An evaluation of cultivated corneal limbal epithelial cells, using cell-suspension culture.
OBJECTIVE
A previous report has described an ocular surface reconstruction method involving the use of cultivated corneal epithelium derived from limbal explants. In the current study, a new culture system was developed involving the in vitro propagation on amniotic membrane (AM) of epithelial cells from enzymatically dissociated limbal epithelium. The purpose of this new method is to produce a cultivated epithelial cell layer that contains stem cells and that is superior to explanted cultivated epithelium. The new cell-suspension technique was compared with the existing explant method.


METHODS
Limbal epithelial cells were dissociated from donor eyes by dispase and seeded on the denuded AM. Small pieces of limbal epithelium were also cultured on denuded AM as explant cultures. The cultivated epithelium was examined by electron microscopy and immunohistochemistry for cornea-specific keratins (K3 and K12).


RESULTS
Both cell-suspension and explant culture methods produced a healthy epithelial cell layer. The cell-suspension culture had significantly (P < 0.001) more desmosomal junctions between the explant-cultured basal cells. In addition, the intercellular spaces between the cell-suspension's basal cells were significantly (P < 0.001) smaller than those between the explant-cultured basal cells. Both types of cultivated epithelium showed positive expression of K3 and K12 keratins. In the cell-suspension culture, expression of K3 and K12 keratins was more prominent in the superficial cells.


CONCLUSIONS
Corneal epithelial cells were successfully regenerated in vitro by a cell-suspension culture system. The suspension-cultured epithelium must include some stem cells and morphologically is significantly superior to explant-cultured epithelium. Thus, this new technique is potentially more suitable for cultivated corneal limbal epithelial transplantation.
Koizumi
Noriko
N
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi-hirokoji, Kamigyo-ku, Kyoto 602-0811, Japan. nkoizumi@ophth.kpu-m.ac.jp
Cooper
Leanne J
LJ
Fullwood
Nigel J
NJ
Nakamura
Takahiro
T
Inoki
Keiko
K
Tsuzuki
Masakatsu
M
Kinoshita
Shigeru
S
D016475:3T3 Cells; D000650:Amnion; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007633:Keratins / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology*; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence
D007633:Keratins