A Novel Method Using Quantum Dots for Testing the Barrier Function of Cultured Epithelial Cell Sheets.
OBJECTIVE
The corneal epithelium provides a barrier to protect the deeper structures of the eye from any particles or pathogens. Cultured epithelial cell sheets are used in transplantation surgery for corneal repair or regeneration. The purpose of this study was to develop a novel method using fluorescent quantum dot nanoparticles for validating the quality and barrier function of cultured epithelial cell sheets.


METHODS
Human function epithelial cell sheets, cultured from oral mucosal or corneal limbal cells, were incubated in either normal calcium-containing medium or medium containing no calcium with a calcium chelator. Also contained in the media were suspensions of two different sizes of quantum dots. Following incubation, analysis of quantum dot penetration was carried out using confocal microscopy.


RESULTS
In contrast to the cell sheets incubated in calcium-containing medium, removal of extracellular calcium resulted in the disruption of tight junctions, compromising the cell sheet's barrier function. This caused a reduction in transepithelial electrical resistance and deeper, more ubiquitous penetration of the quantum dots into the paracellular space and interior of the cell sheet.


CONCLUSIONS
This method provides easy to interpret qualitative and quantitative data on the functionality of a cell sheet's tight junctions, as well as nanoscale and microscale structural information on its surface and interior morphology, and any localized areas of damage or abnormality. This novel technique could be used as part of the validation system for cultured epithelial cell sheets for use in transplantation.
Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan.
Thomas J
TJ
Duncan
Department of Visual Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Japan.
Koichi
K
Baba
Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan.
Yoshinori
Y
Oie
Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan.
Kohji
K
Nishida
D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D016039:Corneal Transplantation; D019573:Epithelium, Corneal / Q000166:cytology*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D018613:Microscopy, Confocal; D008875:Middle Aged; D009061:Mouth Mucosa / Q000166:cytology; D045663:Quantum Dots; D019108:Tight Junctions