September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
The effect of p38 mitogen-activated protein kinase inhibitor on cell density and phenotype of cultivated human corneal endothelial cells
Author Affiliations & Notes
  • Akane Hongo
    Biomedical Engineering , Doshisha University, Kyotanabe, Kyoto, Japan
  • Naoki Okumura
    Biomedical Engineering , Doshisha University, Kyotanabe, Kyoto, Japan
  • Makiko Nakahara
    Biomedical Engineering , Doshisha University, Kyotanabe, Kyoto, Japan
  • Noriko Koizumi
    Biomedical Engineering , Doshisha University, Kyotanabe, Kyoto, Japan
  • Footnotes
    Commercial Relationships   Akane Hongo, None; Naoki Okumura, Doshisha University (P); Makiko Nakahara, None; Noriko Koizumi, Doshisha University (P)
  • Footnotes
    Support  Program for Strategic Research Foundation at Private Universities from MEXT
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5292. doi:
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      Akane Hongo, Naoki Okumura, Makiko Nakahara, Noriko Koizumi; The effect of p38 mitogen-activated protein kinase inhibitor on cell density and phenotype of cultivated human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5292.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We recently began a Japanese clinical trial of a cell-based therapy for treating corneal endothelial dysfunction, yet corneal endothelial cell (CEC) senescence during culture associated with cell density (CD) drop is a serious obstacle in obtaining large quantities of cells for clinical use. This study investigated the feasibility of using SB203580, a selective p38 mitogen-activated protein kinase (p38MAPK) inhibitor, in the cultivation of CECs.

Methods : For CEC cultivation, 16 human donor corneas (mean donor age; 52.9±17.1 years) were used. Each cornea was cut into 2 pieces and cultured in OptiMEM-I based culture medium supplemented with or without 10μM of SB203580. Cell morphology was evaluated by phase contrast microscopy and CD was evaluated post reaching confluence (culture time: 24.7±5.2 days). Expression of function-related proteins Na+/K+-ATPase and ZO-1, and distribution of actin were examined by fluorescein staining. Expression of 36 cytokines and chemokines related to senescence-associated secretory phenotype (SASP) were evaluated by use of the Proteome Profiler Human Cytokine Array Kit (R&D Systems, Inc.).

Results : Phase contrast microscopy showed that CECs were cultured successfully and exhibited a hexagonal and monolayer morphology, both with and without SB203580. However, the mean CD of SB203580-cultured CECs was significantly higher than in CECs cultured without SB203580 (2623.2±657.3 cells/mm2 and 1751.8±627.5 cells/mm2, respectively) (p<0.01). Fluorescein staining showed that function-related markers Na+/K+-ATPase and ZO-1were expressed in SB203580-cultured CECs, yet their expression was partially disrupted in CECs cultured without SB203580. Actin was distributed in the cell cortex similar to its distribution in healthy cells in SB203580-cultured CECs, yet cortical actin distribution was irregular, with stress fibers, in CECs cultured without SB203580. Several cytokines and chemokines, such as IL-6, IL-8, and MCP-1, were suppressed in CECs cultured with SB203580, yet not in CECs cultured without SB203580.

Conclusions : Our data suggests p38MAPK signaling might be activated by culture-condition-related cell stress that induces SASP, and that p38MAPK inhibition enables CECs culture with maintaining high CD and functional phenotype, thus indicating its usefulness for corneal endothelial regenerative medicine.

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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