The effects of Rho-associated kinase inhibitor Y-27632 on primary human corneal endothelial cells propagated using a dual media approach.

Donald T. Tan,Benjamin L. George,Khadijah Adnan,Gary S. L. Peh,Xin-Yi Seah,Heng-Pei Ang,Jodhbir S. Mehta

doi:10.1038/srep09167

Abstract

The global shortage of donor corneas has garnered extensive interest in the development of graft alternatives suitable for endothelial keratoplasty using cultivated primary human corneal endothelial cells (CECs). We have recently described a dual media approach for the propagation of human CECs. In this work, we characterize the effects of a Rho-kinase inhibitor Y-27632 on the cultivation of CECs propagated using the dual media culture system. Seventy donor corneas deemed unsuitable for transplantation were procured for this study. We assessed the use of Y-27632 for its effect at each stage of the cell culture process, specifically for cell attachment, cell proliferation, and during both regular passaging and cryopreservation. Lastly, comparison of donor-matched CEC-cultures expanded with or without Y-27632 was also performed. Our results showed that Y-27632 significantly improved the attachment and proliferation of primary CECs. A non-significant pro-survival effect was detected during regular cellular passage when CECs were pre-treated with Y-27632, an effect that became more evident during cryopreservation. Our study showed that the inclusion of Y-27632 was beneficial for the propagation of primary CECs expanded via the dual media approach, and was able to increase overall cell yield by between 1.96 to 3.36 fold.

Highlights

The effects of Rho-associated kinase inhibitor Y-27632 on primary human corneal endothelial cells propagated using a dual media approach
To determine the optimal concentration of Y27632 to be used in this study, we performed a cell impedance-based assay following the exposure of corneal endothelial cells (CECs) to various concentrations of Y27632
The isolation and propagation of primary human CECs has been described by various research groups using different approaches revolving around the use of a single basal medium, each supplemented with different additives[23,24,25,26,32,35]

Summary

Introduction

The effects of Rho-associated kinase inhibitor Y-27632 on primary human corneal endothelial cells propagated using a dual media approach. The global shortage of donor corneas has garnered extensive interest in the development of graft alternatives suitable for endothelial keratoplasty using cultivated primary human corneal endothelial cells (CECs). When extensive cell-loss of the CE layer occurs beyond a certain threshold such that the functional capacity of the remaining CECs becomes compromised, corneal decompensation will occur This results in cornea edema that will eventually lead to corneal blindness[1]. Donor corneas, even those rejected for transplant due to low corneal endothelial cell counts[20,21], can be set aside for cellular expansion for these alternative approaches This requires the capacity to propagate human CECs in an in vitro setting. The direct inhibition of TGF-b signaling using SB431542 and the use of bone morphogenetic protein-7 to inhibit TGF-b-mediated EMT has been reported to prevent the fibroblastic transformation of expanded CECs26

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Conclusion