Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A physical biomarker of the quality of cultured corneal endothelial cells and of the long-term prognosis of corneal restoration in patients

Abstract

Dysfunction of the corneal endothelium reduces the transparency of the cornea and can cause blindness. Because corneal endothelial cells have an extremely limited proliferative ability in vivo, treatment for corneal endothelial dysfunction involves the transplantation of donor corneal tissue. Corneal endothelium can also be restored via intraocular injection of endothelial cells in suspension after their expansion in vitro. Yet, because quality assessment during the expansion of the cells is a destructive process, a substantial number of the cultured cells are lost. Here, we show that the ‘spring constant’ of the effective interaction potential between endothelial cells in a confluent monolayer serves as a biomarker of the quality of corneal endothelial cells in vitro and of the long-term prognosis of corneal restoration in patients treated with culture-expanded endothelial cells or with transplanted corneas. The biomarker can be measured from phase contrast imaging in vitro and from specular microscopy in vivo, and may enable a shift from passive monitoring to pre-emptive intervention in patients with severe corneal disorders.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Restoration of the human cornea via injection of culture-expanded corneal endothelial cells.
Fig. 2: Course of experiments and analyses in vitro and in vivo.
Fig. 3: Characterization of in vivo corneal endothelium.
Fig. 4: Quality control of in vitro cell sources.
Fig. 5: Classification accuracies of different indicators.
Fig. 6: Collective order for predictive diagnosis.

Data availability

The authors declare that the main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request.

Code availability

All Igor codes in this work are available from the corresponding authors on reasonable request.

References

  1. 1.

    Dawson, D. G., Ubels, J. L. & Edelhauser, H. F. Adler’s Physiology of the Eye 71–130 (Elsevier, 2011).

  2. 2.

    Kinoshita, S. et al. Grading for corneal endothelial damage. Jpn. J. Ophthalmol. 118, 3 (2014).

    Google Scholar 

  3. 3.

    Bourne, W. M. Clinical estimation of corneal endothelial pump function. Trans. Am. Ophthalmol. Soc. 96, 229–242 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Gain, P. et al. Global survey of corneal transplantation and eye banking. JAMA Ophthalmol. 134, 167–173 (2016).

    PubMed  Article  Google Scholar 

  5. 5.

    Rahman, I., Carley, F., Hillarby, C., Brahma, A. & Tullo, A. B. Penetrating keratoplasty: indications, outcomes, and complications. Eye 23, 1288–1294 (2009).

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    Terry, M. A. & Ousley, P. J. Deep lamellar endothelial keratoplasty visual acuity, astigmatism, and endothelial survival in a large prospective series. Ophthalmology 112, 1541–1548 (2005).

    PubMed  Article  Google Scholar 

  7. 7.

    Melles, G. R., Wijdh, R. H. & Nieuwendaal, C. P. A technique to excise the Descemet membrane from a recipient cornea (descemetorhexis). Cornea 23, 286–288 (2004).

    PubMed  Article  Google Scholar 

  8. 8.

    Melles, G. R., Ong, T. S., Ververs, B. & van der Wees, J. Descemet membrane endothelial keratoplasty (DMEK). Cornea 25, 987–990 (2006).

    PubMed  Article  Google Scholar 

  9. 9.

    Tan, D. T. H., Dart, J. K. G., Holland, E. J. & Kinoshita, S. Corneal transplantation. Lancet 379, 1749–1761 (2012).

    PubMed  Article  Google Scholar 

  10. 10.

    2015 Eye Banking Statistical Report (Eye Bank Association of America, 2016).

  11. 11.

    Melles, G. R. J., Ong, T. S., Ververs, B. & van der Wees, J. Preliminary clinical results of Descemet membrane endothelial keratoplasty. Am. J. Ophthalmol. 145, 222–227.e1 (2008).

    PubMed  Article  Google Scholar 

  12. 12.

    Price, M. O., Giebel, A. W., Fairchild, K. M. & Price, F. W. Descemet’s membrane endothelial keratoplasty: prospective multicenter study of visual and refractive outcomes and endothelial survival. Ophthalmology 116, 2361–2368 (2009).

    PubMed  Article  Google Scholar 

  13. 13.

    Schlögl, A., Tourtas, T., Kruse, F. E. & Weller, J. M. Long-term clinical outcome after Descemet membrane endothelial keratoplasty. Am. J. Ophthalmol. 169, 218–226 (2016).

    PubMed  Article  Google Scholar 

  14. 14.

    Wacker, K., Baratz, K. H., Maguire, L. J., McLaren, J. W. & Patel, S. V. Descemet stripping endothelial keratoplasty for Fuchs’ endothelial corneal dystrophy: five-year results of a prospective study. Ophthalmology 123, 154–160 (2016).

    PubMed  Article  Google Scholar 

  15. 15.

    Okumura, N. et al. Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest. Ophthalmol. Vis. Sci. 50, 3680–3687 (2009).

