title: Validation of Na,K-ATPase pump function of corneal endothelial cells for corneal regenerative medicine.
abstract: Tissue-engineering approaches to cultivate corneal endothelial cells (CECs) or induce CECs from stem cells are under investigation for the treatment of endothelial dysfunction. Before clinical application, a validation method to determine the quality of these cells is required. In this study, we quantified the endothelial pump function required for maintaining the corneal thickness using rabbit CECs (RCECs) and a human CEC line (B4G12). The potential difference of RCECs cultured on a permeable polyester membrane (Snapwell), B4G12 cells on Snapwell, or B4G12 cells on a collagen membrane (CM6) was measured by an Ussing chamber system, and the effect of different concentrations of ouabain (Na,K-ATPase specific inhibitor) was obtained. A mathematical equation derived from the concentration curve revealed that 2 mM ouabain decreases pump function of RCECs to 1.0 mV, and 0.6 mM ouabain decreases pump function of B4G12 on CM6 to 1.0 mV. Ouabain injection into the anterior chamber of rabbit eyes at a concentration of <2 mM maintained the corneal thickness, while those over 3 mM significantly increased the corneal thickness. B4G12 cell sheets transplanted into rabbit eyes treated with 0.6 mM ouabain maintained the corneal thickness, while 3.5 mM ouabain significantly increased the corneal thickness. Taken together, pump function >1.0 mV is required to maintain the corneal thickness. These results can be used for standardization of CEC pump function and validation of tissue-engineered CEC sheets for clinical use.
authors.affiliation: 1 Department of Ophthalmology, Keio University School of Medicine , Tokyo, Japan .
authors.forename: Shin
authors.initials: S
authors.lastname: Hatou
authors.forename: Kazunari
authors.initials: K
authors.lastname: Higa
authors.forename: Emi
authors.initials: E
authors.lastname: Inagaki
authors.forename: Satoru
authors.initials: S
authors.lastname: Yoshida
authors.forename: Erika
authors.initials: E
authors.lastname: Kimura
authors.forename: Ryuhei
authors.initials: R
authors.lastname: Hayashi
authors.forename: Motokazu
authors.initials: M
authors.lastname: Tsujikawa
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tsubota
authors.forename: Kohji
authors.initials: K
authors.lastname: Nishida
authors.forename: Shigeto
authors.initials: S
authors.lastname: Shimmura
mesh_terms: D000818:Animals; D002460:Cell Line; D003315:Cornea / Q000166:cytology / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D010042:Ouabain / Q000494:pharmacology; D011091:Polyesters / Q000737:chemistry; D011817:Rabbits; D044968:Regenerative Medicine; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D023822:Tissue Engineering
chemical_list: D004791:Enzyme Inhibitors; D011091:Polyesters; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase