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Development of Cell Analysis Software to Evaluate Fibroblastic Changes in Cultivated Corneal Endothelial Cells for Quality Control

Okumura, Naoki, MD, PhD*; Kobayashi, Keitaro, BS; Ishida, Naoya, BS; Kagami, Takato, BS*; Hiwa, Satoru, PhD; Hiroyasu, Tomoyuki, PhD; Koizumi, Noriko, MD, PhD*

doi: 10.1097/ICO.0000000000001757
Basic Investigation

Purpose: To develop software to evaluate the fibroblastic morphological changes in cultured human corneal endothelial cells (HCECs) as a quality control measure for use in tissue engineering therapy.

Methods: Software was designed to recognize cell borders, to approximate cell shape as an ellipse, and to calculate the aspect ratio of the ellipse as an indicator of severity of the fibroblastic morphological change. Using the designed software, 60 phase contrast images of polygonal HCECs and 60 phase contrast images of fibroblastic HCECs were analyzed. The correlations of the aspect ratio and other parameters (cell density, percentage of cells surrounded by 6 cells, and coefficient of variation) were evaluated.

Results: Cell shapes were recognized based on phase contrast images and were approximated as ellipses by software. The average aspect ratio was significantly higher (34.9% ± 6.1%) in fibroblastic HCECs than in polygonal HCECs (24.4% ± 2.3%) (P < 0.01). The aspect ratio showed a correlation with cell density, with the percentage of cells surrounded by 6 neighboring cells, and with the coefficient of variation (Pearson correlation coefficients, −0.84, −0.38, and 0.66, respectively).

Conclusions: We propose that fibroblastic alteration of HCECs can be evaluated by the cell morphology based on the aspect ratio. Software developed in this study, which can analyze the frequency and severity of fibroblastic alteration, will be useful for nondestructive assessment of cells destined for use in cell-based therapy for corneal endothelial decompensation.

Departments of *Biomedical Engineering; and

Biomedical Information, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.

Correspondence: Noriko Koizumi, MD, PhD, Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe 610-0321, Japan (e-mail:

Supported by the Program for the Strategic Research Foundation at Private Universities from MEXT (N.K. and N.O.).

The authors have no conflicts of interest to disclose.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (

Received March 15, 2018

Received in revised form July 13, 2018

Accepted July 31, 2018

Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.