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Mengyuan Xie, Qiaolang Zeng, Zekai Cui, Yonglong Guo, Shiwei Liu, Peiyuan Wang, Jun Zhang, Jiansu Chen; A new method to construct tissue engineered corneal endothelial layer by spray printing. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2228.
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© ARVO (1962-2015); The Authors (2016-present)
The main treatment for the dysfunction of corneal endothelial cells (CECs) is penetrating keratoplasty or CEC transplantation, which are restricted by factors such as donor corneal scarcity and postoperative side effects. In this study, we used the cell-spray approach to build planar and curvature tissue-engineered (TE) CEC layer in vitro and study the therapeutic potential for corneal CEC dysfunction.
Bovine CECs (B-CECs) were spray-printed onto the surface of planar collagen membrane and cultured for 7 days. The specific antigen expressions and morphology of the B-CECs cells printed on the planar collagen membrane were observed. The transmembrane resistance of the sprayed and non-sprayed B-CECs was measured separately by a transendothelial electrical resistance (TEER) assay to determine the barrier function after printing. Frozen sections of the cell sheets were paved for DAPI staining to observe the distribution of cells on both planar and curvature collagen membrane.
The immunofluorescence staining indicated that Na+-K+-ATPase and ZO-1 phenotypes in both of spray-printed and traditionally cultured B-CEC group were positive (Fig.1 A). The observations under scanning electron microscopy (SEM) and atomic force microscope (AFM) showed that spray-printed and cultured B-CECs had a hexagonal morphology on the planar collagen membrane (Fig.1 B, C). In addition, there is no statistical difference in the TEER between B-CEC spray-printed group and traditional seeding group. The DAPI staining results of the frozen sections revealed that the printed cells could grow uniformly on both of planar and curvature collagen membrane (Fig.2).
B-CECs spray-printed on planar collagen membranes and conventional cultured group have similar characteristic hexagonal structure, cell phenotype, and barrier functions. The conventional inoculation for curved TE construction is challenge, while spray printing allows the cells to grow evenly on the curvature collagen membrane. These results indicate that cell-spray may be a suitable approach to build TE CEC layer for the treatment of corneal CEC dysfunction.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
B-CECs maintained phenotypic (A) and hexagonal morphology (B, C) after spray printed on planar collagen membrane
B-CECs sprayed on curvature collagen membrane formed a uniform layer after culturing for 7 days
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