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Cell and Tissue Banking

, Volume 18, Issue 2, pp 185–191 | Cite as

Considering 3D topography of endothelial folds to improve cell count of organ cultured corneas

  • Clotilde Jumelle
  • Thibaud Garcin
  • Anne Sophie Gauthier
  • Yaël Glasson
  • Aurélien Bernard
  • Yann Gavet
  • Jacques Klossa
  • Zhiguo He
  • Sophie Acquart
  • Philippe Gain
  • Gilles Thuret
Article

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.

Keywords

Cornea Endothelium Cell count Eye bank Organ culture 3D analysis Quality control 

Notes

Acknowledgements

The authors thank Cassandra Dubesset and Chantal Perrache for their technical assistance.

Funding

French National Research Agency for financial support (ANR-12-TECS-004, CorImMo 3D) in partnership with the company Tribvn, Châtillon, France.

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Clotilde Jumelle
    • 1
  • Thibaud Garcin
    • 1
  • Anne Sophie Gauthier
    • 1
  • Yaël Glasson
    • 1
  • Aurélien Bernard
    • 1
  • Yann Gavet
    • 2
  • Jacques Klossa
    • 3
  • Zhiguo He
    • 1
  • Sophie Acquart
    • 4
  • Philippe Gain
    • 1
    • 5
  • Gilles Thuret
    • 1
    • 5
    • 6
  1. 1.Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Federative Institute of Research in Sciences and Health EngineeringJean Monnet UniversitySaint-Étienne Cedex 2France
  2. 2.Ecole Nationale Supérieure des Mines de Saint-EtienneSaint-EtienneFrance
  3. 3.TrybvnChâtillonFrance
  4. 4.Eye Bank, French Blood CenterSaint-ÉtienneFrance
  5. 5.Department of OphthalmologyUniversity Hospital of Saint-EtienneSaint-EtienneFrance
  6. 6.Institut Universitaire de FranceParisFrance

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