Mesenchymal cells and fluid flow stimulation synergistically regulate the kinetics of corneal epithelial cells at the air–liquid interface

  • Kosuke Kawata
  • Shigehisa AokiEmail author
  • Maki Futamata
  • Mihoko Yamamoto-Rikitake
  • Isao Nakao
  • Hiroshi Enaida
  • Shuji Toda
Basic Science



In vivo microenvironments are critical to tissue homeostasis and wound healing, and the cornea is regulated by a specific microenvironment complex that consists of cell–cell interactions, air–liquid interfaces, and fluid flow stimulation. In this study, we aimed to clarify the effects of and the correlations among these three component factors on the cell kinetics of corneal epithelial cells.


Human corneal epithelial–transformed (HCE–T) cells were cocultured with either primary rat corneal fibroblasts or NIH 3T3 fibroblasts. We employed a double-dish culture method to create an air–liquid interface and a gyratory shaker to create fluid flow stimulation. Morphometric and protein expression analyses were performed for the HCE–T cells.


Both the primary rat fibroblasts and the NIH 3T3 cells promoted HCE–T cell proliferation, and the presence of fluid flow synergistically enhanced this effect and inhibited the apoptosis of HCE–T cells. Moreover, fluid flow enhanced the emergence of myofibroblasts when cocultured with primary rat fibroblasts or NIH 3T3 cells. Extracellular signal-regulated kinase and p38 signaling were regulated either synergistically or independently by both fluid flow and cellular interaction between the HCE–T and NIH 3T3 cells.


The cell–cell interaction and fluid flow stimulation in the air–liquid interface synergistically or independently regulated the behavior of HCE–T cells. Fluid flow accelerated the phenotypic change from corneal fibroblasts and NIH 3T3 cells to myofibroblasts. Elucidation of the multicomponent interplay in this microenvironment will be critical to the homeostasis and regeneration of the cornea and other ocular tissues.


Corneal microenvironment Shear stress Cell–cell interaction Wound healing 



We are grateful to Professor S. Aishima and Dr. M. Hashiguchi for useful discussion and for sharing their dataset, and we thank T. Sakumoto, S. Morito, M. Nishida, F. Mutoh, S. Nakahara, and I. Nanbu for their excellent technical assistance. We also thank Mr. K. Tokaichi for refining the English of the manuscript. We also thank Enago ( for the English language review.


This work was supported in part by the Center for Clinical and Translational Research of Kyushu University Hospital (to S.A.), and Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology for Scientific Research (no. 16K09284 to S.A. and no. 18K09451 to H.E.).

Compliance with ethical standards

All animal experiments complied with the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and institutional guidelines for the care and use of animals were followed.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Pathology and Microbiology, Faculty of MedicineSaga UniversitySagaJapan
  2. 2.Department of Ophthalmology, Faculty of MedicineSaga UniversitySagaJapan

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