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Hiroshi Tanaka, Akihisa Yamamoto, Munetoyo Toda, Junji Hamuro, Chie Sotozono, Shigeru Kinoshita, Morio Ueno, Motomu Tanaka; Quantifying the adhesion strength of human corneal endothelial cells in an in vitro model. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5305. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelial cells (CECs) are located at the inner-most surface of the cornea and play an important role in maintaining corneal transparency. When the number of human CECs (HCECs) substantially decrease due to injury or disease, corneal transplantation is required because of the subsequent limited proliferation of HCECs. We are currently developing a new therapy for bullous keratopathy that involves the infusion of cultivated HCECs into the anterior chamber. A key aspect of this novel regenerative therapy is the stable accommodation of HCECs on the Descemet’s membrane which express laminin 511. Here, we present a novel assay to quantify the mechanical strength of single-cell adhesion using pressure waves induced by picosecond laser pulses, thus enabling the critical pressure required for cell detachment to be determined. Using planar lipid membranes displaying laminin 511 E-8 fragment (laminin E-8) at well-defined surface densities (known as supported membranes), we quantitatively evaluated the influence of rho-associated protein kinase (ROCK)-inhibitor Y-26732 on the adhesion function of cultivated HCECs.
Human donor corneal tissues were obtained from an eye bank (SightLife, Seattle, WA), and HCECs obtained from those tissues were re-suspended and cultured until reaching confluence. The cultivated HCECs were classified into one of two subpopulations (CD44- or CD44+) by fluorescence-activated cell sorting and then re-suspended and cultured on the supported membranes functionalized with human laminin E-8 with or without Y-26732 for 1 hour.
The energy needed to detach 50% of the cultivated CD44+ HCECs (P50) from the laminin functionalized supported membranes in the presence and absence of Y-27632 was 12.0 MPa and 14.2 MPa, respectively, and P50 of the CD44- cells in culture media with or without Y-27632 was 14.1MPa and 10.6 MPa, respectively.
Our findings show that the adhesion strength of both CD44- and CD44+ cells was successfully measured by a novel pressure-wave assay. Y-26732 improved the adhesion of CD44- HCECs, but slightly reduced that of CD44+ HCECs. Our experimental findings provide with the first quantitative data of CD44+/CD44- HCECs on in vitro Descemet’s membrane models in the presence and absence of Y-26732.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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