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

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. [ABSTRACT FROM AUTHOR]