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The existence of dead cells in donor corneal endothelium preserved with storage media
  1. Koji Kitazawa1,2,
  2. Tsutomu Inatomi3,
  3. Hidetoshi Tanioka3,
  4. Satoshi Kawasaki4,
  5. Hiroko Nakagawa3,
  6. Osamu Hieda3,
  7. Hideki Fukuoka3,
  8. Naoki Okumura5,
  9. Noriko Koizumi5,
  10. Bernie Iliakis6,
  11. Chie Sotozono3,
  12. Shigeru Kinoshita1
  1. 1 Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  2. 2 Baptist Eye Institute, Kyoto, Japan
  3. 3 Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  4. 4 Department of Ophthalmology, Osaka University, Suita, Japan
  5. 5 Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan
  6. 6 SightLife, Seattle, Washington, DC, USA
  1. Correspondence to Professor Shigeru Kinoshita, Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto 602-0842, Japan; shigeruk{at}koto.kpu-m.ac.jp

Abstract

Aim To investigate the viability of donor corneal endothelial cells (CECs) preserved in storage media by histological examination.

Methods Twenty-eight donor corneas were obtained from SightLife Eye Bank (Seattle, Washington), and redundant peripheral portions of those corneas were used for histological examination after removal of the centre corneal graft for transplantation. To assess cell viability in the corneal endothelium, biostaining experiments were performed using propidium iodide, calcein-AM, Hoechst 33 342, annexin V, anti-vimentin antibody and toluidine blue.

Results Histological analysis of the endothelium showed that the cytoplasm of dead cells had low-intensity fluorescence and that their nuclei stained red, while almost all living cells had green cytoplasm and blue-stained nuclei. The mean dead cell rate in the 28 donor corneas was 4.9%±3.3% (mean ±SD) (range: 0.6%–10.5%). The propidium iodide-positive cells stained positive for annexin V, negative for vimentin and pale for toluidine blue. After the specimens were incubated in a culture medium, the red nucleus dead cells dropped off from the level of the blue nucleus living cells.

Conclusion Our findings showed the existence of dead cells in storage-media-preserved donor corneal endothelium and that they dropped off after incubation, thus suggesting that the decrease of CECs following keratoplasty may be related to the presence of dead cells.

  • corneal endothelium
  • corneal endothelial cell
  • donor cornea
  • storage media
  • keratoplasty
  • corneal transplantation

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Footnotes

  • Contributors ShK: conception and design of the study and obtaining of funding. KK and HT: collection, management, analysis and interpretation of data; and searching the literature. KK, HT and ShK: writing of the article. KK, HT, SaK, TI, HN, OH, HF, NO, NK, BI and ShK: approval of the manuscript. HT: statistical expertise.

  • Funding This study was supported in part by a Grant-in-Aid for scientific research from the Ministry of Education, Science, Culture, and Sports of Japan.

  • Competing interests None declared.

  • Patient consent Tissue used were anonymous.

  • Ethics approval the Committee for Ethical Issues in Human Research of Kyoto Prefectural University of Medicine.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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