.lastname: Klossa
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health Engineering, Jean Monnet University, 10, Rue de la Marandiere, 42023, Saint-Étienne Cedex 2, France.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: He
authors.affiliation: Eye Bank, French Blood Center, Saint-Étienne, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Acquart
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health Engineering, Jean Monnet University, 10, Rue de la Marandiere, 42023, Saint-Étienne Cedex 2, France.|Department of Ophthalmology, University Hospital of Saint-Etienne, Saint-Etienne, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Gain
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health Engineering, Jean Monnet University, 10, Rue de la Marandiere, 42023, Saint-Étienne Cedex 2, France. gilles.thuret@univ-st-etienne.fr.|Department of Ophthalmology, University Hospital of Saint-Etienne, Saint-Etienne, France. gilles.thuret@univ-st-etienne.fr.|Institut Universitaire de France, Bd Saint Michel, Paris, France. gilles.thuret@univ-st-etienne.fr.
authors.forename: Gilles
authors.identifier: http://orcid.org/0000-0003-3486-2875
authors.initials: G
authors.lastname: Thuret
mesh_terms: D002452:Cell Count / Q000379:methods*; D003315:Cornea / Q000166:cytology*; D004728:Endothelium, Corneal / Q000166:cytology*; D005125:Eye Banks / Q000379:methods; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D009924:Organ Culture Techniques / Q000379:methods; D009926:Organ Preservation / Q000379:methods; D011786:Quality Control
keywords: 3D analysis; Cell count; Cornea; Endothelium; Eye bank; Organ culture; Quality control