    PubMed  Article  Google Scholar 

  16. 16.

    Kinoshita, S. et al. Cultured corneal endothelial cell injection therapy for bullous keratopathy. N. Eng. J. Med. 378, 995–1003 (2018).

    CAS  Article  Google Scholar 

  17. 17.

    Toda, M. et al. Production of homogeneous cultured human corneal endothelial cells indispensable for innovative cell therapy. Invest. Ophthalmol. Vis. Sci. 58, 2011–2020 (2017).

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Hamuro, J. et al. Cultured human corneal endothelial cell aneuploidy dependence on the presence of heterogeneous subpopulations with distinct differentiation phenotypes. Invest. Ophthalmol. Vis. Sci. 57, 4385–4392 (2016).

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Hamuro, J. et al. Cell homogeneity indispensable for regenerative medicine by cultured human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 57, 4749–4761 (2016).

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    McCarey, B. E. Noncontact specular microscopy: a macrophotography technique and some endothelial cell findings. Ophthalmology 86, 1848–1860 (1979).

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Tanaka, H. et al. Panoramic view of human corneal endothelial cell layer observed by a prototype slit-scanning wide-field contact specular microscope. Br. J. Ophthalmol. 101, 655–659 (2017).

    PubMed  Article  Google Scholar 

  22. 22.

    McCarey, B. E., Edelhauser, H. F. & Lynn, M. J. Review of corneal endothelial specular microscopy for FDA clinical trials of refractive procedures, surgical devices, and new intraocular drugs and solutions. Cornea 27, 1–16 (2008).

    PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Doughty, M. J. Toward a quantitative analysis of corneal endothelial cell morphology: a review of techniques and their application. Optom. Vis. Sci. 66, 626–642 (1989).

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Okumura, N. et al. Development of cell analysis software for cultivated corneal endothelial cells. Cornea 36, 1387–1394 (2017).

    PubMed  Article  Google Scholar 

  25. 25.

    Veschgini, M. et al. Size, shape, and lateral correlation of highly uniform, mesoscopic, self-assembled domains of fluorocarbon–hydrocarbon diblocks at the air/water interface: a GISAXS study. ChemPhysChem 18, 2791–2798 (2017).

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Ramos, L., Lubensky, T. C., Dan, N., Nelson, P. & Weitz, D. A. Surfactant-mediated two-dimensional crystallization of colloidal crystals. Science 286, 2325–2328 (1999).

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Wickman, H. H. & Korley, J. N. Colloid crystal self-organization and dynamics at the air/water interface. Nature 393, 445–447 (1998).

    CAS  Article  Google Scholar 

  28. 28.

    Brookes, N. H. Riding the cell jamming boundary: geometry, topology, and phase of human corneal endothelium. Exp. Eye Res. 172, 171–180 (2018).

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Park, C. Y., Lee, J. K., Gore, P. K., Lim, C. Y. & Chuck, R. S. Keratoplasty in the United States: a 10-year review from 2005 through 2014. Ophthalmology 122, 2432–2442 (2015).

    PubMed  Article  Google Scholar 

  30. 30.

    Harrison, T. A. et al. Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area. Mol. Vis. 22, 31–39 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Swets, J. A. Measuring the accuracy of diagnostic systems. Science 240, 1285–1293 (1988).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Egan, J. P. Signal Detection Theory and ROC Analysis (Academic Press, 1975).

  33. 33.

    Fawcett, T. An introduction to ROC analysis. Pattern Recognit. Lett. 27, 861–874 (2006).

    Article  Google Scholar 

Download references

Acknowledgements

We thank the Baptist Eye Institute for access to the clinical records, H. Nakagawa for data collection, K. Yoshikawa for stimulating discussions, and J. Bush for reviewing the manuscript. This work was supported by Nakatani Foundation (M.Tanaka and M.U.), JSPS (17H00855 to M.Tanaka; 16K05515 to A.Y.), MEXT (16KT0070 to M.Tanaka), the Highway Program for Realization of Regenerative Medicine of the Japan Agency for Medical Research and Development (S.K.) and the Research Project for Practical Applications of Regenerative Medicine of the Japanese Ministry of Health, Labor and Welfare (S.K.).

Author information

Affiliations

Authors

Contributions

M.U. and M.Tanaka conceived and directed the research. A.Y. and H.T. performed the research and analysis. M.Toda collected the data. C.S. and S.K. supervised the retrospective analysis of clinical research. A.Y., H.T., J.H., S.K., M.U. and M.Tanaka wrote the paper.

Corresponding authors

Correspondence to Morio Ueno or Motomu Tanaka.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yamamoto, A., Tanaka, H., Toda, M. et al. A physical biomarker of the quality of cultured corneal endothelial cells and of the long-term prognosis of corneal restoration in patients. Nat Biomed Eng 3, 953–960 (2019). https://doi.org/10.1038/s41551-019-0429-9

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing