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Kazuya Kakutani, Naoki Okumura, Ryohei Numata, Ursula Schlotzer-Schrehardt, Friedrich E Kruse, Shigeru Kinoshita, Noriko Koizumi; The efficiency of laminin-511 and laminin-521 as extracellular matrix for human corneal endothelial cell culture. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2055.
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© ARVO (1962-2015); The Authors (2016-present)
Cultivated corneal endothelial cell (CEC) transplantation is used to treat endothelial dysfunction, yet the in vitro expansion of human CECs (HCECs) is still a pivotal practical issue. In this study, we investigated the expression pattern of specific laminins in Descemet’s membrane (DM) and the feasibility of using them as a substrate for HCEC culture for clinical applications.
Expression pattern of laminin chains (α1-5, β1-4, γ1-3) in donor-cornea HCECs were examined by reverse transcriptase polymerase chain reaction (RT-PCR). Next, the expression of laminin subunits (α2, α5, β1, β2, γ1) of human DMs with intact HCECs was evaluated by immunostaining. HCECs were cultivated on laminin-511 (α5β1γ1), -521 (α5β2γ1), -211 (α2β1γ1) coated dishes, or non-coated dishes. When the cells reached confluence, immunocytochemical analysis was performed using antibodies for Na+/K+-ATPase and ZO-1 and cell density was measured. Cell adhesion was evaluated by Cell Titer-Glo® assay at 24 hours and cell proliferation was analyzed by BrdU ELISA at 48 hours.
mRNA expression of laminin chains for α5, β1, β2, β3, γ1, and γ2 was detected in HCECs. Immunostaining showed that laminin chains of α5, β1, β2, γ1 were expressed in the DM. The HCECs cultivated on laminin-511 and -521 (expressed in DM) showed a hexagonal morphology and a staining profile characteristic of Na+/K+-ATPase and ZO-1 at the plasma membrane, while the HCECs cultivated on laminin-211 (not expressed in DM) and non-coated dishes showed less hexagonality. Cell densities were significantly higher in HCECs cultivated on laminin-511 and -521 coated dishes compared to those on non-coated dishes (2162.0±3.9, 2367.5±1.3 and 1015.5±23.2 cells/mm2, respectively, p<0.01). Cellular adhesion of HCECs was significantly promoted in HCECs cultivated on laminin-511 and -521 coated dishes compared to that on non-coated dishes (151.3±3.8% and 153.1±3.1%, respectively, p<0.01). BrdU incorporation was significantly enhanced in HCECs cultivated on laminin-511 or -521 coated dishes compared to that on non-coated dishes (261.9±5.1% and 324.0±3.6%, respectively, p<0.01).
Our findings indicate that laminin-511 and 521 are expressed in DM and that those laminins might provide an ideal niche for HCECs and be applicable for the in vitro expansion of HCECs.
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