Validation of Na,k-ATPase pump function of corneal endothelial cells for corneal regenerative medicine

Shin Hatou, Kazunari Higa, Emi Inagaki, Satoru Yoshida, Erika Kimura, Ryuhei Hayashi, Motokazu Tsujikawa, Kazuo Tsubota, Kohji Nishida, Shigeto Shimmura

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)901-910
Number of pages10
JournalTissue Engineering - Part C: Methods
Volume19
Issue number12
DOIs
Publication statusPublished - 2013 Dec 1

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Regenerative Medicine
Endothelial cells
Adenosine Triphosphatases
Endothelial Cells
Pumps
Ouabain
Rabbits
Membranes
Polyesters
Anterior Chamber
Tissue Engineering
Stem cells
Tissue engineering
Collagen
Standardization
sodium-translocating ATPase
Stem Cells
Tissue
Cell Line
Injections

ASJC Scopus subject areas

  • Biomedical Engineering
  • Bioengineering
  • Medicine (miscellaneous)

Cite this

Validation of Na,k-ATPase pump function of corneal endothelial cells for corneal regenerative medicine. / Hatou, Shin; Higa, Kazunari; Inagaki, Emi; Yoshida, Satoru; Kimura, Erika; Hayashi, Ryuhei; Tsujikawa, Motokazu; Tsubota, Kazuo; Nishida, Kohji; Shimmura, Shigeto.

In: Tissue Engineering - Part C: Methods, Vol. 19, No. 12, 01.12.2013, p. 901-910.

Research output: Contribution to journalArticle

Hatou, Shin ; Higa, Kazunari ; Inagaki, Emi ; Yoshida, Satoru ; Kimura, Erika ; Hayashi, Ryuhei ; Tsujikawa, Motokazu ; Tsubota, Kazuo ; Nishida, Kohji ; Shimmura, Shigeto. / Validation of Na,k-ATPase pump function of corneal endothelial cells for corneal regenerative medicine. In: Tissue Engineering - Part C: Methods. 2013 ; Vol. 19, No. 12. pp. 901-910.
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