Feasibility of a cryopreservation of cultured human corneal endothelial cells.

Transparency of the cornea is essential for vision and is maintained by the corneal endothelium. Consequently, corneal endothelial decompensation arising from irreversible damage to the corneal endothelium causes severe vision impairment. Until recently, transplantation of donor corneas was the only therapeutic choice for treatment of endothelial decompensation. In 2013, we initiated clinical research into cell-based therapy involving injection of a suspension of cultured human corneal endothelial cells (HCECs), in combination with Rho kinase inhibitor, into the anterior chamber. The aim of the present study was to establish a protocol for cryopreservation of HCECs to allow large-scale commercial manufacturing of these cells. This study focused on the effects of various cryopreservation reagents on HCEC viability. Screening of several commercially available cryopreservation reagents identified Bambanker hRM as an effective agent that maintained a cell viability of 89.4% after 14 days of cryopreservation, equivalent to the cell viability of 89.2% for non-cryopreserved control cells. The use of Bambanker hRM and HCECs at a similar grade to that used clinically for cell based therapy (passage 3-5 and a cell density higher than 2000 cells/mm2) gave a similar cell density for cryopreserved HCECs to that of non-preserved control HCECs after 28 days of cultivation (2099 cells/mm2 and 2111 cells/mm2, respectively). HCECs preserved using Bambanker hRM grew in a similar fashion to non-preserved control HCECs and formed a monolayer sheet-like structure. Cryopreservation of HCECs has multiple advantages including the ability to accumulate stocks of master cells, to transport HCEC stocks, and to manufacture HCECs on demand for use in cell-based treatment of endothelial decompensation.

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

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.

Involvement of Nectin-Afadin in the Adherens Junctions of the Corneal Endothelium.

PURPOSE: The cell-cell adhesion molecules, cadherins and nectins, are involved in the formation of adherens junctions. However, involvement of nectins in the corneal endothelium has not yet been established. This study investigated the involvement of nectins in adherens junctions of the corneal endothelium. METHODS: Nectin and cadherin expression in the corneal endothelium was evaluated by real-time polymerase chain reaction. Colocalization and direct binding of nectin-1 and N-cadherin to anchoring proteins (afadin and ß-catenin, respectively) were determined by immunostaining and immunoprecipitation. The effect of afadin and N-cadherin knockdown on apical junctions was evaluated by immunostaining. RESULTS: Real-time polymerase chain reaction confirmed nectin-1, nectin-2, nectin-3, nectin-4, and afadin expression in the corneal endothelium. Immunofluorescence staining showed colocalization of nectin and afadin at the basal side of the tight junction (where adherens junctions typically locate) and immunoprecipitation confirmed direct binding of nectin to afadin. N-cadherin, P-cadherin, VE-cadherin, and OB-cadherin messenger RNAs were expressed in the corneal endothelium. N-cadherin and ß-catenin colocalized at the cell-cell border, where they directly bound and formed a cell-cell adhesion complex. N-cadherin knockdown disrupted the normal expression pattern of zonula occludens protein-1 and afadin, but afadin knockdown had no effect on the expression pattern of zonula occludens protein-1 and N-cadherin. CONCLUSIONS: We believe this to be the first report of conservation of the nectin-afadin system in the corneal endothelium and its involvement in the formation of adherens junctions. N-cadherin, as a member of the cadherin family, is also essential for the formation and maintenance of cell-cell adhesion mediated by nectins and tight junctions in the corneal endothelium.

Activation of the Rho/Rho Kinase Signaling Pathway Is Involved in Cell Death of Corneal Endothelium.

Purpose: Rho kinase (ROCK) pathways control fundamental cell functions, making ROCK an important therapeutic target in several pathophysiologic conditions. The purpose of this study was to investigate whether inhibition of ROCK can suppress apoptosis of the corneal endothelium and to determine the role of ROCK signaling in regulating apoptosis. Methods: The effects of inhibitors of ROCK or myosin light chain (MLC) were evaluated in cultured monkey corneal endothelial cells (MCECs) irradiated with ultraviolet (UV) (100 J/m2) to induce apoptosis. Annexin V and TUNEL staining and Western blot for apoptosis-related proteins and focal adhesion complexes were then performed. RhoA activation was further evaluated by pull-down assays. ROCK inhibitor and caspase inhibitor effects on apoptosis were also evaluated in MCECs treated with ethylene glycol tetraacetic acid (EGTA) to induce MLC phosphorylation. Results: ROCK or MLC inhibition suppressed the caspase-3 cleavage and Annexin V and TUNEL expression typically seen during UV-mediated apoptosis of MCECs. The apoptotic stimulus activated RhoA and then induced phosphorylation of MLC via ROCK activation. EGTA-mediated phosphorylation of MLC was sufficient to induce the loss of cell contact with the substrate and subsequent apoptosis. Western blot showed that ROCK inhibition upregulated the expression of the focal adhesion complex in adhered cells, following UV stress. Conclusions: Apoptotic stimuli activated Rho/ROCK/MLC phosphorylation in the corneal endothelium, and subsequent actomyosin contraction induced apoptosis by loss of cell adhesion. ROCK inhibition suppressed MLC phosphorylation and subsequent cell death, and it counteracted the loss of cell adhesion by activating the focal adhesion complex.