valuation the use of proximal tubule cells as autograft materials for corneal endothelium
Date Issued
2012
Date
2012
Author(s)
Chang, Shao-Hsuan
Abstract
The corneal endothelium is physiologically the most important monolayer of the cornea and plays a pivotal part on the maintenance of corneal transparency and is not known to regenerate in vivo. Several corneal transplantations have been greatly restricted due to the global shortage of donor corneas, whereas the demands for corneal grafts have been gradually increasing. Furthermore, Non-immunologic endothelial decompensation and allograft endothelial rejection may lead to progressive graft failure. In the present study, we developed a novel method for primary culture of rat proximal tubule cells (PTCs) as an alternative for graft material. We evaluated the effects of chitosan membrane on the primary cultured rat PTCs by comparing with collagen surface. Rat PTCs cultured on chitosan surface displayed a prominent expression of proliferation marker and ERK activation. 8 days after reaching confluence, PTCs showed specific characteristic of transporting epithelia, the formation of domes, which mimic the onset of differentiation. Dome formation is thought to parallel tubular differentiation and morphogenesis in vivo and represents the result of active water transport. The transepithelial electrical resistance evaluation revealed that differentiated PTCs established on chitosan surface exhibited high capacity for transepithelial fluid transport. Moreover, Immunofluorescence revealed that the Na+-K+ ATPase, which is critical for the proper physiological function of the corneal endothelium, was ubiquitously expressed and localized to the cell-to-cell borders. These cultured PTCs were harvested as intact monolayer cell sheets with chitosan film which prepared via simple process. Immunostaining for ZO-1, Na+-K+ ATPase was positive in the cell sheet. Scanning electron microscopy indicated that these cells were primarily cobblestone-shaped morphology with numerous microvilli. Rat PT cell sheets were placed onto the posterior surface of the transplanted rabbit cornea. However, the results of xenograft transplantation showed the fabricated sheets failed of maintaining proper stromal hydration in vivo. Our results implicate that chitosan membrane would be a valuable tool for studying the function and development of proximal tubule cells, as well as supporting the value of harvested cell sheets for clinical applications.
Key word: corneal endothelium, proximal tubule cells, chitosan, dome formation, pump function
Subjects
corneal endothelium
proximal tubule cells
chitosan
dome formation
pump function
Type
thesis
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