Ex vivo construction of rabbit corneal endothelial cell sheets on a porcine descemet membrane graft

Liu,Chunyu; Tang,Shenfei; Niu,Guozhen; Zhang,Juan; Huang,Xinyu; Zhang,Yushan; Bi,Yanlong

doi:10.3892/etm.2019.7573

Ex vivo construction of rabbit corneal endothelial cell sheets on a porcine descemet membrane graft

Authors:
- Chunyu Liu
- Shenfei Tang
- Guozhen Niu
- Juan Zhang
- Xinyu Huang
- Yushan Zhang
- Yanlong Bi
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Affiliations: Department of Ophthalmology, Tongji Hospital Affiliated with Tongji University School of Medicine, Shanghai 200333, P.R. China
Published online on: May 10, 2019 https://doi.org/10.3892/etm.2019.7573
Pages: 242-252
Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to investigate the feasibility of a new graft construction method using rabbit corneal endothelial cells (RCECs) and a porcine descemet membrane (DM) carrier. RCECs were isolated and the experimental group was treated with Y‑27632, whereas the control group were cultured in medium without Y‑27632. RCEC morphology was observed using an inverted microscope, and cell proliferation and apoptosis were detected by flow cytometry. To confirm the presence of RCECs, reverse transcription‑quantitative PCR was used to detect gene expression levels of Na+‑K+‑ATPase, aquaporin 1, collagen α2 (IV), collagen α1 (VIII) and keratin‑12. Histocompatibility testing was used to detect porcine DM antigenicity. A DM‑RCEC graft was constructed, and morphology was observed using alizarin red‑trypan blue and haematoxylin and eosin staining. Cell membrane potential was measured to evaluate the physical function of the DM‑RCEC graft. Complex graft tension was measured using a modified tension detector and compared with fresh porcine DM‑endothelium complex. In vitro‑cultured RCECs formed a monolayer with a polygon morphology and cobblestone‑like arrangement. In vitro‑cultured RCECs exhibited typical RCEC characteristics before and after transplantation. The proliferation rates of the experimental and control groups were 62.68 and 34.50%, respectively (P<0.05); the apoptosis rates of the experimental and control groups were 8.99 and 35.68%, respectively (P<0.05). There was no antigenicity observed with the porcine DM. The action potential amplitude of the experimental and control groups was over ‑80 mV, reflecting normal RCEC physiological function. The tension measurements of the experimental and control groups were 20.0248±1.048 and 20.5013±0.657 g, respectively (P>0.05). Taken together, the results of the present study demonstrated that Y‑27632 enhanced RCEC proliferation. In addition, the findings revealed the successful ex vivo construction of a RCEC sheet on a porcine DM graft.

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