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Eun-Soon Kim, Soyoung Hong, Changmo Hwang, Jae Yong Kim, Hungwon Tchah; Corneal surface regeneration using human conjunctiva epithelial cells-cultured biodegradable membrane scaffold. Invest. Ophthalmol. Vis. Sci. 2016;57(12):884.
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© ARVO (1962-2015); The Authors (2016-present)
We attempted to reconstruct damaged ocular surface by human conjunctiva epithelial cells-cultured biodegradable membrane scaffolds. We evaluated feasibility of stratified corneal epithelial cell using biodegradable membrane scaffold as epithelium substitute, stacked conjunctival monolayer cell to form multilayer epithelial cell and applied them to the damaged ocular surface, and proposed a method of stratified corneal epithelial cell culture using biodegradable membrane scaffold in order to efficiently form a multilayered cultivated epithelial cell tissue,.
Human cadaveric donor-oriented conjunctiva epithelial cells were seeded at a density of 5*105 cells/sheet on the biodegradable membrane scaffold, bonded with empty transwell (12 well) and pre-coated with 1 mg/ml collagen. In terms of sheet preparations, a 10% (w/v) solution of Polycaprolactone (PCL) in 1, 1,1,3,3,3-Hexafluoro-2-propanol (HFIP) and 15% (w/v) of Poly (DL-lactide-goglycolide) (PLGA) in HFIP were mixed at a 4:1 volume ratio and electrospun at a flow rate of 2 mL/h and DC voltage of 18 kV. The electrospun polymer mats were collected in the form of a random mesh on the collector. As for formation of cell layer and maintenance of phenotype, live/dead assay and actin (Rhodamine phalloidin) staining with DAPI staining were carried out 2, 4 and 7 days after seeding. MUC1 staining was performed at 10 days.
During cell culture on the sheet, we observed stable cell morphology such as cell viability, cell adhesion, and epithelial cell marker using with laser scanning microscopy (Zeiss, LSM 780) and ZEN software. The actin-staining intensity was about 4-fold higher at 7 days after seeding, compared with that at 2 days
We found the feasibility of stratified epithelial cells-cultured biodegradable membrane scaffold for damaged ocular surface treatment. However, further assessment needs to be carried out to show the generation of transparent epithelial sheet during regeneration of corneal epithelium.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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